3 7 7

mu /fa.

ANALYSIS OF THE Trypanosoma brucei GENOME AND IDENTIFICATION AND CHARACTERIZATION OF A GENE FAMILY ENCODING

PUTATIVE EF-HAND CALCIUM-BINDING PROTEINS

DISSERTATION

Presented to the Graduate Council of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

James H. DeFord, B.S., M.S.

Denton, Texas

May, 1998

DeFord, James, H. Analysis Of The Trypanosoma brucei Genome And

Identification And Characterization Of A Gene Family Encoding Putative

EF-Hand Calcium-Binding Proteins. Doctor of Philosophy (Biology), May, 1998,

171 pp., 4 tables, 17 figures, bibliography, 159 titles.

The flagellum of Trypanosoma brucei contains a family of antigenically

related EF-hand calcium-binding proteins which are called the calflagins.

Genomic Southern blots indicated that multiple copies of calflagin genes occur

in T brucei. All of the copies were contained in a single 23 kb Xho\-Xho\

fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded

calflagin sequences. Two new members of the calflagin family were found from

genomic clone sequences. The deduced amino acid sequences of the

genomic clones showed the calflagin genes were arranged tandemly along the

genomic fragments and were similar to previously described calflagins. The

calflagin genes were related by two unrelated 3' flanking sequences. An open

reading frame that was unrelated to any calflagin was found at the 5' end of the

2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three

EF-hand calcium-binding motifs and one degenerate EF-hand motif. In

general, variability among the T. brucei calflagins is greater than related

proteins in T. lewisii and T. cruzi. This variability results from amino acid

substitutions at the amino and carboxy termini, and duplication of internal

segments.

Trypanosoma DNA was sequenced by M13 shotgun cloning techniques

to expedite the discovery of novel targets. 455 sequences were generated

representing approximate 1% of the T. Brucei genome. The sequences were

evaluated for putative genes and non coding elements against the nr (non-

redundant) and GSS databases at the National Center for Biotechnology

Information using BLAST family software. 84 sequences are reported with

significant homology to database sequences.

Several sequences were found to be of particular interest and are

discussed separately. Putative sequences for: a putative Pyruvate: ferredoxin

oxidoreductase (dbGSS no. B13566); a putative Fe-superoxide dismutase

(dbGSS no. B13733); and a non-coding sequence (dbGSS no. B13593) with

homology to a region 3' to human apoptosis genes are reported.

In conclusion the identification of novel targets via shotgun cloning

methods is a viable method that is augmented with each submission to the

database.

3 7 7

mu /fa.

ANALYSIS OF THE Trypanosoma brucei GENOME AND IDENTIFICATION AND CHARACTERIZATION OF A GENE FAMILY ENCODING

PUTATIVE EF-HAND CALCIUM-BINDING PROTEINS

DISSERTATION

Presented to the Graduate Council of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

James H. DeFord, B.S., M.S.

Denton, Texas

May, 1998

ACKNOWLEDGEMENTS

I would like to express my thanks and gratitude to my major professor, Dr.

Robert Benjamin for his support and advice in both my research and career

decisions. I would also like to express special thanks to the following persons

and institutions for their help and assistance in this project. To Dr. Larry Ruben

at Southern Methodist University for his expert advice, guidance, and use of his

lab. To Dr. Glen Evans at the McDermott Center for Human Growth and

Development at the University of Texas Southwestern Medical Center at Dallas

for the opportunity to learn use some of the most sophisticated DNA equipment

in the world. And to Michelle for her untiring help and belief in both the project

and myself.

TABLE OF CONTENTS

List Of Tables vi

List Of Figures vii

Abreviations ix

1. Introduction 1.1 African trypanosomes. 1 1.2 South american trypanosomes. 4 1.3 Rationale for research expediency/epidemiology. 6 1.4 Calcium. 7 1.5 Calcium binding proteins. 11 1.6 Calf lag ins. 16 1.7 Calcium/calcium binding protein interactions on trypanosomal

metabolism and development. 19 1.8 Calcium: effects on trypanosome killing and treatment. 27 1.9 A shotgun approach to identification of novel targets. 30

2. Materials and Methods 2.1 Amplification and isolation of genomic fragments. 33 2.2 Isolation of plasmids (analytical scale). 35 2.3 Agarose mini gel analysis. 36 2.4 Gel photography. 37 2.5 Digesting dna with restriction endonucleases. 38 2.6 Large scale isolation of recombinant plasmids. 38 2.7 Preparative scale digestion of cloned dna fragments. 42 2.8 Preparative scale isolation of cloned dna fragments detected by

uv shadowing techniques. 42 2.9 Electroelution and dna recovery from excised from gels. A1 2.10 Screening isolated dna fragments for restriction sites. 50 2.11 Subcloning into m13mp and pucvectors. 52 2.12 TSS competant cells. 53 2.13 Transformation of dh5 alpha F'with recombinant m13mp 18/19. 54 2.14 Transformation of dh5 alpha F or F cells with recombinant

plasmid. 54 2.15 Preparation ofm13mp single stranded template for sequencing. 55 2.16 Nucleotide sequencing double-stranded and single-stranded

templates by the dideoxyribonucleotide method. 57 2.17 Preparation and electrophoresis of polyacrylamide sequencing

gels. 59 2.18 Autoradiography and sequence analysis. 65

2.19 Detection of genomic sequences using radio labeling techniques: southern blotting. 65

2.20 Radio labeled probes: end labeled; random primed. 67 2.21 Hybidizations. 67 2.22 M13 library construction: isolation , size fractionation and

preparation of genomic dna. 6 8 2.23 M13 library construction: vector preparation and ligation. 70 2.24 M13 library construction: transformation of competant cells. 71 2.25 M13 library sequencing and analysis. 72 2.26 Cosmid library construction: vector preparation. 73 2.27 Cosmid library construction: isolation and preparation of

genomic dna. 74 2.28 Cosmid library construction: ligation of genomic dna tovector. 75 2.29 Pakaging, titering, and plating the cosmid library. 78 2.30 Cosmid library finger printing. 79

3. Results 3.1 Detection and isolation of genomic clones. 80 3.2 Restriction site analysis and subcloning of genomic clones. 85 3.3 Sequence analysis of genomic clones pB1.7 and pB2.5. 85 3.4 Comparisons of coding and flanking sequences to other calflagin

clones. 96 3.5 Analysis of a cloned T. brucei repeated element. 104 3.6. Detection of novel T.brucei sequences using M13 shotgun

sequencing. 107 3.7 M13 Sequences of particular interest. 113 3.8 Cosmid library analysis. 114

4. Discussion 132

References 142

LIST OF TABLES

1. Labeling Mix Table. 60

2. Sequenase Dilution Table. 60

3. Sequencing Reaction Table. 61

4. Trypanosoma brucei: Shotgun Genomic Sequence Survey. 108

List Of Figures

IA. Electrophresis stand and buffer pump assembly for vertical agarose gel electrophoresis. 45

IB. Assembled cassette for vertical acrylamide gel electrophoresis. 45

2. Principle of UV shadowing for the visualization of DNA

bands in agarose gels. 48

3A. Sau3A I partial digestions of genomic T. brucei DNA 76

3B. Preparation of scos seq 3 cosmid vector 76

3C. Conversion of ligation products to high molecular weight recombinant concatamer DNA 76

4. Southern blot of T. brucei DNA probed with 32P-labeled

cDNA, pTB44A. 81

5A. Recombinant plasmid pB2.5. 83

5B. Recombinant plasmid 1.7. 83

6. Restriction digested genomic clones pB1.7 and pB2.5. 86

7. Restriction map and sequencing strategy of the genomic clonepB1.7. 88

8. Restriction map and sequencing strategy of the genomic clone pB2.5. 90

9. The complete nucleotide sequence of the genomic clone pB1.7. 92

10. The complete nucleotide sequence of the genomic clone pB2.5. 94

11. Common features of calflagin clones pB2.5, pB1.7, pBTB44A, and pTB17. 98

12A. The sequence and the translation of the carboxyl end of and ORF in the genomic clone Tb-24. 100

12B. A Kyte and Doolittle hydropathy plot of the 5' open reading frame in TB-24. 100

13. Comparisons of calflagin protein sequences from T. brucei,

T. cruzi, and T. lewisi. 102

14. Analysis of a Trypanosoma brucei repeated element. 105

15A. Identification of genes involved in regulation of oxidative stress inT. brucei. 115

15B. A comparison between a translated region of T. brucei and Fe-superoxide dismutase from T. cruzi. 115

16. Comparison between coding and non-coding regions of T. brucei and a human apoptosis gene sequence. 117

17. Analysis of random cosmid clones from T. brucei. 119

LIST OF ABREVIATIONS

BAP bacterial alkaline phosphatase BLAST basic local alignment search tool bp base pairs CaBP calcium binding proteins cDNA copy DNA CIAP calf intestinal alkaline phosphatase DMSO dimethyl sulfoxide dpm disintegrations per minute DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EST expressed sequence tags EtBr ethidium bromide ETOH ethanol g gravity GSS genome sequence survey k thousand kb kilobases IPTG isopropyl-1-thio-B-D-galactopyranoside kDa kilodalton LB Luria-Bertani mb megabases mcs mutiple cloning site ml milliliter mm millimeter mM millimolar MW molecular weight NCBI National Center for Biotechnology Information nm nanometer nr non-redundant data base (NCBI) PFO pyruvate: ferredoxin oxidoreductase ORF open reading frame rpm revolutions per minute RT room temperature SDS sodium dodecyl sulfate TEMED NNN"N"-tetrametylethylene-diamine TLF trypanosome lytic factor VSG variable surface glycoprotein X-gal 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside

1. Introduction

1.1 African trypanosomes.

African trypanosomes are protozoan parasites that infect a variety of animals.

Within the family Trypanosomatidae are a number of genera. Two of these,

Leishmania and Trypanosoma, present major health and economic problems

for humans and their livestock. Members of the genus Trypanosoma cause two

distinct disease processes limited to, but not restricted to, two geographical

areas. One of these processes known as "sleeping sickness" is found in vast

areas of central Africa. The other is known as Chagas disease and is found in

Central and South America.

The economic impact of African trypanosomiasis (the disease) is mainly

caused by three organisms that are essentially indistinguishable from each

other serologically. The organisms, T. brucei brucei,T. brucei gambiense, and

T. brucei rhodesiense, are separated by their clinical features and geographical

locations. T. rhodesiense is the cause of East African or Rhodesian sleeping

sickness, and is the most virulent of the three affecting both humans and

animals. T. gambiense affects only humans and is found in Central and West

Africa. T. brucei is endemic to all Africa except North Africa. It is apparently non-

infective for humans and its mortality on native game is low. However, it is

devastating to domestic cattle and other economically important animals.

Several species of Glossina (tsetse flies) are vectors of human and

animal trypanosomiasis. Both sexes of the flies transmit the disease and are

2

infective for life (2-3 months). The tsetse fly becomes infected when taking a

blood meal from an infected host. There are a wide variety of reservoir hosts for

the T. rhodesiense and T. brucei forms. Trypomastigotes (blood forms) are

ingested by the fly and transformation to the procyclic trypanomastigote form

takes place in the midgut of the fly. These forms continue to divide for ten days

before migrating to the salivary glands of the fly and transforming into

epimastigotes. The epimastigotes are transformed into metatrypanosomes,

which is the infective stage for humans and animals. While the minimum

infective dose of trypanosomes is 300-500, the tsetse fly is capable of injecting

40,000 or more (1). The metatrypanosomes will transform into trypomastigotes

in the subcutaneous tissues, and a primary chancre will mark the site of

inoculation. Dissemination into the blood and lymph of the new host completes

the life cycle. Other modes of transmission include eating infected raw meat and

mechanical transmission by biting flies.

The pathology is related to fluctuating parasitemias due to antigenic

variation of the trypanosomes. Two classes of antibody are directed against

either cellular antigens (IgG) or surface Ags (IgM). Within a given population of

trypanosomes in an infected host, a subpopulation escapes detection by host

antibody. The undetected subpopulation of trypanosomes are variants that

have different antigenic surface glycoproteins (VSGs). As the original

population is destroyed, a new subpopulation begins to multiply until it to is in

turn detected and destroyed by host immune response. As before, another

subpopulation arises taking the place of the population before it.

In studies involving experimental infections, one hundred

immunologically distinct variants were found (2). T. rhodesiense infections

3

produce acute sleeping sickness with parasitemias as high as 100 K

trypanosomes per milliliter. These peak parasitemias can last 2-3 days,

occurring at irregular intervals from 2-10 days. T. gambiense infections produce

much lower parasitemias, resulting in a more chronic form of infection.

After inoculation, the cardinal signs of inflammation occur at the bite site.

On or about the sixth day, the trypanosomal chancre starts to develop. If the

chancre is recognized for what it is and treatment is begun, recovery is usually

without incident. Suramin is the drug of choice. If treatment is delayed, flu-like

symptoms develop. The appearance of enlarged lymph nodes, particularly the

posterior cervical nodes (Winterbottom's sign), and spleen enlargement are

characteristic but variable. Symptoms including, headache, stiff neck, insomnia,

and depression are manifestations of central nervous system (CNS)

involvement. Treatment at this stage is more difficult in that only

chemotherapeutic agents that can cross the blood brain barrier are effective.

Melarsoprol in combination with Suramin can be effective but is highly toxic. As

the CNS involvement progresses, a generalized meningoencephalitis with

cerebral edema develops. CNS function deteriorates resulting in abnormal

reflexes, esthesia, ataxia, psychosis, and eventually convulsions and coma.

The heart and lungs are affected by a general pattern of edema which in

combination with deteriorating respiratory reflexes results in pneumonia.

Pneumonia, sepsis, or both are the leading causes of death in most cases. If

untreated, death can occur in a few weeks to nine months for T. rhodesiense

and months to years for T. gambiense. Drug prophylaxis is limited with a risk of

cryptic infection and side effects. Development of a vaccine has been

hampered by the previously described antigenic variation of the parasite (1).

1.2 South American trypanosomes.

Trypanosoma cruzi is essentially an intracellular parasite transmitted by vectors

in the order Hemiptera, family Reduviidae, subfamily Triatominae (triatomids) of

which there are nine genera. These are also more commonly known as

conenose bugs, wheel bugs, and kissing bugs.

Infection is usually transmitted at night when these bugs

characteristically feed. The bug bites near the mouth or eyes of a sleeping

victim (hence the name kissing bug), and defecates as it takes a blood meal.

The feces contain the parasites that must be rubbed into the bite site by the

victim if infection is to occur. The bite itself is not painful, but begins to itch after

a short time, thus promoting the infective process.

Infections also occur without contact with the bug. In areas where

thatched roofs exist, bugs live in the roofs and defecate upon sleeping people

who then become infected by rubbing feces into mucus membranes. In rural

areas where people are close to their livestock, the risk of infection increases

because of the many types of reservoir hosts (e.g. dogs, cats, rats, bats, sloths,

and other primates). The trypomastigote form of the parasite is the infective

form for mammals, entering the bite site or mucus membranes and infecting a

wide variety of host cells. Upon entering the cell, the parasites transform into the

non-flagellated amastigote form and begin to divide. After several divisions,

they transform back into trypomastigotes, rupture the cell, and distribute

throughout the host. They infect a variety of tissues including: central nervous

system, gastrointestinal, and cardiac tissue.

Triatomids become infected when they take a blood meal from an

5

infected host, and remain infective for life. The trypomastigotes migrate to the

midgut of the bug, transform into epimastigotes, and divide, producing

thousands of organisms which transform into metacyclic trypomastigotes. The

trypomastigotes migrate to the hindgut where they are expelled with the feces.

Edema occurring at the bite site is known as a primary chagoma or

Romana's sign. It is usually located on the face and contains intracellular

amastigotes. This can be diagnostic, especially in young children who are

frequent targets of infection. An incubation period of 4-5 days is followed by the

acute stage which is often asymptomatic and frequently misdiagnosed. It is

characterized by the spread of parasites and accompanied by an intense

inflammatory reaction lasting up to four months. Those individuals that survive

the acute stage enter the chronic stage which results in the slow destruction of

invaded cells. This can lead to heart enlargement with possible ventricular

aneurysm and or megacolon and megaesophogus.

Chronic Chagas disease is quite variable with some persons remaining

asymptomatic their entire lives. Others remain healthy for years before

developing symptoms of chronic Chagas (1). Commonly, infected persons

have abnormal electrocardiograms. Diagnosis in the acute stage is made by

finding trypomastigotes in the blood. In the chronic stage diagnosis is made by

finding amastigotes in a muscle biopsy. Serological test are also available but

are variable during the acute stage. Treatment is difficult. Nifurtimox can help

during the acute stage but it is not as effective during the chronic stage and has

serious side effects. There is also some evidence that the trypanosomes have

become resistant.

1.3 Rationale for research expediency/epidemiology.

Trypanosomes cause death and disease across vast areas of sub-Saharan

Africa and Central and South America. In endemic areas the loss of human life,

productivity, and livestock cannot be over stated.

African trypanosomiasis (the disease), also known as sleeping sickness,

is endemic to an area approximately the size of the United States. This area is

virtually free of human beings and their livestock. The area is limited

geographically by the distribution of the Tsetse fly, the vector of African

trypanosomiasis. Mortality rates climb dramatically when people and their

livestock migrate into these areas for political or economic reasons. Currently, it

is estimated that 50 million people in 36 sub-Saharan countries are at risk with

50,000 new cases reported annually (3). This is considered to be grossly

underestimated due to logistical problems in diagnosis. The disease is

invariably fatal if not diagnosed and aggressively treated in the early stages.

South American trypanosomiasis, known as Chagas' disease, is

endemic to areas as far south as Argentina and as far north as the southern

border states of the U.S. In endemic areas, approximately 20% of the

population is at risk, resulting in 7 million infected persons (4). Some estimates

indicate the number of infected persons at between 10 and 12 million

individuals (5). Ten percent of infected persons die in the acute phase or

develop serious myocardiopathy in the chronic stage of the disease (5). While

vector-transmitted Chagas' disease largely occurs below the Tropic of Cancer,

sporadic cases have been reported in the U.S. with increasing frequency.

There are at least two reasons for this:

7

1 .Infected vectors and nondomestic reservoir host appear to be

abundant in North America, though the mode of transmission and epidemiology

does not often involve humans.

2. Political and social unrest in endemic areas have increased the

number of infected people in the U.S. resulting in cases of Chagas' disease

transmitted by blood transfusion (6).

Louis V. Kirchoff has estimated that 100,000 immigrants in the U.S. may

be chronically infected with the vast majority asymptomatic (7). In a study of

205 immigrants from El Salvador and Nicaragua living in Washington, DC, Five

percent were found to harbor the parasite. Data from South America indicate

that approximately 25% of recipients receiving blood from infected persons

could also become infected. Blood recipients can be protected from infection

by treatment of the donated blood with gentian violet (1:4000 dilution), which

kills the trypomastigotes.

These two forms of trypanosomes are spreading geographically with

morbidity and mortality rates expected to climb by millions in the next few years.

To combat this, vector control programs, early diagnosis, and treatment have

shown some limited success. There has also been a renewed interest in

identifying novel aspects of the biology, biochemistry, and molecular biology of

trypanosomes that might be exploited to develop new targets for vaccines or

chemotherapy (3).

1.4 Calcium.

We all began life with a wave of calcium sweeping through the cytoplasm of the

8

egg from which we came at the moment of fertilization. Calcium is no less

important to all living things where it initiates the advent of many physiological

processes under strict homeostatic control. Techniques, such as video-imaging

of flourescent indicator dyes at the level of a single cell, has revealed that the

stimulus-induced intracellular calcium signal is remarkably organized both in

time (calcium spikes) and space (calcium waves), suggesting that the method of

delivery of the calcium pulse into the cell is of primary importance in eliciting a

physiological response (8).

In most eukaryotic cells calcium functions as an intracellular signaling

molecule, a so called "second messenger". The first messenger being

hydrophillic molecules communicating at the surface of the cell. These first

messenger molecules interact with cell surface receptors to produce a variety of

cellular activities. These activities are stimulated either by extracellular

molecules interacting with membrane bound enzymes or by interacting with

gated ion channels. In either event the concentration of intracellular mediators

changes which results in a signal that initiates an intracellular response.

Adenylate cyclase is an example of a membrane bound protein that is

activated by extracellular molecules. It is not activated directly by extracellular

molecules but by other activated proteins. When a signaling molecule (first

messenger) binds to the plasma membrane receptor it activates another

membrane bound protein, G protein . The activated G protein activates

adenylate cyclase. One First messenger receptor protein can activate many G

proteins thereby greatly amplifying the adenylate cyclase response. Adenylate

cyclase is composed of two subunits. One has catalytic activity which converts

ATP to cyclic-AMP (cAMP), the other is a regulatory protein that activates the

9

catalytic unit when it binds GTP and inactivates it when it hydrolyzes GTP.

Cyclic-AMP in turn acts as a second messenger to activate cAMP-dependent

protein kinases. These enzymes are tetramers which are composed of two

regulatory and two catalytic subunits. The cAMP dependent kinases and their

associated phosphorylases modulate many physiological and metabolic

activities. Some of these activities are calcium dependent (discussed later) and

known to be initiated by: many types of hormones, neurotransmitters, seratonin

and histamine, certain prostaglandins, and other extracellular first messengers.

An alternative to modulating intracellular mediators by activating cell

surface proteins like adenylate cyclase, is for extracellular molecules to activate

cell surface proteins which interact with gated ion channels. The alteration of

intracellular mediator concentration produces transient action potentials, as

seen in electrically active cells or large influxes of ions that mediate intracellular

responses. Action potentials generated in neural and muscle cells are not

accompanied by large changes in intracellular ion concentration, nor is the

initial influx an intracellular signal. The intracellular signal occurs at the nerve

terminal where calcium ion channels are opened. Calcium then acts as a

second messenger to elicit an exocytotic release of neurotransmitters. When

greater influxes of calcium enter the cell, an intracellular response can be

initiated. An enormous calcium ion gradient is available across the plasma

membrane of cells to mediate a variety of intracellular activities. Where as the

concentration of internal and external calcium is more or less equal, eg.,

approximately 10'3 M, the concentration of free cytosolic calcium is 10 "7 M or

less, so that most of the internal calcium is bound to other molecules or

sequestered in cellular organelles. This gradient must be maintained if calcium

10

is to serve as an intracellular signal.

Membrane bound ATPases use ATP hydrolysis energy to pump calcium

out of the cell or into cellular organelles. These calcium pumps are sometimes

coupled to the movement of other ions like sodium to maintain homeostatic

gradients and membrane potentials. Endoplasmic reticulum (ER) and

particularly sarcoplasmic reticulum of muscle cells remove large amounts of

calcium from the cytosol. Mitochondria maintain high levels of lumenal calcium

by removing it from the cytosol in a process that is driven by inner mitochondrial

membrane potentials. Calcium is freely permeable to the outer mitochondrial

membrane. This process directs the positively charged calcium ions through

gates where the lumenal negative charges are generated during the electron

transfer steps of oxidative phosphorylation. Calcium, pumped out of cells by

ATPases, and in some cases coupled to reactions that pump other ions into the

cell while pumping calcium out.

When extracellular molecules stimulate second messenger activity by

opening voltage regulated gates or by activating certain membrane bound

enzymes, the cytosolic concentration of intracellular mediators change. For

calcium generated activities this means a cytosolic rise from 10-7 M to 10-5 M.

The proteins that respond must be able to do so at these levels since cytosolic

calcium concentrations rarely get above 10-5 M. Calcium binding proteins

(CaBPs) like calmodulin and others, not only sequester calcium at low levels,

but they are highly specific. They can distinguish calcium ions from other

similar ions such as magnesium even though magnesium is generally

maintained at cytozolic levels of 10-3 M, and can be as much as a thousand

fold higher than Calcium levels.

11

1.5 Calcium binding proteins.

CaBPs are proteins that undergo a conformational change when they bind

calcium. These changes mediate cellular functions through their actions on

other proteins. Troponin is one of the most understood CaBPs. It functions as a

complex of three proteins (T, I, and C) that have a close association with actin

and myosin myofilaments, particularly actin. When troponin C binds calcium it

undergoes a conformational change which allows myosin cross-bridges to bind

with actin subunits. It does this by altering the position of tropomyosin on the

actin filament as part of a classic myofibril contraction. While troponin C is the

best understood CaBP, it appears to have evolved from another ubiquitous

CaBP, calmodulin. Calmodulin has been found in all plant and animal cells

that have been examined indicating that it is highly conserved throughout

evolution (9,10,11,12,13). It has been implicated in regulating a wide variety of

cellular activities (14). It is the fourth member of a family of calcium-binding

proteins to be characterized by crystallography, the others are: parvalbumin

(15), intestinal CaBP (16), and Troponin - C (17,18).

Calmodulin is a small dumbbell shaped protein of 148 amino acid

residues (Mol. wt. 16.8K) with two calcium binding domains (4 total) at each

globular end of the dumbbell. The globular ends are completely separated by

an amphipathic 8 turn alpha helix. The binding of calcium by calmodulin

causes a conformational change in the molecule allowing the proteins to

interact with the alpha helix, although it exhibits significant alpha-helicity even

in the absence of calcium binding (19,20,21,22). Calmodulin has no enzyme

activity in itself, but regulates the activity of other enzymes in response to

12

cytosolic calcium levels. The calcium binding domains in this family of CaBPs

were described first by Kretinger, et al. (23) and is the basis for the family's

characteristics. Calmodulin and other members of this family, including

trypanosomal calflagins are calcimedins. Calcimedins are molecules that

reversibly expose hydrophobic residues in the presence of Calcium. Three

families have been proposed to compose the Calcimedins they are:

(1) Annexins - A group of related proteins that are involved in regulating

calcium channels and calcium dependant endo- and exocytotic processes.

They have tandem arrays of 70 residues repeated 4 or 8 times interspersed

with a 17 residue consensus sequence "the endonexin" within each repeat.

(2) Protein Kinase C Isoenzymes - A group of calcium dependent enzymes

which can phosphorylate a number of proteins with different cellular functions.

These enzymes are interesting in that they are activated cooperatively by the

products of a triphosphorylated inositol phosopholipid (PIP2). This molecule is

cleaved into inositol triphosphate, a molecule that transiently releases calcium

from its various compartments and diacylglycerol, which transiently activates

the calcium dependent protein kinase C isoenzymes. In a similar process to

the cyclic AMP pathway mentioned earlier, an activated receptor is thought to

activate a G protein (Gp) which in turn activates an enzyme (phosphoinositide-

specific phospholipase C) that cleaves PIP2. The protein Gp has been shown to

be activated by many different membrane receptors.

(3) E F-hand calcium-binding proteins. Kretsinger etal. described a helix loop

helix calcium-binding motif which was called an E F-hand because it resembles

a hand, where the thumb and index finger are extended to represent the two

alpha helices, and the rest of the fingers closed to form the Calcium binding

13

loop (24).

Calmodulin is an EF-hand CABP that contains four calcium-binding

domains composed of seven helices (7-19, 29-39, 46-55, 65-92,102-112,119-

128,138-148) with a total helix content of 63% (25,26,27). Helix IV is the

backbone of the molecule, the interactive site for calmodulin regulated proteins,

and the shared helix between Calcium binding domains 2 and 3.

COOH-

NH-Ac

Ca++

Of interest is that Calmodulin target proteins share very little binding domain

sequence homology (28). It has been suggested that the backbone alpha helix

is more flexible than previously thought (29,30). Evidence using small angle x-

ray scattering (SAXS) techniques and nuclear magnetic resonance Imaging

(NMR) shows that it is possible to cross-link N and C terminal domains, and that

the central backbone is extremely flexible in aqueous solution. While the

flexibility of calmodulin is somewhat reduced by calcium binding, it seems to

enhance its affinity for target proteins (31). The flexibility of calmodulin domains

is thought to promote the "Best fit" around the various targets. The domains

14

have been described as like a flexible tether (32). When the central linking

region (helix IV) of Calmodulin bends to accommodate the target, it loses some

of its helical structure in the process. The two domains then embrace the target

peptide to stabilize the complex. This is in contrast to relatively recent theory

that allows for the target proteins to embrace the central alpha helix of

calmodulin. Hydrogen bonding between the central helix and target peptides is

minimal (only one) and the complexes are largely held together via side chain

interactions. Another unusual feature of the Calmodulin target interaction is that

the target itself assumes an alpha helix conformation (28). This is a very rare

type of interaction and is the reason why target proteins can have very little

binding domain homology. While it will be sometime before intracellular

interactions between Calcium, calmodulin, and their target proteins are fully

understood, we can take what we do know about this ubiquitous calcimedin

and extend and apply that knowledge to other members of the E F-hand family.

In trypanosomes, calcium second messengers involving cellular

functions other than motility, are associated with their flagellum. The flagellum

is a complex structure that contains desmosome like structures for adhesion, an

axoneme for flagellar motility, a structural paraxial rod, and a flagellar pocket

where particulate and soluble substances enter the trypanosome (33,34).

Endocytosis of substances at the flagellar pocket are required for normal growth

and may be regulated by calcium. Coppens etal. (35) reported on low density

lipoprotein (LDL) receptors in T. brucei bloodstream forms. They had

previously shown thatT. brucei takes in host LDL through its flagellar pocket

(36). This presumably occurs as a requirement for membrane synthesis

(37,38). Where as cholesterol is the major sterol of trypanosome blood form

15

membranes, it has been reported that they are unable to synthesize it de novo

(37,39). Coppens et al. (35) report that LDL is required for growth in T. brucei.

They demonstrated that growth can be inhibited by low levels of available LDL

or by antibodies against LDL receptors. Growth can be restored in both cases

by addition of excess LDL. Endocytosis of LDL was characterized in part as

being almost completely dependent on calcium. The pocket may also be

involved in host recognition (36). Vesicals within the flagellar pockets are

similar to clatharin-coated vesicles of other eukaryotic cells. A molecule having

a molecular weight similar to the heavy chain of clatharin was observed (40)

but, was not serologically cross reactive with mammalian clatharin. The

vesicles bud from the flagellar pocket membrane and discharge their contents

into an intracellar tubular system which connects with structures similar to the

multivesicular bodies and lysosomes of mammalian cells (34,41). Within the

flagellar pockets are located a variety of receptors through which extracellular

molecules can stimulate intracellular signals. Calmodulin has been localized to

the flagella of trypanosomes and is no doubt involved in various cellular

processes including regulation of metabolism, growth, and development as it is

in mammalian cells. These receptor mediated processes might provide an

excellent target for a vaccines or chemotherapeutic agents if calmodulin were

not so highly conserved (even between mammals and trypanosomes).

In mammalian hosts African trypanosomes possess a continuous

variable surface glycoprotein coat (VSG) that can be altered antigenically and

biochemically to evade host immune responses (42,43,44). These VSG's have

in the past, been targets of much vaccine research. It now looks like

trypanosome VSG's are not going to be useful as vaccine targets based on

16

their antigenic diversity (45). New targets for vaccines and chemotherapeutic

agents are being sought. The endocytotic interface between host and

trypanosome has been suggested as a new avenue of vaccine attack (41). The

clatharin-like protein that is not cross reactive with mammalian clatharin might

be a possible site for a vaccine. Another group of proteins for possible vaccine

or chemotherapeutic agent research, has the potential at least of hitting a

number of regulatory targets. These are a group of E F-hand Calcimedins

found thus far, exclusively in trypanosomes. They are the Calflagins (46) and

like calmodulin, have been localized to the flagella (7,47). The calflagins may

also be involved in cellular activities such as growth, metabolism, and

development, similar to calmodulin. If this is the case, then they might make

good targets for vaccines or chemotherapeutic drugs, since they are not cross

reactive with mammalian calcimedins (7, 47, 48).

1.6 Calflagins.

Calflagins were first reported in 1985 by Lizardi, et. al and Gozalez, ef a/.(48)

who were searching for a vaccine to Trypanosoma cruzi by cloning genes that

coded for major surface antigens. A positive clone (IF8) was isolated by

screening expression libraries using IgG from sera of chronic, as well as, acute

chagas patients. IF8 was found to contain a single open reading frame which

was used for a homology search of protein sequences. Remarkably good

homologies were found with a number of E F-hand containing calcium binding

proteins such as parvalbumin, troponin C, calmodulin, and catalytic myosin light

chain. Hybridization studies of nick-translated IF8 probed to T. cruzi DNA

17

showed that it belonged to a group of tandemly arranged genes, and that there

was at least 30 copies of the basic 940 bp repeat unit. They showed that the

gene is transcribed in a polycistronic manner, and that the resultant mRNA is as

abundant as B -tubulin mRNA (about 5% of the total poly A mRNA). The

relative abundance of this calcium binding protein has stimulated further

interest in possible regulatory roles similar to those of calmodulin.

The T. cruzi calflagins were further characterized by Engman, et. al. in

(7) and later by Ouaissi et.al. (49). They were also selecting T. cruzi calflagin

cDNA clones with sera from infected animals. They expressed calflagin cDNA

in E. coli and showed that it could be detected repeatedly with different sera

and different expression libraries. The protein was localized to the

trypanosome flagella using mouse antisera, and it was shown that recombinant

as well as native proteins bound calcium. They demonstrated that the protein

has a low calcium binding capacity, <2 mol. Ca++/mol. protein, and a high

calcium binding affinity Kd <50u M Ca++ which is consistant with other E F-

hand calcium binding proteins. This suggests that the protein might be involved

in trypanosome motility due to its location and the role calcium plays in the

motility of other organisms. They proposed the name FCaBP for flagellar

calcium binding protein.

Lee, et. al. (50), reported that while screening African trypanosome cDNA

clones for evolutionarily conserved genes that encode cell surface proteins,

they found a cDNA clone (Tb17) with an open reading frame that shared

significant homology to the E F-hand calcium-binding proteins of T. cruzi

previously identified by Lazardi and Engman. When this clone was probed

back to genomic DNA several other clones encoding related proteins were

18

found, isolated and later sequenced (51)

In 1992, Wu, et. al. (47) reported a similar 44kDa protein from T. brucei

and described a family of flagellar E F-hand calcium binding proteins they call

the predominant calcimedins of T. brucei. Of interest is the differences between

the family of calcimedins described by Wu, et. al. (51) in T. brucei, and the

tandemly repeated genes described by Lazardi, et. al.(48), and Engman, et.

al.(7), in7. cruzi. Wu, et. al.(51), point out that the differences in gene

organization between T. brucei and T. cruzi indicate that at least some of the

calflagins might function in rolls other than motility. They characterized the

organization of these genes in T. brucei as comprising a family of proteins

consisting of a 44KDa protein (Tb-44) and a 23-26 KDa cluster that probably

includes the gene Tb-17 described by Lee et al (50). They isolated the protein

Tb-44 to the trypanosome flagella by immunofluorescence and further

characterized the proteins of Tb-44 and 23-26 KDa cluster. Partial sequence

analysis and calcium-dependent phenyl-Sepharose binding experiments

(defines calcimedins as binding to specific hydrophobic matrices in a calcium

dependent manner), demonstrated that the proteins were calcimedins of the

E F-hand type. Antibody cross-reactivity and immunoblot analysis indicate that

these proteins probably comprise a family of related proteins and that they were

not members of the annexin family.

The genomic organization of the calflagin family of proteins is currently

under investigation, and it has been shown by Wu,etai. (51) that multiple

calflagin family members occur in T. brucei where as only a single member

occurs in T. cruzi. Evaluation of the calflagin gene organization in

T. Brucei suggests evolutionary origins of calflagin variants. Of interest are

19

variants not found in T. cruzi, suggesting that at least some calflagin family

members are not required for motility, but serve some other functions. Currently

however, none of calflagin members has a known function.

1.7 Calcium/calcium binding protein interactions on trypanosomal metabolism

and development.

Evidence that Calcium is interactive with proteins like calmodulin and the

calflagins in a way that regulates trypanosomes metabolically or

developmentally has remained elusive though indirect evidence is continuing

to be compiled. Much of the evidence is gleaned from other protozoa and

mammalian investigations. This can involve some sweeping assumptions. For

instance, metazoan organisms evolved from protozoa and retained much of

their calcium metabolism. There can be further risk in comparing other

protozoa with trypanosomes since trypanosomes seem to have more than their

share of unique features. None the less, circumstantial and direct evidence is

continuing to be sought for the role calcium plays in the development and

metabolism of trypanosomes and the family Trypanosomatidae in general.

It has been shown that the cellular complement of calcium binding

proteins varies during the trypanosome life cycle (52, 53), and that calmodulin

does not represent the majority of calcium binding activity (54, 55, 56). This

suggests that trypanosomes respond to calcium signals differently during their

life cycle. Several calcium dependent proteins have shown stage specific

activation by calcium.

Paindavoine et al. (57) report on a group of cyclase glycoproteins in

20

T. brucei that are calcium dependent and developmental^ regulated. They

showed that four transcripts for adenylate and guanylate cyclases have been

found in the bloodstream forms of T. brucei. These transcripts have partial

sequence homologies to known eukaryotic adenylate and guanylate cyclases

(57,58). One of these genes ESAG 4 (expression site associated gene 4) was

found to be developmental^ regulated and transcribed only in the blood stream

form, while the other three transcripts were also found in the procyclic (insect)

form. ESAGS are a well documented group of genes (23,24,44,58,59,60,61).

ESAGs are transcribed on large multicistronic transcripts along with the

telomeric VSG (variable surface glycoprotein) gene expression site. Differential

expression of ESAGs in trypanosomes was also demonstrated by Gram and

Barry (62) who showed that ESAGs could be expressed with VSG in slender

bloodstream forms or with out VSG expression in procyclic forms. Mathews

etal. (63) showed that ESAGs are not present in the telomeric expression site

during the metacyclic trypanosome stage. Paindavoine et al. (57) showed

ESAG 4 and related glycoproteins of 150kDa appear to be trans membrane

proteins localized exclusively to the flagellum. They showed that when ESAG 4

and another related glycoprotein (GRESAG 4.1) complement a Saccharomyces

cervisiae mutant for adenylate cyclase, only ESAG 4 was activated by calcium,

the others being inhibited by it.

Another group of calcium dependent proteins found to be

developmental^ regulated in trypanosomes are protein kinase C isoenzymes.

As mentioned earlier, these are calcimedins that are calcium dependent and

known to phosphorylate a number of proteins with different functions in

mammalian cells. Keith et al. (64) report on protein kinase activities in T. brucei.

21

They identified protein kinase C enzymatic activity and isolated it to either

slender blood forms or procyclic insect forms. Using DEAE cellulose they

separated proteins based on their ability to phosphorylate histone in the

presence of calcium and diacylglycerol. Peaks of activity differed between

lysates of T. brucei bloodstream and procyclic forms. Isoelectric focusing and in

situ protein kinase assays further characterized the two groups as having

different properties. The authors conclude that the bloodstream kinases are

either more numerous or more stable than the procyclic forms. One of the

bloodstream kinases was remarkable in that it shared many characteristics with

mammalian protein kinase C. The authors report that this protein kinase C was

shown by Western blot analysis to contain an epitope that is recognized by

monoclonal antibody raised against mammalian protein kinase C. They

conclude that the bloodstream form of T. brucei has a protein kinase C

homologue which is not found in procyclic forms.

Parsons et at. (65) report on the role of protein kinases in organisms that

diverged early in the eukaryotic lineage. They report that primitive eukaryotes

possess a large complement of protein kinases, and that they play a pivotal role

in proliferation and differentiation in protozoa. They examined the activity of

renaturable protein kinases during the life cycle of T. brucei. They

demonstrated that six of eight renaturable protein kinases were regulated

during the trypanosome life cycle. Based on these finding and a previous work

where the authors demonstrated that tyrosine phosphorylation is stage specific

in T. brucei(66) they conclude that protein phosphorylation networks are

important in regulating the cyclical development of T. brucei. Tyrosine

phosphorylation is a key regulator of cell proliferation and development in

22

higher eukaryotes (66). In further support of this hypothesis they site the

observation that a strain of T. brucei defective for the slender to stumpy

transformation had substantially decreased activity in two of the stage specific

renaturable protein kinases. It was suggested by these authors that a proper

study of specific protein kinases could be done through genomic manipulation,

and that the protein kinases described in their paper would be targets of

particular interest once the relevant genes were cloned.

Some of the best evidence that calcium/calcium-binding protein

interactions are involved in the development and regulation of trypanosomes is

where calcium dependent gene products are associated with a certain

developmental stages or located on a transcriptional units with other regulatory

genes. Wong et al. (46) investigated transcription of housekeeping genes in T.

brucei. They characterized the expression of two ubiquitin genes up stream of a

EF-hand superfamily member gene and a calmodulin gene cluster. Ubiquitin is

part of the proteolytic machinery responsible for selective protein degradation in

eukaryotes. It is easy to speculate that genes transcribed with the ubiquitin

genes might also have some house keeping function. Wong etal. (46) reported

that the genes are linked and transcribed polycistronically. They suggest that

all four genes are under the control of a single distant upstream promoter. They

showed that RNA molecules that span the intergenic regions can be detected

by PCR. They conclude that there is coordinated transcription of three different

genes, each of which encode a protein with a potential regulatory function. The

coordinated production of ubiquitin with two calcium binding proteins implicates

calcium as being involved in the regulation of trypanosome metabolism. The

authors suggests that regulation of transcription initiation plays an important

23

role in the coordinated expression of housekeeping genes in trypanosomes.

As evidence that calcium plays an important role in the regulation of

trypanosome development and metabolism continues to be compiled, it

becomes as important to consider calcium homeostasis in trypanosomes as it is

to consider calcium dependent proteins. In general calcium homeostasis is

regulated by the concerted action of the plasma membrane, endoplasmic

reticulum, and mitochondria. As mentioned earlier, low levels of free ionic

calcium are maintained in the cytosol (10-5 M) in eukaryotic cells. These

delicately balanced calcium levels are critical to the physiology of the cell and

are maintained by high affinity ATPases (67). It has been shown that members

of the family Trypanosomatidae are able to maintain very low ionic calcium

concentrations (50 nM) (68,69,70,71). Information on how this is accomplished

is sketchy at best, but it has been suggested (72,73) that high affinity calcium

activated ATPases could have a role in regulating intracellular calcium levels

and calcium transport in these protozoa. High affinity ATPases have been

implicated in the transport of calcium across plasma membranes and

maintenance of cytosolic calcium levels in of some parasitic kinetoplastids (L

braziliensis, L. donovani, T. cruzi, and T. rhodesiense).

Benaim and Romero (69) demonstrated the presence of a calcium pump

in plasma membrane vesicles of L braziliensis promastigotes. This pump is

capable of maintaining low levels of cytoplasmic calcium. They describe a high

affinity calcium ATPase (Km 0.7uM) that saturates at 5 uM. They report that

maximal calcium-ATPase activity is attained when equimolar amounts of

magnesium-ATP are present in the medium. This suggests to them that the

magnesium-ATP is the substrate for the calcium-ATPase. This ATPase was

24

stimulated by calmodulin (70-80% with 5ug/ml) and inhibited by trifluoperazine.

Their work is the first to describe such a pump in L braziliensis. They showed

that the vesicles accumulated calcium against a gradient and released it after

addition of a calcium inonophore (A23187). Calcium uptake and transport were

completely inhibited by vanadate (20uM). They conclude that kinetic

parameters of the enzyme are compatible with long term maintenance of

intracellular free calcium levels in these parasites.

Ghosh et al. (72) describe a calcium dependent ATPase in

L. donovani. It appears to be involved in calcium transport and maintenance of

cytoplasmic calcium levels. The enzyme is composed of two subunits, MW

51,000 and 57,000 and has a total molecular weight of 215,000, indicating a

tetrameric structure. The authors report that this enzyme is significantly different

from mammalian types and it is apparently some what different from the

ATPases reported by Benaim etai (68,69,74) in L braziliensis and T.cruzi.

The ATPase described by Benaim and Romero (69) maximized enzyme activity

with equimolar amounts of ATP-Mg, this enzyme reported by Ghosh etai (72) is

inhibited by magnesium at higher concentrations. Ghosh etai. (72) generally

describe this enzyme as a high affinity ATPase with an extremely low Km

(35nM) which is further lowered (10-15nM) in the presence of calmodulin. The

enzyme is sensitive to vanadate and to a lessor degree trifluoperazine.

Labeling studies of the enzyme and membrane suggest that it is an integral

membrane protein that has its catalytic site on the inner face. The authors

envision that the enzyme is a pump involved in the extrusion of calcium, but

they maintain that this idea will remain speculative until the enzyme is

integrated on liposomes and ATP-driven calcium movement is detected. Their

25

report and others is strongly suggestive to the authors of a role for calcium in

the physiology of these parasites. They conclude that the localization,

orientation, and high affinity of the calmodulin sensitive calcium ATPase is

suggestive that calcium movement plays some role in the life cycle of

Ldonovani.

Similar ATPase investigations have been reported in trypanosomes.

Benaim et al. (74) also reported on a calmodulin activated ATPase in

T. cruzi. This ATPase is reported to be involved in calcium transport by plasma

membrane vesicles. Unlike other similarly described parasitic protozoan

ATPases where magnesium was inhibitory (72,73) or stimulatory, but not

required (69) the calcium-ATPase described here has an absolute requirement

for magnesium. An investigation of calcium transport by plasma membrane

vesicles using Arsenazo III as a calcium indicator showed that vesicles

accumulated calcium only when magnesium was present. Calcium was

released by the vesicles in response to the calcium ionophore A23187. The

enzyme was completely inhibited by vanadate at low levels (10uM). A Km of

0.3 uM for free calcium indicated a high affinity enzyme, but activity and

transport were further stimulated by calmodulin from endogenous and

mammalian sources. Trifluoperazine and calmidazolium inhibited calmodulin-

stimulated ATPase activity. The authors conclude that their observations

support the existence of a maganesium-depenent calcium pump in these

parasites.

Lastly but probably the first to report on ATPases in parasites of the

family Trypanosomatidae was John McLaughlin (73). He described a calcium-

ATPase in the blood stream forms of T. rhodesiense. The ATPase besides

26

having no magnesium requirement was inhibited by submicromolar levels of

magnesium. Cell fractionation studies localized the ATPase to the surface

membrane. An enriched surface membrane fraction was shown to have

ATPase activity using very low levels of free calcium (200 nM). He investigated

a range of ATPase modulators and found only vanadate had any effect (47%

inhibition). Enzyme activity was unaffected by calmodulin or calmodulin

antagonist (Chlorpromazine and trifluoperazine). He concluded that the kinetic

properties of the calcium-ATPase are compatible with a role in calcium

regulation in the bloodstream forms of T. rhodesiense.

It is interesting to speculate as to why some of these ATPases require, or

are stimulated by, magnesium and others are inhibited by it. One clear

separation of the two groups are the author's descriptions of the ATPase

location in the cell. Beniam et al. (68,69,70) described ATPases from plasma

vesicles of two different organisms, L braziliensis promastigotes, and T. cruzi

epimastigotes. These ATPases required, or were stimulated by, magnesium. In

contrast Ghosh et al. (72) and McLaughlin (73) described ATPases on surface

membranes of L donovani promastigotes and T. rhodesiense bloodstream

forms respectively. As other ATPases are discovered in similar protozoan

parasites it will be interesting to associate their magnesium requirements with

the description of their ATPase location. Of the four T. rhodesiense was the

only organism whose ATPase was not stimulated by calmodulin. Some

eukaryotic ATPases have been found to be evolutionarily related. Calcium

ATPases in the sarcoplasmic reticulum of muscle cells are one of the best

understood calcium pumps. It was found via cloning and sequencing

techniques that these pumps are evolutionarily related to the large catalytic

27

subunit of the Sodium-potasium-ATPase (pump). Similar molecular studies of

these organisms might add to their related evolutionary history as well as

contributing to their biology.

1.8 Calcium: effects on trypanosome killing and treatment.

In ways not fully understood calcium/calciun binding protein interactions

appears to be very important to the metabolism, physiology, and development

of parasitic protozoans in the family Trypanosomatidae. It may also be

important to the treatment of these infections by targeting or suplementing

various components of calcium metabolism.

Since 1902 normal human serum has been shown to have cytotoxic

effects on T. brucei (75). The trypanocidal activity was further characterized by

D'hondt et al. (76) who reported that calcium played a role in the trypanocidal

activity of HDL as an essential cofactor. They showed that normal serum, when

dialyzed, lost trypanocidal activity, but regained it after the addition of calcium.

Calcium was also found to stimulate lysis of nondialysed serum. Magnesium

was reported to have a negligible effect. Calcium could not restore heat

inactivated serum (45 mln. @ 60 °C) indicating that calcium is dependent on

some other factor for this activity. The authors presented evidence that alpha-2-

M (a protein ligand for calcium) might be involved as a calcium carrier for the

activity since serum treated with antialpha-2-M showed no trypanocidal activity.

Several D-sugars were also shown to inhibit trypanocidal activity when added

to the medium. It was thought that they might compete for a trypanocidal factor's

binding site. Glycerol was shown to have the opposite effect even in resistant

28

clones of T. rhodesiense. Rifkin (77,78) showed that high density lipoprotein

(HDL) in the serum of humans and baboons was cytotoxic to T. brucei. Rifkin

found that rabbit and rat serum had no cytotoxic effects. It was shown that HDL

induced trypanocidal activity, could be lost by digestion with phospholipase.

This was an important finding in that it showed the importance of phospholipids

in the biological activity of HDL Trypanocidal activity by HDL could also be

inhibited by adding rabbit or rat HDL to the medium. Purified phospholipids or

phosphatidylinositol could also inhibit trypanocidal activity when added to the

medium, with the latter being the most effective. Trypanocidal HDL was

characterized as having low phosphoinositol content and high

phosphatidlycholine content. As recently as July 1994 a mechanism for the

trypanocidal activity of HDL was proposed by Hager et al. (79). This work was

based on the previous work by Rifkin referenced earlier and Hajduk (Hajduk

et a/. 1989, Hajduk etal. 1992). The mechanism was reported to be based on a

minor (<1%) subclass of human and presumably baboon serum HDL called

trypanosome lytic factor (TLF). Briefly the mechanism as reported involves TLF

binding at high affinity to the flagellar pocket of the trypanosome. TLF becomes

associated with a coated pit (CP) (34) and internalized in coated vesicles (CV).

These small vesicles were thought to contain an inactive form of TLF since it

has no effect on the plasma membrane. TLF is found to be active in a

lysosomal compartment of the cell. Studies where the pH was raised indicate

that TLF must pass though an acidic environment in order to become active.

Large lysosome-like vesicals are shown to contain TLF before lysis. The

mechanism involved in the disruption of these large vesicles is still speculative,

however studies involving leupeptin (a thioprotease inhibitor) inhibited lysis by

29

69% but did not block endocytosis of TLF. The authors speculate that an

alteration in the pH environment could activate the enzyme. Alternatively TLF is

composed of two linked apolipoprotein oligomers (L-l, L-lll), which maybe

activated by modification to the protein or lipid component. The authors

conclude that TLF is a unique natural killing factor in that it not only has to be

internalized, but also requires a putative activation step. Hajduk's group has

recently showed (82) that TLF is composed of two apolipoproteins

(haptoglobin-related protein, paraoxonase-arylesterase) that exhibit peroxidaes

activity. They contend that TLF is endocytosed bound to hemoglobin and

targeted to the lysosome where it reacts with H2O2 to cause peroxidation of the

lysosomal memebrane and ultimately the death of the cell. Wheather or not

calcium or calcium/calcium binding protein interactions are envolved in TLF

killing as reported by D'hondt etal. (76) or if they are envolved with internalizing

HDL as it has with LDL (described earlier) remains unclear, but it is cause for

speculation and further studies in this area.

In the area of treatment calcium has been shown to enhance drug

therapy. The synergistic effects of calcium on trypanocidal compounds were

described by Clarkson and Amole (83). They reported that the synergistic

effects of serum on the drug combination of salicylhydroxamic acid (SHAM) and

glycerol were due to diffusible calcium ions. They were able to remove the

synergistic activity by dialysis or by adding chelating agents (EGTA, EDTA) to

the serum. They further showed that a buffer containing calcium could mimic

the synergistic activity of the serum. Neither magnesium or zinc showed any

synergistic activity. Trypanosome killing by melarsoprol was also enhanced by

calcium even more than it enhanced the SHAM plus glycerol killing. The

30

authors suggested that a mechanism may involve activation of phospholipases

by an increase in intracellular calcium which would result in lysophospholipids

causing membrane damage and cell lysis. Calcium has been shown to

influence other antiprotozoan compounds as well. In Plasmodia antimalarial

compounds maybe related to their interactions with the parasites calmodulin

(84), and in leishmania phenothiazine antileishmanials are thought to inhibit

calmodulin activity in pathogenic and vector forms (85).

Calcium whether acting alone or in concert with calcium binding proteins

appears to play a pivotal role in the biology, evolution, and control of

protozoans in the Trypanosomatidae family. To further the understanding of

the role calcium plays in these organisms, the molecular genetic analysis of a

group of flagellar calcium binding proteins of the EF-hand type was undertaken

in T. brucei. These proteins called "calflagins" (51) are thought to be

representative of the type of targets that may ultimately control these organisms.

1.9 Shotgun approach to identification of novel targets.

In recent years the indentification of novel targets and genes of a variety of

organisms has come from the random (shotgun) sequencing of genomic and

cDNA fragments (86,87,88,89). Seeking genes in this way not only contributes

to our knowledge of an organism but also contributes to the database, which is

proportional to the chance that a given shotgun sequence will contribute to a

larger contiguous sequence and ultimately the entire genome. This scenario

plays well to the strategy for understanding trypanosome biology because of

the high density of genes on the larger chromosomes and polycistronic

31

transcripts which code related genes of various cellular functions. Furthermore

difficulties in applying standard molecular techniques to trypanosomes and

their relatively small genome of 4 x 107 bp (90) makes them an attractive

candidate for whole genome sequencing. Trypanosomes are ancient

organisms that present unusual features of genomic organization not found in

other eukaryotes. In

T. brucei the nuclear organization consists of a diploid genome of seven pairs

of non-condensing homologous chromosomes (0.3 - 5.0 mb) and

approximately 100 minichromosomes (50 -150 kb) of unknown ploidy (91).

The chromosomes can not be stained but have been characterized by pulse-

field gradient-gel electrophoresis. It was shown by this method that karyotypes

can vary dramatically even within strains of a given species (92,93,94,95,96).

The minichromosomes contain few if any genes and are composed mainly of

tandemly arranged 177 bp repeats (97). Approximately 15% of the

trypanosome genome is of a repeated nature due to the minichromosomes and

telemeric repeats. Trypanosomes disperse their genetic material clonally but

sexual recombination has been reported, including: recombination of haploid

nuclei (98); haploid and diploid nuclei (99); and diploid nuclei (100). A unique

mitochondrial structure called the kenetoplast, is found in trypanosomes which

contains unusual DNA in the form of concatonated mini and maxi circles. Most

of the mitochondrial transcripts are incomplete, but are reconciled by small RNA

molecules (guide RNA) that act as templates in a process called "RNA editing"

(101). Other unique features related to the molecular biology of trypanosomes

include: Polycistronic transcription (102) and transplicing of precursor RNAs

(103,104).

32

In recent years, random (shotgun) sequencing has been applied to

Trypanosoma brucei (105,106) and entered into the EST (expressed sequence

tag) and the GSS (genome sequence survey) databases of the National Center

for Biotechnology Information (NCBI). In searching these sequences, many

interesting and potentially advantagous genes can be found by comparing

them to other related sequences using the BLAST software. Most EST and

GSS sequences in the database are derived from a variety of shotgun cloning

techniques. Recently, El-Sayed and Donelson (105,106) compared the

efficiency of sequencing random genomic fragments with random cDNAs of T.

(brucei) rhodesiense. They concluded that sequencing random DNA

fragments is as efficient in identifying novel genes as sequencing cDNA clones

due to the high density of Trypanosome genes.

In this report a method of M13 shotgun cloning was used to generate 455

Trypanosoma brucei genomic sequences totaling more than 300,000 bases.

These sequences were evaluated for putative genes and non-coding elements

against the nr (non-redundant) and GSS databases at the National Center for

Biothechnology Information using BLAST family software.

Many hopeful inroads into the biology of trypanosomes lie with the

discovery of new genes that may code unique targets for chemotherapeutic

agents or vaccines. Molecular genetic analysis of T. brucei will ultimately

reveal sequences involved in the development, survival, and pathogenicity of

these parasites and may be extended to include other similar parasites. This

study begins with the genomic organization of the Calflagin gene family and

was expanded to encompass a basic analysis of the entire T. brucei genome.

2. Materials and Methods

2.1. Amplification and isolation of genomic fragments.

Two clones Tb-1.8 (1.8 Kb) and Tb-24 (2.5 Kb) were previously

subcloned by Lee (50) and were made available in an ampicillin resistant

plasmid vector pl)C18 . The fragments were ligated into the BamH I multiple

cloning site of the vector. Nanogram quantities of Tb-1.8 and Tb-24 were

amplified by transformation of competent E.coli cells. Competent cells were

prepared by growing overnight cultures of E. coli strains DH5 alpha and/or DH5

alpha F* in YT (8 g Difco™ Bactotryptone, 5 g yeast extract, 5 g NaCI) or LB (10g

Difco™ Bactotryptone, 5 g yeast extract, 10 g NaCI) culture media. Inoculums

were taken from either frozen log phase cultures (50 ul) or colonies from fresh

agar plates. Inoculated broths were incubated in a New Brunswick™

shaker/incubator at 37° at 250 rpm. 500 ul of the overnight culture was then

added to 50 ml of YT or LB culture media in a 250 ml flask and returned to the

shaker incubator. The optical density (OD) was monitored at 550 nm with a

Beckman™ DU40 spectrophotometer until the culture reached an optimal

absorbance of 0.45. The culture was then divided in half and placed into two

30 ml Corex™ tubes and placed on ice for 10-20 min. The culture was then

centrifuged for 10 minutes at 2200 rpm (1000 x g) at 4 °C. The supernatant was

discarded and the pellet was resuspended in 2.5 ml sterile fresh (within 2

weeks) TSS solution (0.85 g Difco™ Bactotryptone, 0.43 g yeast extract, 0.85 g

NaCI, 5.0 ml 5% DMSO, 10 g of 10% PEG 8000 MW, 50mM MgCl2 (pH 6.5),

34

and ddH20 up to 100 ml). The competent cells were place on ice until ready for

transformation. Optimally they were used within 6 hours. Cells were

transformed by placing nanogram quantities of vector plasmid (10-25 ul) in the

bottom of a microfuge tube. To this was added 200 ul of DH5 alpha competent

cells. The tubes were then placed on ice for 10 minutes. The cells were then

heat shocked in a water bath at 42 °C for 2 minutes. One ml. of YT or LB media

was added to the tubes and they were placed in a shaker/incubator at 37 °C for

one hour at 250 rpm . Transformed cells were screened on ampicillin plates

using blue/white selection (107). Ampicillin plates (8.0 g Difco™ tryptone, 5.0 g

yeast extract, 5.0 g NaCI, 15.0 g agar, 50 ug/ml ampicillin, and ddH20 up to one

liter) were prepared for visualizing transformed cells by spreading 10 ul of 100

mM IPTG (isopropyl-b-D-thiogalactopyranoside) and 50 ul of 2% X-gal (5-

bromo-4-chloro-3-indoyl-b-D-galatoside) (25mg in 1.25 ml dimethylformamide)

over the surface of the plates and allowing the solutions to soak in for a few

minutes. Optimum plating densities were determined by plating volumes of 10,

50, and 100 ul of transformed cell culture. The transformed cells were spread

onto the surface of the ampicillin plates and allowed to soak in for a few minutes

before incubation. Controls included: Competent cells without plasmid

(nontransformed) on selective media to test antibiotic killing and competent

cells transformed with varying amounts of plasmid (.01-1.0 ug) on ampicillin

plates without X-gal or IPTG to test transforming efficiency and the level of

competence. Blue colonies (carry plasmids with no DNA inserted into the

multiple cloning site (MCS)) were noted on non-control plates as a measure of

ligation efficiency. White colonies (carry plasmids with DNA inserted into the

MCS, thus inactivating the lac Z gene) were inoculated into 5 ml tubes of YT or

35

LB broth containing 50 ug/ml ampicillin and incubated over night in a shaker/

incubator at 250 rpm and 37 °C.

2.2 Isolation of plasmids (analytical scale).

Positive cells (white colonies) were cultured overnight, harvested, and

analytical scale plasmid DNA isolation performed by standard alkaline lysis

"rapid prep" procedures as outlined in the monograph by Maniatis et al. (107),

which is a modification of a procedure described by Birnboim and Doly (108)

and Ish-Horowicz and Burke (109). 3.0 ml of the overnight culture was

centrifuged at 10,000 x g for one minute. The remaining 2 ml of culture was

stored at 4 °C or combined with 80% glycerol and stored at -70°C. The

supernatant was discarded and the pellet was resuspended by vortexing in ice

cold "Solution A" (50.0 mM glucose, 10.0 mM EDTA

(ethylenediaminetetraacetate, disodium form), 25.0 mM Tris-HCI pH 8.0, and

6.0 mg/ml lysozyme (added just prior to use). This solution was allowed to set

at room temperature for 5 minutes. Next was added 200 ul of freshly prepared

"Solution B" (0.2 N NaOH, 1.0% SDS (sodium dodecyl sulfate)). The tube was

then inverted gently two or three times and placed on ice for 5 minutes. To this

solution was added 150 ul of ice-cold potassium acetate, pH 4.8. The solution

was mixed by inverting two or three times and stored on ice for five minutes.

The solution was then centrifuged for 5 min at 10,000 x g and the supernatant

transferred to a new tube. An equal volume (450 ul) of phenol:chloroform was

added to the supernatant and thoroughly vortexed. The solution was then

centrifuged at 10,000 x g for 2 minutes and the supernatant was transferred to a

36

fresh tube (phenol layer discarded). Two volumes of ice-cold 100% ethanol

was added to the supernatant and mixed by vortexing. The samples were then

placed in an iced ethanol bath for 5 minutes, then centrifuged at 10,000 x g for

10 minutes at 4°C. The supernatant was then discarded and the pellet was

washed with 1 ml of ice cold 70% ethanol by gently inverting the tube once or

twice. The samples were then centrifuged once again at 10,000 x g for 10

minutes at 4°C. The supernatant was removed from the samples and the

pellets were further dried in a vacuum desiccator for approximately 5 minutes.

Care was taken not to over dry the samples which would have made

resuspending them more time consuming. The dry samples were resuspended

in 30 ul of TE buffer (10.0 mM Tris base pH 8,1.0 mM EDTA) and stored at

-20°C. DNA samples were analyzed by agarose minigel electrophoresis for the

presence and size of plasmid DNA.

2.3 Agarose mini gel analysis.

1 % agarose gels were cast by melting one gram agarose/100 ml 1 X

TBE (10.8 g tris base, 5.5 g boric acid, 4 ml 0.5 M EDTA, and ddH20 up to 1

liter) in a microwave oven. The agarose solution was weighed prior to heating

and rehydrated afterwards. The hot agarose solution was poured into open

ended plexiglass trays with the ends sealed with masking tape. A well form

was then placed square to and near one end of the tray and the agarose was

allowed so solidify at room temperature. In preparation for electrophoresis of

"rapid prep" plasmid DNA, the gel was placed in an electrophoresis tank

(masking tape removed) and covered (1-2 mm) with 1 X TBE solution. Typically

37

a convenient volume (25 ul) of a sample was prepared by adding 3 ul of rapid

prep DNA to 5 ul Loading Buffer (25 % glycerol, 0.5% SDS, 0.1% bromophenol

blue leading dye, 0.1 % xylene cyanol lagging dye, 50.0 mM EDTA) and 17 ul

of ddH20. The samples plus a size marker (usually Hind III digested lambda

DNA) were placed into the agarose slots and electrophoresed at 80 volts for

one hour. To visualize the plasmid DNA the gel was stained for 30 minutes with

an ethidium bromide (EtBr) solution (1 mg / liter ddH20) and destained for 30

minutes with ddH20. Ethidium bromide fluoresces orange in UV light. The EtBr

intercalates between DNA bases. Caution: EtBr is considered a potent

mutagen and extreme care is taken when working with this substance including

the use of gloves and protective Benchcoat™. "Rapid prep" samples showing

bands of a size consistent with the plasmid plus the insert were noted for further

treatment and / or photographed.

2.4 Gel photography.

A UV radiation source (Fotodyne™ transilluminator) was used in

conjunction with a Polaroid™ MP-4 camera system. Gels were placed on the

transilluminator and visualized with UV. The gels were then centered and

brought into focus with the camera. The camera was then loaded with

Polaroid™film Type 55 Positive/Negative (ISO 50). A red filter was placed over

the aperture, and the aperture setting was typically adjusted to 4.5. Higher

aperture settings were used with faint bands. At an aperture of 4.5 the film was

exposed for 1 minute and developed according to package instructions.

38

Negatives were treated by soaking in 9% sodium sulfite and kept as part of the

data.

2.5 Digesting DNA with restriction endonucleases.

The inserts were removed from the plasmid DNA by BamH I restriction

endonuclease, and samples were analyzed by agarose mini gel

electrophoresis. 1- 5 ug of plasmid DNA was typically digested in a convenient

volume (typically 20 ul for mini gel analysis) with no more than 10%/unit volume

restriction endonuclease and the appropriate 10X reaction buffer. One unit of

enzyme was used to digest 1 ug DNA for 1 hr. Protocols were essentially the

same for other low molecular weight DNA fragments. Twice the amount of

enzyme was used to digest genomic DNA. Digested samples were analyzed

by mini gel electrophoresis and positive clones showed banding patterns

consistent with the size of the plasmid and the insert. These clones were noted

on the selective antibiotic plate from which they came in preparation of

obtaining large quantities of the insert for sequencing and other analysis.

2.6 Large scale isolation of recombinant plasm ids.

Large scale ultra pure isolation of recombinant plasmids was performed

on positive isolates. Up to two liters of each E. coli clone was prepared for

isolation by inoculating a 25-50 ml LB broth culture and incubating overnight in

a shaking (250 rpm) incubator at 37 °C. 10-50 ml of the overnight culture was

used to inoculate 1 or more 2800 ml Fernbach™ flasks containing one liter LB

39

broth each. Some cultures containing plasmid vectors whose copy numbers

were less than that of pUC vectors were monitored at 550 nm until they reached

0.8-1.0 (log growth phase) at which time 2 ml of 20 ug/ml chloramphenicol in

95% ethanol was added to increase plasmid yields. Cultures were incubated

overnight in a shaking (250 rpm) incubator at 37 °C. The cells were collected

by centrifugation in a precooled (4 °C) Sorvall™ centrifuge with a GS3 rotor.

Approximately 500 ml/bottle was centrifuged at 6000 rpm for 6 minutes. The

cells were resuspended in 20 ml 0.15 M NaCI and placed in 45 ml Oakridge™

style tubes. Each tube contained the pellet equivalent of 750-1000 ml of

culture. These tubes were centrifuged in a precooled (4 °C) Sorvall™

centrifuge with a SA600 rotor at 6000 rpm for 5 minutes. From this point on all

work was done on ice in a cold room at 4 °C. The pellet of each tube was

thoroughly resuspended in 10 ml 50 mM Tris, 25 % sucrose, pH 8.0. Two ml of

5mg/ml lysozyme was added to each tube and mixed by gently inverting the

tubes 2-3 times. The tubes were then placed on ice for 5 minutes. The addition

of 4 ml 0.25 M Na2 EDTA, pH 8.0, followed and the tubes were again mixed by

inverting and allowed to sit on ice for 5 minutes. Five milliters of 5 M NaCI and

2 ml of room temperature 10% SDS was then added and the tubes mixed by

gentle inversion after each addition. The tubes were then balanced and left on

ice in the cold room for 90 minutes. The tubes were then centrifuged at 16,350

rpm for 45 minutes in a SA600 rotor. The supernatant was then poured into a

graduated cylinder and one volume of isopropanol was added to it. This

solution was then poured into a GSA centrifuge tube and placed in a dry ice

ethanol bath for twenty minutes or left over night at -70 °C. The frozen contents

of the GSA centrifuge tubes were then thawed in a tap water bath. The tubes

40

were then centrifuged in a GSA rotor for 20 minutes at 8000 rpm. The pellet

was resuspended in 15 ml/liter of culture TE (10mM Tris, 1mM EDTA, pH 8.0) by

slowly stirring on a stirring plate. Care was taken to avoid foaming of the

solution. A concentration of 20 ug/ml DNase free RNase was added to the

solution. Larger pieces of pellet were broken up by repeated pipetting.

Approximate yields of plasmid were determined by mini gel analysis.

Undissolved material was removed by centrifugation at 4 °C in a ™Sorvall

centrifuge using a SA600 rotor at 10,000 rpm for 10 minutes. To the

supernatant was added 5.3 g CsCI/5 ml TE. The solution was mixed gently to

avoiding the introduction of bubbles. Four hundred microliters of 10 mg/ml EtBr

(ethidium bromide) was added per 8 ml of solution under reduced lighting to

prevent DNA cleavage. The plasmid preparation was then introduced into Til

270 ultracentrifuge tubes, one per liter of culture. The tubes were balanced to

within 0.03 g by adding a balancing solution of either TE/CsCI (heavier) or

mineral oil (lighter). Great care was taken to insure that the tubes were full to

the top to prevent the tubes from collapsing during centrifugation. The tubes

were crimped shut with Ultracrimp™ caps and crimping device. The tubes were

centrifuged in a Dupont™ OTD65 ultracentrifuge for 40 hours at 36,000 rpm.

The tubes were then removed from the rotor and clamped into a ring stand.

Bands were visualized with a hand-held UV source. Care was taken to work in

subdued light to prevent photo-activated cleavage of the DNA by EtBr.

Protein

• • <4— Chromosomal DNA Plasmid

J ^ ~ \ RNA

41

Protective eye ware was also used through out the experiment where UV light

was used. EtBr stained plasmid DNA bands were extracted from the centrifuge

tubes while the tubes were clamped in a ring stand. A 20 gage needle was

inserted in the top of the tube near the cap to act as a vent. Another 20 gage

needle attached to a 3 ml syringe was inserted directly into the lower portion of

the plasmid containing band and the contents withdrawn. In some cases the

bands containing the plasmid DNA were subjected to another 40 hour

centrifugation as previously described to further remove contaminating

materials. After centrifugation, the plasmid DNA was placed into 15 ml conical

tubes and extracted with water-saturated isobutanol in subdued light to remove

the EtBr. The EtBr was removed with the top isobutanol phase (water-saturated

isobutanol is the upper phase of the mix). Several extractions were necessary

to remove all of the EtBr as monitored by a visually clear isobutanol layer.

When the isobutanol layer was clear two volumes of ddH20 and 9 volumes of

100% ethanol were added to the plasmid preparation and the solution was

thoroughly vortexed . The solution was then poured into 30 ml Corex™ tubes

and centrifuged at 4 °C in a Sorvall™ centrifuge at 8000 rpm for 10 minutes.

After centrifugation the supernatant was poured off completely and the pellet

was redissolved in 300 ul of 0.3 M sodium acetate and transferred to a 1.5 ml

microfuge tube. The DNA was extracted with 1 ml of 100% ice cold ethanol and

placed in a dry ice ethanol bath for two minutes. The DNA was then pelleted by

centrifugation (10,000 x g for 10 min. ® 4 °C). The supernatant was then

removed and the pellet was washed with 1 ml 70% ethanol by inverting the

tube one time (care was taken not to disturb the pellet while adding the

ethanol). The DNA was collected by centrifugation (10,000 x g for 5 minutes at

42

4 °C). The samples were then placed in a vacuum desiccator for 2 minutes to

remove residual ethanol. Care was taken not to completely dry the pellet in

order to facilitate later resuspension. The pellet was resuspended in 1 ml of TE

buffer or ddH20/liter of culture (0.5 ml if the pellet was divided into two tubes).

The absorbance was scanned from 220 nm to 320 nm (Beckman™ DU-40

spectrophotometer) to give an estimate of the yield and purity.

2.7 Preparative scale digestion of cloned DNA fragments.

100-200 ug of recombinant plasmid DNA (100-200 ul) was prepared for

fragment isolation. The plasmid DNA was digested in 1 ml microfuge tubes with

restriction enzymes. Both plasmids containing T. brucei calflagin sequences

were digested with Bam HI restriction enzyme. The digestion mixture was

completed in a volume of 250 ul and contained 25 ul of 10 X reaction buffer

(New England Biolabs) and 250 units of restriction enzyme (New England

Biolabs). Digestions were allowed to proceed for at least 3 hours at an optimal

temperature for the enzyme (37 °C for BamH I). The digested plasmid DNA was

purified (buffer salts removed) by ethanol precipitation and analyzed by mini gel

electrophoresis (see Agarose mini gel analysis).

2.8 Preparative scale isolation of cloned DNA fragments detected by UV

shadowing techniques.

The vector was separated from genomic DNA fragments by vertical

agarose gel electrophoresis. The technique required the assembly of an

43

agarose gel cassette. Glass plates (20 x 20 cm and 20 x 22 cm) were

thoroughly cleansed with Windex ® and then wiped with non-denatured 95%

ethanol. The plates were then wiped with a solution of 1%

dimethyldichlorosilane under a fume hood and allowed to dry for a few minutes.

The plates were then baked briefly in a drying oven at a warm setting. Prior to

assembly of the cassette, the plates were inspected visually at low angle and

wiped with a clean dry Kimwipe® to remove any dust particles. The cassette

was assembled by placing the two siliconized surfaces together, and placing

between them two Delrin® 20 x 1.0 x 0.15 cm spacers flush with the edge along

the long axis of the plates. A cellulose sponge (1.0 cm x 18.5 cm) saturated n

TBE buffer was placed between the two plates perpendicular to the long axis

and flush with the edge of the plates leaving the 22 cm plate to overhang at the

opposite end. The plates were then clamped firmly together with three medium

sized binding clips. Care was taken to insure that there was no space between

the Delrin® spacers and the sponge. Excess buffer from the sponge was

poured out from between the plates. The cassette was then placed into the

lower buffer well of a vertical agarose gel stand (which circulates the buffer from

the lower to the upper buffer wells) and clamped into place with the

overhanging 22 cm plate facing outward (Fig. 1 A and B). Care was taken to

insure the cassette was in a true vertical position. Two neoprene spacers (1 cm

x 2 cm x 0.6 cm) were placed between the 22cm plate and the gel stand forming

the upper buffer chamber. A small amount of vacuum grease was placed

between the top of the Delrin spacers and the neoprene spacers. A well

forming comb was inserted into the cassette such that the top of the slots of the

comb were above the level of the 20 x 20 cm plate. The size of the comb was

44

chosen according to the amount of DNA to be loaded, generally 500 ug DNA to

a 7-8 cm well. 1X TBE electrophoresis buffer was placed in the lower buffer

well up to the top of the sponge. The gel was cast while the cassette was in the

gel stand. 10-12 ml of melted agarose was pipetted into the cassette down one

edge with care being taken to avoid the introduction of bubbles. The level of

TBE in the lower buffer chamber was then raised to the level of the agarose, but

not exceeding it, forming a plug in the bottom of the cassette. After the agarose

had solidified the remainder of the cassette was filled with molten agarose. The

gel was allowed to solidify completely and the well forming comb was carefully

removed. Both the upper and lower buffer chambers were filled to capacity and

the circulating pump turned on to test for leaks. The DNA was then loaded into

the wells with agarose loading buffer (see Mini Gel Analysis). Care was taken to

insure that the samples were distributed evenly across the wells. The gel was

run at 80 volts for 4-5 hrs depending on the size of the fragment that was to be

isolated. The bands were detected by UV shadowing techniques. After the

DNA had electrophoresed for the desired length of time the gel was transferred

45

7 1 _ X L i l i r L i J T

poer r„, Buffer Chamber Z

Agarose Gel

. Sample Wells

•Delrin Spacer

Lover Buffer Chember

-Binding Clip

PUMP

i m r o f c w

^ / / / / / / / / / ^ ^ ^ ^ ^ ^

#WAA{ IAAAA0/ mwm.

front view side view

B 3/4 Inch Offset - [

0.5 Inchx 1.5 mm Delrin™ Spacer

Cellulose Sponge

Delrin Comb

Medium Clip

46

Figure 1 A Electrophresis stand and buffer pump assembly for

vertical agarose gel electrophoresis.

Figure 1 B Assembled cassette for vertical acrylamide gel electrophoresis.

47

to a thin layer chromatography (TLC) plate impregnated with an ultraviolet

indicator. A shortwave (260 nm) hand held UV lamp was held over the gel.

The plate fluoresced except where the DNA was present to absorb the UV light

which presented a shadow or darkened area on the gel. (Fig. 2). Those bands

corresponding to known desired fragment sizes were excised from the gel and

prepared for electroelutlon.

2.9 Electroelution and DNA recovery from excised from gels.

The excised bands were placed into cut lengths of dialysis tubing

according to the size of the gel slice. A dialysis bag clip was placed at one end

of the bag and the bag was filled with a quantity of 0.25X TBE buffer. Another

clip was placed at the other end of the bag and care was taken to remove all the

bubbles. The dialysis bag with the gel slice inside was then placed horizontally

in an electrophoresis chamber and covered with .25X TBE buffer. The level of

buffer was adjusted so it just covered the bag. Electroelution proceeded at 80

volts for 2-4 hours depending on the size of the fragment. When the

electroelution was complete the current was reversed for approximately one

half a minute to dislodge the DNA from the side of the dialysis tubing. The

DNA/TBE buffer was removed with a siliconized Pasteur pipette. The gel was

washed in the tubing with an additional volume of 0.25X TBE. Electroeluted

samples were extracted twice with one volume each TE saturated Phenol. The

aqueous DNA solution was then extracted with one volume of ether. The upper

ether layer was removed and the samples were allowed to sit under a fume

48

sz O)

0)

o •r >

oo L.

L. CQ

< Z Q O) c

c o U fmwm

CD CD

Q)

C CD O CO CD L. D 3

49

Figure 2. Principle of UV shadowing for the visualization of DNA bands in

agarose gels.

50

hood while residual ether volitalized for a few minutes. The samples were

centrifuged at 10,000 x g to remove any residual agarose and the supernatant

was transferred to a fresh tube. The samples were then placed in a vacuum

desiccator to reduce the volume to a more workable level (250 ul). The DNA

was then precipitated by adding one tenth volume (approximately 25 ul) of 3 M

sodium acetate and 3 volumes of 100% EtOH. Each sample was then vortexed

and placed in a dry ice ethanol bath for five minutes. The DNA was then

pelleted by centrifugation at 10,000 x g for 10 minutes. The supernatant was

removed and the pelleted DNA sample was gently washed (inverted once) with

1 ml of 70% ethanol and recentrifuged for an additional 5 minutes at 10,000 x g.

The supernatant was again removed and the pelleted DNA sample was placed

in a vacuum desiccator for five minutes to remove residual EtOH and H2O. The

DNA pellet was then resuspended in 25 ul of TE buffer by vortexing, and

alternately heating (65°C) and freezing the sample. A small fraction of the

sample (1.5 ul) was prepared for mini gel analysis (see Agarose Mini Gel

Analysis) to verify DNA fragments. The analysis was carried out with a tf/'nd III

cut lambda ladder for size reference. Once the fragments were verified they

were screened for restriction sites that would allow for subcloning.

2.10 Screening isolated DNA fragments for restriction sites.

Isolated DNA fragments were cut with various restriction enzymes

(described earlier) and electrophoresed on agarose gels (see sections 2.3 and

2.5) or 6% non-denaturing polyacrylamide gels (described below). Non-

denaturing polyacrylamide gels were used to identify small DNA fragments

51

(100-500 bp), which are beneficial in subcloning with some vectors. Digested

DNA fragments (1-2 ug) were combined with 2.5 ul of 5X nondenaturing

polyacrylamide gel electrophoresis loading buffer (0.25% bromophenol blue,

0.25% xylene cyanol, 0.5% SDS (sodium dodecyl sulfate), 25.0% glycerol) and

heated to 65 °C for five minutes. Samples not used immediately were stored at

-20 °C. A 20 x 20 x 0.15 cm cassette was assembled to contain the gel during

electrophoresis. The nondenaturing polyacrylamide gel solution was made by

mixing the following: 12.0 ml acrylamide stock solution (40.0 g electrophoretic

grade acrylamide, 1.0 g bisacrylamide, ddHteO to a final volume of 100 ml); 8.0

ml 10X nondenaturing TBE buffer; 60.0 ml ddH20; and 0.1 g ammonium

persulfate. The solution was filtered into a 250 ml vacuum flask and then

degassed until no bubbles were visible. Note: It is important to remove radical

scavengers like O2 because they will inhibit the polymerization process. After

degassing, 20 ul of TEMED (N,N,N\N\-Tetramethylethylene-diamine) was

added to the solution with gentle swirling of the flask. The solution was then

poured into the cassette prior to polymerization. Once the gel had polymerized

it was pre-electrophoresed for one hour at 300 volts. Samples and reference

markers (pBR322 plasmid cut with Hae III, Hpa II, or Hinf I restriction enzymes)

were loaded on to the gel with a 25 ul Hamilton® microsyringe. The gel was

electrophoresed at 250 volts until the bromophenol blue dye (leading dye)

migrated to about 80% of the gel's length. On completion of the electrophoresis

the gel was stained with EtBr for 5-10 minutes and then destained for 10

minutes. Some gels were photographed for later reference (see Gel

Photography).

52

2.11 Subcloning into M13mp and pUC vectors.

After the samples were screened for restriction sites using a variety of

restriction enzymes, the fragments were analyzed for further subcloning (110)

and sequencing. The restriction fragments for subcloning were ligated into

M13mp18/19 virus vectors or pUC 18/19 plasmid vectors. The variants of each

vector (18/19) have multiple cloning sites in opposite orientations permitting

sequencing of both DNA strands. Insertion of foreign DNA into the multiple

cloning site of either vector causes the inactivation of the amino-terminal

fragment of the beta-galactosidase gene which can be detected visually (white

plaques or colonies) when the presence of a synthetic inducer, isopropylthio-B-

D-galactoside (IPTG) and a substrate, 5-bromo-4-chloro-3-indoyl-l3-D-

galactoside (X-gal) is present in the media. Vectors were prepared by digesting

with restriction endonucleases complementary to the target DNA fragments.

Vectors were digested such that DNA fragments could be subcloned in either

direction or cloned unidirectionally. The vectors were further treated with

bacterial or calf intestinal alkaline phosphatase (BAP, CIAP) which removes

the 5' phosphate and prevents closing of the restriction digested ends. The

phosphatase treatment involved combining 100 ng of linearized vector with 100

units of bacterial alkaline phosphatase enzyme in a volume of 50 ul diluted with

TE buffer. The digested and phosphatase treated vector was then extracted

with phenol and precipitated with ethanol to remove enzyme and buffer salts.

The target DNA and the vector were then brought together in a ligation mixture

at a ratio of 1:1 - 3:1 (although other ratios were used to maximize

53

transformants). The ligation was contained in a volume of 10-20 ul and

contained: approximately 100 ng of vector DNA; 100 ng of target DNA; 5X DNA

ligase reaction buffer (250.0 mM Tris-HCL (pH 7.6), 25%(w/v) PEG 8000, 50.0

mM MgCl2, 5.0 mM rATP, 5.0 mM DTT (dithiotheitol)), T4 DNA ligase (0.1 BRL

unit for sticky ends, 1.0 unit for blunt ends), and sterile water up to volume. The

mixture was incubated at room temperature for 4 hours. The reaction was

stopped with 1/5 volume 0.25 M EDTA and sterile water up to a final volume of

30 ul.

2.12 TSS competent cells.

DH5 alpha F or P cells were inoculated into a 5 ml. culture of YT or LB

broth and grown overnight at 37 °C and 250 rpm in a New Brunswick™

shaker/incubator. 500 ul of the overnight culture was used in inoculate 50 ml

YT or LB broth in a 250 ml flask. The culture was returned to the shaker

/incubator and monitored until the absorbance 550 nm reached 0.45. The

culture was then divided into two sterile tubes and placed in an ice bath for 10-

20 minutes. The culture was then centrifuged at 1000 x g for 10 minutes at 4

°C. The supernatant was then discarded and the pellet resuspended in 2.5 ml

of sterile (filtered or autoclaved) TSS (0.85 g Bacto™ tryptone, 0.43 g yeast

extract, 0.85 g NaCI, 5.0 ml DMSO, 10.0 g PEG 8000, 50 mM MgCl2 (pH 6.5),

ddH20 to 100 ml). Each tube was then placed on ice until ready for use or

stored at 4°C for up to two weeks.

54

2.13 Transformation of DH5 alpha F with recombinant M13mp 18/19.

Recombinant M13mp RF forms were used to transform DH5 alpha F'

cells by the TSS competent cell method. Transformation was visualized as a

plaque in a lawn of cells. Plaques containing recombinant phage appear as

clear while plaques without recombinant phage appear blue. 300 ul of TSS

competent cells (described above) were added to a 1.5 ml microfuge tube with

approximately 10 ul ligation mixture (25-50 ng recombinant DNA). This mixture

was placed on ice for 30 minutes and then heat shocked for 2 minutes at 42 °C.

The cells were then added to a tube containing: 3 ml YT soft agar (10 g Bacto™

peptone, 8 g NaCI, 6 g Bacto™ agar/ liter) stored at 42 °C; 50 ul 2% X-gal, 10 ul

100mM IPTG, and 200 ul of lawn cells. The entire contents was swirled briefly

and poured on to a YT agar plate. The contents was spread evenly over the

plate by tilting the plate as necessary. The top agar was allowed to solidify at

room temperature and then placed at 37°C overnight.

2.14 Transformation of DH5 alpha F or F cells with recombinant plasmid.

1.0 - 0.01 ug of recombinant plasmid in a volume of 10 - 25 ul was

placed into the bottom of a 1.5 ml microfuge tube with 200 ul TSS competent

cells (described above) and placed on ice for 10 minutes. The cells were then

heat shocked in a 42 °C water bath for 2 minutes. 1 ml of sterile YT or LB broth

was then added to each tube and incubated at 37°C in a shaker/incubator at

55

250 rpm for one hour. During the incubation period LB/ampicillin plates were

prepared by spreading 10 ul of 100 mM IPTG and 50 ul 2% X-gal on to each

plate with a sterile hockey stick. At the end of the incubation period transformed

cells were spread onto the plates in varying amounts (10, 50,100 ul) and

incubated overnight at 37 °C. Plasmid was isolated by "rapid prep" protocols

(see Isolation of plasmid 2.2)

2.15 Preparation of M13mp single stranded template for sequencing.

The plaques containing the recombinant phage (clear) were removed

from the plate with sterile Pasteur pipette by driving the end of the pipette into

the plaque and pulling out the plaque along with a plug of agar. The plugs

were placed in 5 ml of YT media along with 100 ul of overnight DH5 alpha F

culture. The culture with the plaques were shaken at 250 rpm for 5 hours at 37

°C. After the incubation period 3.6 ml of phage infected culture was transferred

to 5 ml polypropylene tubes and centrifuged for 8 minutes at 1000 x g in a

microcentrifuge. The supernatants were transferred to new sterile 5 ml

polypropylene tubes where they could be stored at 4 °C, or used. The pellet

contained Replicative Form DNA (RF DNA) and could be saved for a small

scale preparation of the clone. To proceed with the isolation of the single

stranded template for sequencing, the supernatants were centrifuged again in

the same manor as before and transferred to sterile tubes to ensure that all

bacterial debris was removed. To the clean supernatants was added 900 ul of

a solution containing 20% PEG 8000 (polyethylene glycol) and 2.5 M NaCI.

The tubes were covered with Parafilm® and thoroughly vortexed. The tubes

56

were then set aside at room temperature for 15 minutes. Following the

incubation the parafilm was removed and the tubes were centrifuged for 10

minutes at 10,000 x g to pellet the phage particles. The supernatant was

removed completely with a drawn out pasteur pipette and the phage pellets

resuspended in 100 ul of TES buffer (20 mM Tris-HCI, pH 7.5,10 mM NaCI, 0.1

mM Na2 EDTA) by thoroughly vortexing. The solution was then transferred to

1.5 microfuge tube containing 100 ul of TE-saturated phenol and vortexed 20

seconds. The solution was then centrifuged for 2 min at 1000 x g followed by

the removal of 80 ul of the phenol layer. The solution was then vortexed for 20

seconds with 100 ul of chloroform and again centrifuged for 2 minutes to

separate the organic and aqueous phases. 80 ul of the upper aqueous phase

was then transferred to sterile 1.5 ml microfuge tubes. To these tubes was

added 9 ul of 3 M sodium acetate (300 mM final concentration) followed by

vortexing and 200 ul of 100% EtOH followed by more vortexing. The tubes

were then place in a dry ice ethanol bath, or -70 °C freezer to precipitate the

DNA. The DNA was then pelleted by centrifugation at 1000 x g for 10 minutes.

The 100% ethanol was removed and the pellet was then washed with 70%

ethanol by inverting the tube once and then recentrifuging the pellet for 5

minutes at 1000 x g. The Ethanol was again removed and the pellet was dried

in a vacuum desiccator for 2 minutes to remove any residual ethanol and H2O.

The viral DNA pellet was resuspended in 30 ul of TE buffer by alternately

vortexing, centrifuging, and placing the tube in a 65 °C water bath until the

pellet was thoroughly dissolved. Three microliters of the sample was analyzed

by mini gel electrophoresis (see 2.3). Recombinant phage with at least 300 bp

of insert will have reduced mobility compared to nonrecombinant M13mp DNA.

57

(108,109,110)

2.16 Nucleotide sequencing of double-stranded and single-stranded templates

by the dideoxyribonucleotide method.

DNA sequencing reactions were performed on genomic sub-clones as

described in the Sequenase® version 2.0 DNA sequencing kit obtained from

US Biochemical (111,112,113). Double-stranded templates (plasmid) were not

initially treated the same way as single-stranded templates. From the primer

annealing step on, however, the sequencing reaction was the same for both

templates.

To prepare for double stranded sequencing, approximately 4 ug of clean

plasmid DNA was dissolved in 30 ul of sterile water. The DNA was denatured

with 3 ul of 2 N NaOH at room temperature for five minutes. The samples were

then ethanol precipitated by vortexing with 120 ul 100% ethanol followed by the

addition of 5 ul of 3 M sodium acetate (pH 5.0) and more vortexing. The DNA

was pelleted at 1000 x g for 10 minutes at 4 °C. The supernatant was carefully

removed and the pellet was washed with 70% ethanol by inverting the tubes

once and then repelleting the DNA the same as before. The supernatant was

again removed and the pellet was dried in a Speed Vac™ for 5 minutes. The

denatured DNA pellet was then resuspended in 8 ul of sequencing water,

primer solution, and 2 ul of reaction buffer, all supplied in the sequencing kit.

The primer was annealed to the template by placing the DNA primer mixture in

a 37 °C water bath for 45 minutes.

58

To prepare for single-stranded templates, a primer solution (all

components supplied in the kit) was typically made for up to 12 samples (13 ul

of primer, 26 ul reaction buffer, 26 ul sterile water, total 65 ul). Five microliters of

the mixture was then added to 5 ul of the single-stranded template preparation

and placed in a 65 °C water bath for 2 minutes. The samples were then

removed and the primer was allowed to anneal to the template by placing the

tubes in a tray of 65 °C water, which was then allowed to cool slowly (30-40

minutes) at room temperature. At this point the procedures for both double-

stranded and single-stranded sequencing were the same. While the templates

were annealing a series of termination tubes were set up. One set consisted of

four different colored .5 ml microfuge tubes with 2.5 ul of one kind of termination

mix per color (ddGTP, ddATP, ddTTP, ddCTP). The Labeling Solution was

also prepared at this time. The solution consisted of 0.1 M DTT, distilled water,

and labeling mix, all supplied in the sequenase kit, and 3E>S-ATP, supplied

separately, for a total volume of 45.5 ul/12 samples. The Sequenase™ enzyme

was diluted with dilution buffer with or without pyrophosphatase according to

the number of samples (26.4 ul for 12 samples). Both sequenase and labeling

mixes were adjusted according to the number of samples to be sequenced

(Table 1. A and B). After the annealing, 3.5 ul of the 35S-ATP labeling mix was

added to each of the annealed template samples. For each reaction 2 ul of the

diluted sequenase was added to the annealed templates and allowed to

incubate at room temperature for 4 minutes. While the incubation was taking

place, the termination tubes (G, A, T, C) were placed in a 37 °C water bath. On

completion of the 4 minute incubation, 3.5 ul of the enzyme reaction mixture

was transferred to each tube in a set of termination tubes and mixed by

59

pipetting up and down a few times. The termination tubes were then returned to

the water bath and incubated for an additional 20 minutes. At the end of the 20

minute incubation period the reaction was stopped by adding 4 ul of formamide

stop solution to each of the G, A, T, C tubes. The tubes were then stored at -20

°C until ready for use. The samples were heat denatured in a 90 °C water bath

for 2 minutes and then plunged into an ice bath prior to loading onto

sequencing gels. Multiple samples, up to 12, were "staged" during the reaction

procedure according to a time table (Table 2.).

Many of the genomic subclones were further subcloned to obtain

fragments more suitable for sequencing or to obtain sequence from the

opposite strand. Another sequencing strategy was to employ the use of

oligonucleotide primers in the sequencing reactions (Biosynthesis, Lewisville,

Tx) to extend the sequence from larger subclones where restriction sites were

not present for subcloning.

2.17 Preparation and electrophoresis of polyacrylamide sequencing gels.

Sequenase reaction products were analyzed by electrophoresis on 6%

denaturing polyacrylamide gels. One hundred milliters of 6 % polyacrylamide

gel solution (42.4 g urea, 20 ml 30 % acrylamide/1 % bisacrylamide, 10 ml 10X

TBE buffer, ddH20 up to 100 ml) was prepared in a 250 ml beaker by placing

the solution on a stirring plate and covering the beaker with Parafilm® until the

urea was dissolved (20 minutes) during which time the cassette was usually

assembled.

60

Table 1.

LABELING MIX TABLE

Samples I 2 3 4 5 6 7 8 9 10 11

DTT 1.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

Label Mix 0.4 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8

H2O 4.6 4.8 6.4 8.0 9.6 11.2 12.8 14.4 16.0 17.6 19.2

35S 0.5 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Table 2.

SEOUENASE™ DILUTION TART F

Samples 1 2 2 4 5 6 7 8 q 10 n

Enzyme

Dilution 3.5 5.3 7.0 6.5 8.1 9.7 11.3 13.0 14.6 16.2 19.5

Buffer

Pryophos- 0.5 0.5 0.5 0.63 0.75 0.9 1.0 1.13 1.25 1.4 1.5

phatase Sequenase 0.5 0.7 1.0 1.3 1.5 1.8 2.0 2.3 2.5 2.8 3.0

61

Table 3.

SEQUENCING REACTION TABLE

TIME ACTION

-1 Place all termination tubes for templates 1-4 @ 37 °C 0 Add 2ul diluted sequence to template #1, mix, place @ RT 1 Add 2ul diluted sequence to template #2, mix, place @ RT 2 Add 2ul diluted sequence to template #3, mix, place @ RT 3 Add 2ul diluted sequence to template #4, mix, place @ RT 4 Dispence 3.5ul of template #1 to each GATC tubes @ 37°C 5 Dispence 3.5ul of template #2 to each GATC tubes @ 37°C 6 Dispence 3.5ul of template #3 to each GATC tubes @ 37°C 7 Dispence 3.5ul of template #4 to each GATC tubes @ 37°C 8 Dead time/catch up

9 Place all termination tubes for templates 1-4 @ 37 °C 10 Add 2ul diluted sequence to template #5, mix, place @ RT 11 Add 2ul diluted sequence to template #6, mix, place @ RT 12 Add 2ul diluted sequence to template #7, mix, place @ RT 13 Add 2ul diluted sequence to template #8, mix, place @ RT 14 Dispence 3.5ul of template #5 to each GATC tubes @ 37°C 15 Dispence 3.5ul of template #6 to each GATC tubes @ 37°C 16 Dispence 3.5ul of template #7 to each GATC tubes @ 37°C 17 Dispence 3.5ul of template #8 to each GATC tubes @ 37°C 18 Dead time/catch up

19 Place all termination tubes for templates 1-4 @ 37 °C 20 Add 2ul diluted sequence to template #9, mix, place @ RT 21 Add 2ul diluted sequence to template #10, mix, place @ RT 22 Add 2ul diluted sequence to template #11, mix, place @ RT 23 Add 2ul diluted sequence to template #12, mix, place @ RT 24 Dispence 3.5ul of template #9 to each GATC tubes @ 37°C 25 Dispence 3.5ul of template #10 to each GATC tubes @ 37°C 26 Dispence 3.5ul of template #11 to each GATC tubes @ 37°C 27 Dispence 3.5ul of template #12 to each GATC tubes @ 37°C 28 Add 4ul of stop solution to all template 1-4 tubes 37 Add 4ul of stop solution to all template 5-8 tubes 46 Add 4ul of stop solution to all template 9-12 tubes

62

plates are thoroughly cleaned. Cleaning was carried out first with Windex® and

then with 95% ethanol. The inside of the cassette plates were then siliconized

by wiping them down with 5% dichloromethylsilane dissolved in heptane under

a fume hood and allowed to dry (5 minutes). The plates were occasionally

subjected to a drying oven and "baked" at 200 °C for 10 minutes. The plates

were then returned to the rubber stoppers siliconized side up and inspected at

low angle for dust particles, which were removed with a Kimwipe®. The two

plates were then placed one on top of the other, siliconized sides towards each

other and offset lengthwise by 13 mm (0.5 inch). Two Delrin® spacers (51 cm

X 13 mm X 0.25 mm) were placed between the plates and flush with the

lengthwise edges. A third spacer (45 cm X 1.3 cm X 0.25 mm) was placed

between the plates perpendicular to the first two and flush at one end (this will

eventually be the top of the cassette). The cassette was then clamped together

with large binding clamps (three to a side). The spacer junctions were

inspected for potential leaks.

Gel casting: After the urea was dissolved the polyacrylamide solution

was filtered through a Buchner funnel containing a piece of Whatman™ No.1

qualitative filter paper or equivalent and collected in a 250 ml Erlenmeyer

vacuum filtration flask. The Buchner funnel was then removed and 0.12 g of

solid ammonium persulfate was added to the filtered solution. The solution was

then degassed under vacuum for 3-5 minutes with gentle swirling to remove all

the bubbles and gasses. The solution remained under vacuum while a funnel

was created at the open end of the cassette with warmed plastocene®.

Immediately before adding the acrylamide solution to the cassette 25 ul of

TEMED was added to the solution and swirled gently with out introducing

63

bubbles to the solution. The acrylamide solution was then added to the

cassette by carefully pouring the solution down one side of the cassette with the

aide of the plastocene® funnel. When the cassette was 3/4 full it was placed

horizontally on four stoppers and the acrylamide solution was allowed to flow

out the end of the cassette. A small spacer (20 x 13 X 0.25 mm) was then

placed between the plates at the open end of the cassette and the acrylamide

was allow at least one hour to solidify. If the gel was not to be used right away

the open end of the cassette was covered with plastic wrap to prevent drying.

The gels were used within 24 hrs.

Preelectrophoresis and loading of acrylamide gels: The plastic wrap and

short spacer were removed from one end of the cassette and the binding clips

and long spacer were removed from the other end. The cassette was then

placed into a sequencing electrophoresis gel stand such that the bottom of the

cassette (while the gel was being poured) was at the top of the stand and such

that the more elevated of the offset plates was facing away from the stand. All

sequencing gel electrophoresis was carried out in a heated box constructed

with wood and plexiglass. The temperature was elevated to between 30-35 °C

with a small space heater. End spacers were placed vertically between the

plates extending the existing vertical spacers to the top of the glass. Neoprene

spacers took up the space between the top of the offset plate and the stand.

The cassette was then clamped into the stand with large binders such that the

plates completed the formation of the upper buffer chamber and with the bottom

of the cassette in the lower buffer chamber. Both chambers were then filled with

1X TBE sequencing buffer and leaks that may occur in the upper chamber were

addressed using vacuum grease. Air bubbles were removed from the exposed

64

areas of the gel in the upper and lower chambers with a bent Pasteur® pipette.

The electrical leads were connected to a power supply and the gel was

preelectrophoresed at 1700-2300 volts (50 amps constant power) for one hour.

The glass plates were monitored for excessive heat (>55 °C) and the voltage

was adjusted accordingly. When the preelectrophoresis was complete the

power was turned off and the upper and lower buffer chambers were again

cleared of bubbles and any urea that might have diffused into the buffer above

the gel. Prior to loading, the samples were placed in a water bath at

90 °C for two minutes and then placed into an ice bath until loading. Shark's

tooth well-forming combs were inserted between the plates and spaced such

that the gel would be oriented from left to right. Two microliters of each sample

(G,A,T,C) were drawn into a Hamilton® microliter syringe with a 32 gage needle

and placed between the spaces of the shark's tooth combs. Care was taken not

to introduce bubbles into the chamber. The syringe was rinsed in ddH20 after

each loading. At least one space was kept between sequencing reactions.

Generally 12 sequencing reaction were run on a single gel. Electrophoresis

continued at approximately 2300 volts or 50 constant amps according to the

migration of the xylene cyanol component of the sequencing reaction mixture

(cm). Gel runs of 35, 70, and 120 centimeters allowed reads approaching 500

bases. When the electrophoresis was completed the power was turned off and

the cassette was removed from the stand and placed flat on a bench top. The

plates were then separated by inserting a single edge razor blade between the

two plates and carefully separating them. The plates were separated such that

the gel was left intact on one of the plates. If the gel appeared to be stuck to

both plates, techniques such as gently blowing on the gel or the delicate use of

65

a pasture pipette was employed to preserve as much of the gel as possible.

Once the plates were separated a 17 x 14 inch piece of 3 MM Whatman®

paper was placed on the gel even with the bottom edge. The over hanging gel

was then trimmed away with a razor blade. The paper was then carefully lifted

away from the plate with the gel stuck to it and placed gel side up on the bench.

A piece of plastic wrap was then stretched over the gel and paper and the

excess trimmed away. The gel sandwiched between plastic wrap and watman

paper was then dried on a slab drier at high heat for one hour. After drying the

plastic wrap was removed and the gel and paper were briefly (1- 2 minutes)

submerged in a solution of 12% methanol 10% acetic acid to remove excess

urea. The gel was again covered with plastic wrap and dried on a slab drier at

high heat for two hours.

2.18 Autoradiography and sequence analysis.

Dried gels were placed into film cassettes (plastic wrap removed) gel

side up. A piece of Kodak XAR-5 autoradiographic film was placed on top of

the gel in a dark room. The film was exposed from one to several days

depending on the dpm counts emitting from the gel. Nucleotide sequence

analysis was determined for both genomic fragments using the Macintosh®

DNA analysis software program DNAsis® (Hitachi).

2.19 Detection of genomic sequences using radio labeling techniques:

Southern blotting.

66

Labeled DNA fragments or oligonucleotide probes were employed to

screen genomic digest and various libraries using methods originally described

by Southern (114). DNA from genomic digests or cloned fragments were

separated by gel electrophoresis. Some gels were stained with EtBr and

photographed. Gels were then placed in a denaturing solution (0.5 M NaOH,

0.5 M NaCI, for 30 minutes. During gel denaturation 12 cm X 22 cm Biodyne®

nylon membranes (Gibco BRL Life Technologies Inc.) were prewetted in ddH20

then soaked in the denaturing solution described above. Two thin (.25 inch

each) sponges were also presoaked in denaturing solution. The DNA was

transferred from the gel to the nylon membrane by capillary action. The transfer

was conducted in a shallow cafeteria type tray and assembled in the following

manner. The two sponges were placed in the center of the tray, on top of them

were placed four pieces of blotting paper (Schleicher & Schuel), two pieces of

GB002 and two pieces of GB004. The gel was placed on top of the blotting

paper well side down (the DNA runs closer to the bottom of the gel). A notched

and labeled nylon membrane was placed on the gel taking care to avoid

bubbles between the gel and the membrane. Two more pieces of GB004

blotting paper were placed on the membrane followed by a one inch stack of

paper towels cut to size. The assembly was weighted with a 0.5 inch thick

piece of plexiglass on top. The tray was then filled with denaturing solution to

the level of the first sponge. The paper towels were changed out at 30 minute

intervals (x 2) and then at one hour intervals (x 2). At the end of three hours,

the assembly was carefully dismantled. The membrane was gently washed in

2.5 X SSPE to remove any residual agarose. The membrane was then allowed

to dry overnight or placed in a warm oven for a few hours or UV crosslinked to

67

the filter with a hand held UV source or commercial membrane crosslinker at

300 milijoules of UV.

2.20 Radiolabeled probes: end labeled or random primed.

Small (oligonucleotide) probes used in hybridizations were radio labeled

at their 5' ends by using T4 polynucleotide kinase to transfer the alpha-

phosphate from a [gam ma-32 P] ATP to the free 5' hydroxyl group of the probe

(115). Approximately 0.1 ug of oligonucleotide probe was combined in a

microfuge tube with 2 ul of [g-32P] ATP (3000 Ci/mmol, DuPont), 2.5 ul 10 X

kinase buffer (500mMTris-HCI pH 7.4, 50mM MgCl2, 20mM DTT, and 1.0 mM

spermidine), and 1.5 ul 10U/ul T4 polynucleotide kinase. The contents of the

tube were mixed gently and incubated at 37 °C for 45 minutes. The reaction

was stopped by heating to 65 °C for five minutes. Unincorporated nucleotides

were removed by Nensorb™ 20 nucleic acid purification cartridges (NEN

Research Products Bioresearch Systems, Dupont) or NucTrap® probe

purification columns (Statagene).

For larger probes such as cloned sequences, where fewer moles of

probe required more extensive labeling, random priming was employed using

the Life Technologies® Random Primers DNA Labeling System (Gibco BRL)

according to packaged instructions. Labeled probes were cleansed of

unincorporated nucleotides by the same method used for end labeling (above).

2.21 Hybridizations.

68

Screening denatured DNA fragments crosslinked to nylon membranes

with radio labeled probes was carried out in a hybridization vessel. The

membrane was added to the vessel and prehybridized in 5 ml. of Life

Technologies® DNA Typing Grade Hybridization Buffer (Gibco BRL) for 30 min

at 65 °C. During the prehybridization the probe was denatured by heating at

100 °C for 5 minutes. The probe was then added to the hybridization vessel.

The vessels was resealed and put in motion for 12-16 hours at 65 °C. After

hybridization, the hybridization solution was removed and the membrane was

washed in the hybridization vessel with varying concentrations of SSC (0.1 X -

2X), 0.1% SDS until the dpm signal emitting from the membrane was between

1- 2 k. The membranes were then wrapped in plastic and analyzed by

autoradiography using an intensifying screen at -70 °C.

2.22 M13 library construction: isolation, size fractionation and preparation of

genomic DNA..

Genomic DNA from Trypanosoma brucei brucei antigenic type M110

(116) was used in this study. Genomic DNA was isolated by methods

previously described by Medina-Acosta and Cross (117). Genomic DNA was

digested with either Xba I or Xho I and size selected in a 0.8% agarose gel

between 10-14 Kb.

The DNA was placed into Hospitak "up-mist" medication nebulizers with

200 ul 10x TM buffer (100 mM Tris; 50 mM MgCfe), 1.6 ml 40% glycerol, and

ddH20 up to 2 ml. The nebulizers were actuated on ice with 10 lb of N2 for 1

minute. The chambers were then briefly centrifuged and the samples divided

69

into four 1.5 ml microfuge tubes of approximately 490 ul each. The DNA was

then precipitated with 50 ul of 3 M NaAcOH and 1 ml 100% EtOH. The samples

were resuspended in 30ul H20 per tube and then combined (120 ul total

volume).

The ends of nebulized DNA were repaired (blunted) in a preparation

containing:

30 ul of the nebulized sample

5 ul 10x polynucleotide kinase buffer

5 ul 10 mM rATP, 7.5 ul 2 mM dNTP

5 ul Klenow fragment (2 U/ ul)

3 ul T4 polynucleotide kinase (10U/ul).

The preparation was incubated at room temperature for 10 minutes followed by

incubation at 37° for 50 minutes. Following incubation 13 ul of 5X agarose gel

electrophoresis loading buffer (25% glycerol, 0.5% SDS, 0.1% bromophenol

blue, 0.1% xylene cyanol, 50 mM EDTA) was added in preparation for size

selection. Nebulized and end repaired DNA was size selected in a TAE 0.8%

agarose gel. Samples were electrophoresed at 80V for approximately 1 hour

concurrently with a 1 kb ladder (Gibco BRL). DNA was visualized with ethidium

bromide. The DNA was size selected from approximately 1.5 kb to 3.0 kb by

removing the unwanted lower MW end of the gel, reversing the current, and

running the DNA against a small piece of dialysis membrane embedded in the

gel lane at the 3 kb marker. The DNA was then "pushed" into the membrane by

increasing the voltage to 200V for about one minute. The wet membrane was

then carefully removed from the gel and placed into a 1.5 ml. microfuge tube

with 10 ul of H20 (total volume 15-20 ul). The membrane was crushed into the

70

bottom of the tube with a pipette tip and the tube was place in a 65 °C water

bath for 10 minutes to remove the DNA from the membrane.

2.23 M13 library construction: vector preparation and ligation.

RF M13 vector was prepared for ligation by digesting with SmaI

restriction endonuclease followed by treatment with calf intestinal alkaline

phosphatase (CIAP). The reaction was set up as follows:

5 ug RF M13 vector

5 ul (50 U) Sma I

20 ul 10X buffer

17Q Ml cWH#

200 ul total volume

The digestion was incubated at room temperature for at least one hour.

Fifty units of CIAP was added to the digestion followed by further incubation at

37 °C for one hour. The enzyme activity was stopped by heating 65°C for 10

minutes. The vector was extracted with phenol and precipitated in ethanol. The

vector was resuspended in 100 ul ddH20. Ligations were set up in a total

volume of 10 ul containing:

6 ul (3-4 ug) end repaired and size selected genomic DNA

2 ul (100 ng vector [Sma I/CIAP])

1 ul T4 ligase (400 U/ul)

1 ul T4 ligase buffer

10 ul total volume

71

Ligations were incubated overnight at room temperature. Following incubation

90 ul H2O was added to each ligation in preparation for transformation of

competent cells.

2.24 M13 library construction: transformation of competent cells.

5-10 ul of the ligation mix was used to transform Epicurian Coli ® XL2-

blue MRF' ultracompetent cells (Statagene). Competent cells were thawed on

ice. 100 ul of cells was placed into prechilled Falcon® (12 x 75 mm

polypropylene) tubes on ice. 1.7 ul of 1.4 M beta-mercaptoethanol was added

to the cells in the bottom of the tube. The tubes were shaken on ice every 2

minutes for 20 minutes. The ligation mix was added to the cells and allowed to

sit for 30 minutes with shaking every five minutes. The cells were then heat

shocked at 42°C for 45 seconds then placed back on ice for two minutes. The

cells were then returned to the bench at room temperature for 30 minutes. To

each sample of transformed cells was added:

6 ml top agar

80 ul 2% X-gal

16 ul 100 mM IPTG

200 ul XL2-blue MRF' lawn cells.

Transformants were selected using blue/white screening techniques

(118,119,107) on YT agar plates. Clear recombinant plaques were taken with a

sterile toothpicks and grown up overnight at 37 °C with shaking in 800 ul 4YTG

(10 g tryptone, 17 g yeast extract, 5 g NaCI, 2 g dextrose, ddH20 up to one liter)

with 80 ul of overnight XL 2-blue MRF'cells. Following incubation, the cells

72

were pelleted and 600 ul of the supernatant was transferred to a deep-well

microtiter plate. DNA was precipitated by adding to 180 ul of 20% PEG

8000/2.5 M NaCI to the supernatant and inverting several times. After a ten

minute incubation at room temperature the plates were centrifuged at 3000 rpm

(Beckman GS-6R centrifuge, GH-8 rotor, microtiter carrier) to pellet the DNA.

The plates were then inverted onto paper towels and spun again at 300 rpm for

10 seconds to remove excess PEG. The phage pellets were resuspended in

20-40 ul of Triton-TE extraction buffer (0.5% Triton X-100/1X TE) at 90 °C in

preparation for sequencing. The remaining suspensions (not sequenced) were

stored in 96 well microtiter plates at -20°C.

2.25 M13 library sequencing and analysis.

Enzymatic extension reactions for sequencing were done in Perkin

Elmer thermal cyclers (Cetus Corp.) using AmpliTaq® DNA polymerase and

ABI PRISM™ dye terminator (FS enzyme) or dye primer (CS+ enzyme) Ready

reaction kits following the manufacturer's instructions. Samples were

sequenced by single passage through an automated ABI Prism 377 DNA

sequencer. Sample sequences were filtered for vector and low compositional

complexity sequences (120). Sequences were analyzed with a basic local

algorhythm search tool (BLAST) with and without filters (120,121,122,123,

124, 125) at the National Center for Biotechnology Information (NCBI). The

sequences were "BLASTed" in-batch via E-mail using TBLASTX formatting.

Each sequence was then submitted to the Genome Sequence Survey (GSS)

database. "Nearest Neighbors" to the GSS sequences were reported by NCBI

73

and were analyzed for inclusion in the study. All sequences under

consideration were then reBLASTed for the most updated alignments using

BLASTP or BLASTN formatting at the NCBI website. Sequences were

considered individually for inclusion based on homology, statistical (P) values,

length, and the repetitive nature of the sequences.

2.26 Cosmid library construction: vector preparation.

Cosmid libraries were constructed in a modified Supercos 1 (Statagene)

cosmid vector. The vector was modified (site directed mutagenesis) to hold

approximately 1 kb more insert by removing the amp resistance portion of the

vector and part of the pUC multiple cloning site containing Xho I and Hind III

restriction sites. The vector (scos-seq3) was modified in this way by Dr. Lisa

McDaniel at the University of Texas Southwestern Medical Center for use in the

Human Genome Project and others. 13 ug of the vector was linearized with

Xba I at nucleotide 1435 in the following manner.

10 ul vector (13ug)

10 ul enzyme (100 U)

20 ul buffer (10X)

160 ul ddH2Q

200 total volume

The digestion was carried out at 37° C for 1 hour. The vector was then treated

with CIAP (50 U) for 1 hour at 37° C. The reaction was stopped by phenol

extraction and precipitated with ethanol. The DNA was resuspended in 175 ul

ddH20. The digestion was visualized on a 0.8% agarose gel stained with EtBr

74

(Fig. 3 B2) The cos ends of the vector were exposed by digesting with BamH I

at 37 °C for two hours in the following reaction:

175 ul vector

20 ul buffer

5 ul BamH I (50 U)

200 ul total volume

The vector was then extracted with phenol-chloroform-isoamyl (25:24:1),

extracted with chloroform, precipitated with 100% ethanol, washed with 70%

ethanol, and resuspended in 40 ul of ddH20. Two separate bands were

predicted (1.1 kb and 6.5 kb) and detected on 0.8 % agarose gel (Fig. 3 B4).

2.27 Cosmid library construction: isolation and preparation of genomic DNA..

Genomic DNA from bloodstream form Trypanosoma brucei M110 (116)

was used in this study. Trypanosomes were harvested from rodent blood as

described previously (126). Genomic DNA was obtained by LiCI precipitation

of detergent solubilized trypanosomes (117). The optimal digestion time was

determined for 5 ug of high molecular weight genomic DNA with 0.1 U ofSau

3A I enzyme (Fig. 3A) Digestions were carried out at 37° C in the following

reaction:

10 ul (5 ug) DNA

4 ul 10X buffer

1 ul (.1U) enzyme

25 Ul ddHzQ

40 ul total volume

75

The reaction was stopped in 8 ul aliquots with an equal volume of phenol-

chloroform at time intervals of 1, 2, 4, 8, and 16 minutes. The DNA was then

precipitated in ethanol and resuspended in ddhteO. The results of the partial

digestions were visualized on a 0.3% agarose gel stained with EtBr. The

digestion time containing the largest amount of 40 kb DNA fragments was

determined to be three minutes under the conditions described above. The

above digestion was then repeated with 10 ug DNA, 15 ul ddh^O for three

minutes. The reaction was stopped with a phenol extraction (200 ul phenol-

chloroform) and 160 ul ddhteO. Residual phenol was removed with a

chloroform extraction and the DNA was precipitated with ethanol. The DNA

was then resuspended in 40 ul ddH20 and visualized on 0.3 % agarose gel

with EtBr (Fig. 3A). The partially digested DNA was then treated with CIAP, 26

U at 37° C for one hour, to avoid cloning multiple smaller inserts. The DNA was

again extracted with phenol and chloroform, precipitated in 100% ethanol,

washed in 70% ethanol, and resuspended in 40 ul ddH20.

2.28 Cosmid library construction: ligation of genomic DNA and vector.

Genomic DNA fragments were cloned into the Xba I site of the scos-seq

3 vector with T4 DNA ligase in the following reaction:

8.0 ul genomic DNA (4 ug)

5.0 ul Xba I-CIAP and BamH I digested vector (1.5 ug)

1.5 ul buffer

1,0 ul T4 DNA ligase

15.5 ul total volume

76

4 5 6 7

1 2 4 8 16 Minutes

B

4.0 '*

12.2

1.0.

4.0 3.0_

2.0_ 1.6_

¥ *» ;JWi4W

£ivv

** ^ Ca>"

77

Figure 3. Panel A shows digestion times for 5 ug of High molecular weight

genomic DNA with 0.1 U ofSau 3A I enzyme. The reaction was stopped at time

intervals of 1, 2, 4, 8, and 16 minutes. The optimal digestion time containing the

largest amount of 40 kb DNA fragments was determined to be three minutes.

Molecular weight markers are shown in lanes land 7. The DNA was

visualized on 0.3 % agarose gel stained with EtBr. Panel B shows 13 ug of the

vector linearized with Xba I at nucleotide 1435 (lane 2) and the same vector

DNA digested with Bam H I to expose the cos ends of the vector (lane 4). Two

bands were predicted (1.1 kb and 6.5 kb) and detected on 0.8 % agarose gel. 1

kb size markers are shown in lanes 1 and 3. Panel C shows the conversion of

the ligation components to high MW concatamers was visualized on 0.3%

agarose gel stained with EtBr (lane 2). 1 kb size markers are shown in lane 1.

78

The ligation was carried out at room temperature for 24 hours. The conversion

of the ligation components to high MW concatamers was visualized on 0.3%

agarose gel stained with EtBr (Fig. 3C).

2.29 Packaging, titering, and plating the cosmid library.

The ligated DNA was then packaged into phage particles using

Stratagene's Gigapack® II XL packaging extract (cat. # 200217) according to

the instruction manual. 0.5 ml SM buffer (prepared according to package

instructions) was added to the packaged DNA followed by extraction with 20 ul

chloroform. The supernatant containing the recombinant phage was stored at

4 °C until used. XL 1 Blue MRF' cells (Stratagene) were prepared as host cells

for the phage. A 50 ml culture containing 10 mM MgSCU and 0.2% (w/v)

maltose was prepared from a single colony. The culture was incubated

overnight at 30 °C (prevents over growth) with shaking. The cells were pelleted

at 500 x g for 10 minutes at 4 °C. The cells were resuspended in 10 mM

MgSC>4 to an OD600 of 0.5. 1:10 and 1:50 dilutions of the packaged cosmid

library were prepared in SM buffer. 25 ul of each dilution was combined with

25 ul of the XL1-Blue cells described above in a microfuge tube and incubated

for 30 minutes at room temperature. 200 ul of LB broth was added to the tube

and incubated for 1 hour at 37 °C with gentle shaking every 15 minutes to

initiate kanamycin resistance expression. The cells were then pelleted for one

minute and resuspended in 50 ul of fresh LB broth. The cells were then spread

on to 150 mm LB agar plates containing 50 ug/ml kanamycin using a sterile

hockey stick spreader and incubated over night at 37 °C. Single colonies were

79

taken and inoculated into 12 x 75 mm polypropylene tubes containing 5 ml LB

broth and 50 ug/ml kanamycin. Tubes were incubated at 37 °C with vigorous

shaking overnight. 0.5 ml of each sample was combined with 0.5 ml glycerol

and placed into corresponding wells of four groups of 14, 96 well (200 ul/well)

microtiter plates. One group of plates was stamped onto membranes by a

Biomek™ robot in 4 x 4 arrays (384 samples/7.5 x12 cm membrane). Cosmid

DNA of the remaining 4.5 mis of culture was isolated by alkaline lysis "rapid

prep" (108) (see: Isolation ofplasmids (2.2)).

2.30 Cosmid library finger printing.

"Rapid prep" cosmid DNA was resuspended in 25 ul TE (pH 8.0) with 25

ug/ml heat-treated RNase. Two ul of each sample was digested with EcoR I (20

U) and visualized on EtBr stained 0.8 % agarose gels with a Hind Ill-lambda

ladder. EcoR I restriction patterns of recombinant cosmid DNA was

photographed for later analysis. The remainder of the recombinant DNA was

placed in 96 well microtiter plates and stored at -20 °C.

3. Results

3.1. Detection and isolation of genomic clones.

The predominant calcemedins in T. brucei comprise a family of putative E

F-hand calcium binding proteins. The proteins were reported (47) to consist of a

44 kDa protein and a cluster of 23-26 kDa proteins. E F-hand calcium-binding

proteins of T. Cruzi had been previously reported by Lazardi (48), Gonzalez

(127), Engman (7), and Ouaissi (49). A clone in T. brucei was discovered by

Dr. Lee at Columbia University, New York, New York who reported (50) that

while screening African trypanosome cDNA clones for evolutionarily conserved

genes that encode cell surface proteins, they found a cDNA clone (Tb-17) with

an open reading frame that shared significant homology to previously described

E F-hand calcium-binding proteins of T. cruzi. Dr. Ruben at Southern Methodist

University, Dallas, Texas obtained the clone Tb-17 and used it to screen a

lambda Zap (Stratagene) cDNA library provided by Dr. K. Stuart, Seattle

Biomedical Research Institute, Seattle, Washington. A positive clone, pTB44A,

was considered for further examination and sequencing . A full length pTB44A

was used to probe restriction digested trypanosome DNA, strain M110

(Fig. 4) and several genomic fragments were detected. BamH\ fragments 1.7

and 2.5 were selected for further examination. Dr. Lee also probed BamH I

digested genomic trypanosome DNA strain 427-60 with the TB-17 clone and

obtained similar BamH\ fragments. The two fragments, 1.7 and 2.5, were then

excised from the gel by Dr. Lee and cloned into the BamH\ site of pUC19 (Fig. 5

81

T

E c ( 0 a

DO • H U B

* H S

S B

1 1 m

m » «

mim Wmmm

mm „ ,.-^m

82

Figure 4. T. brucei genomic DNA was digested with several restriction enzymes

and electrophoresed on a 1% agarose gel. A Southern blot of the gel was then

probed with a full length 32P labeled cDNA, pTB44A. Autoradiography

detected several homologous genomic fragments. Approximate size markers

are shown in kilobases.

83

Pvull 3.14 Hindlll 2.96 Pstl 2.95 Hindi 2.94 BamHI 2.94

vull Qs,

coRI 0.40 imal 0.42

BamHI 0.42

2.5 kb Genomic Fragment

B coRI 0.40 trial 0.42

BamHI 0.42

1.7 kb Genomic Fragment

BamHI 2.10 Hindi 2.11 Pstl 2.12 Hindlll 2.13

Pvull 2.31

84

Figure 5 A,B. Recombinant plasmids pB2.5 (A) and pB1.7 (B) were the result of

cloning BamH\-BamH\ genomic fragments corresponding to Southern transfer

and autoradiography bands of 1.7 kb and 2.5 kb (see Fig. 4) into the pUC 19

vector.

85

A,B)- Positive size fractionated clones were detected by screening a genomic

BamH\ library with the Tb-17 cDNA probe.

3.2. Restriction site analysis and subcioning of genomic clones.

In order to investigate the genomic organization of these genes the clones were

examined for restriction sites in preparation for subcioning and sequencing.

Restriction site analysis of TB-17 revealed dueling EcoRI sites more or less

central in that clone, clones were therefore digested with BamHI, EcoRI and

BamHI/EcoRI double digestions (Fig. 6). Overlapping subclones were

constructed from both strands of the genomic clones using M13 and pUC

vectors. Where restriction sites were unavailable oligonucleotide primers were

generated from sequence ends to fill in gaps. The sequencing strategy for

pB1.7 is shown (Fig. 7). All subclones for pB1.7 were constructed using M13

bacteriophage vector and sequenced from single stranded templates. pB2.5

subclones were constructed in both M13 and pUC vectors. Further subclones

were also generated from pB2.5 by digesting with two additional restriction

enzymes Hinc II and Pvu II (Fig. 8) to facilitate sequencing.

3.3. Sequence analysis of genomic clones pB1.7and pB2.5.

The sequence generated from subcloned genomic fragments of pB1.7

and pB2.5 was determined by Sanger dideoxynucleotide sequencing using

U.S. Biochemical sequencing kit version 2.0. Overlapping fragments were

86

ffl LU

CC o o

£ X X E E « CO CD GQ

r-*

T~ T" CQ ffi Q. a

cq m

£ m m OQ

87

Figure 6. 1.0% agarose gel stained with ethidium bromide of restriction enzyme

digested genomic clones pB1.7 and pB2.5. Hind III cut lambda and Hinf I cut

pBR322 are shown as size markers with approximate kilobase sizes are shown

at left.

88

a s a a a g s a s s ® * * l l = 3

1 s 1 «

i i s i j ]

nm 3 * * 8

S 8 !

i

w

4 \ laaBX w A « * W •Hi W 0

Icoltl Kind I '

- r -L A leoXLl

N A 0 * M % W 0

o P1

M s ft T3 03

O r« S ? §

fc« S ©

s w 0 &

BaoBI

89

Figure 7. Restriction map and sequencing strategy of the genomic clone pB1.7.

The darker shaded area labeled Tb-1.7g represents a calflagin open reading

frame. Clone numbers are shown under the arrows. Oligo primers used in

sequencing are shown below.

90

I w YyuXI

1

"O CO ro cn

lull"

91

Figure 8. Restriction map and sequencing strategy of the genomic clone pB2.5.

The darker shaded area labeled Tb-24 represents a calflagin open reading

frame. Clone numbers shown under arrows represent M13 clones and clone

numbers above arrows represent pUC clones. A putative 5' open reading

frame of another unidentified calflagin is shown at the 3' end of the clone. The

3' open reading frame is unidentified. Oligo primers used in sequencing are

shown below.

92

<30 00 10

C4

M

vp CO CO

o CM © in

oo oo uO

C4 P*

o r r-

o r* oo

o o o o o © © o <o

a o r i N ( n i , t n « «

K > 0

0

3 *

§ w

§ ° 8 * g«

Si* IH

ST

1 *

8 O

S Q 0

0

3 * < g o

8 ° o

S* b CD c/> •<

8 «< 0

8 - s O

B § °

< Q 0

0

S* § 0 .

i* 5 CO B

S *

9 O CO B

8 °

B B &« B

0 5 °

t U b

S* u

£M

8 *

0 c« o

O M

B

0

3 *

£ fa

a o O W B

*c m o

b %«

S Q O

l! Q 0

O OH U

O O & O

Sw

§ *

b 0 u B

8 a : u

B

s w

B ad O

0 < w a

a n b

o

8«

0 g M

B < a o

o

e * 0 g *

u

< b w <

i ° Sin b

u 0 o B

I 0

! a

B B Hi O 0 .

S >

8 w o

8 oc

o B fe B

0

o

< sc

8 ° 6-i

I °

I * § *

8«

H B O

5 a 0

2 « 0

*1 u

9

IS

2 * § o e>

frt

r a ^

S5" 8 -fr> 8 °

0

5 * H 0

H

| « 0

C J

%K < w 0

^ ° B KM

U £H *3 U

U H M <

o g ° EH g Q

0

3 * S :H 6H

0 . s »

^ >» U < Q 0

8 O EH

H H tw B

EH Hi O

§ 0 s 2

0 « O O

1 ° § 01

i> EH

g - 3 W 0

0 H *1 O

t> to U K m H

ht EH O

0 < U 0

g « 0 ^ EH > 0

8 *

1 ° § H

SM

i ° § *

b 8 ° § 3 1

114

< «

0 B B

0

8 *

b

8 * % w 0

<5 {§ w

oi

0 g «

a

H

H

g B

i -

0

3 *

3 *

B>

1 m

8 o

u

B h

^ M 0

B fi«

^ 6-1

%>

! >

I 5 5

EH U M fri

3 °

S z

a < Q

0

u Jer* H <

0

I 8

R Cu 6-»

0

S H

0

ti O Pi U

h >

I °

SQ

1 °

8^

i z

i ^ i ° § °

8 °

S58

<

8 °

% m o

< w

1 ° Ej < a 0 c~»

8 01

S a 0

8 °

go

I > 0 < OI O 0 ,

K*

8 8 H S 3

0 0

0 0

g 3 5 3

I I I

2 0 0 B Ei

0 0

0 0

< U

o o N

< I

93

Figure 9. The complete nucleotide sequence of the genomic clone pB1.7. The

1.7 kb Bam HI-Bam HI genomic fragment was cloned into pUC19 and

sequenced to completion. The ORF for the calflagin Tb-1.7g is indicated by the

single-letter amino acid code.

9 4

o o o o o o o o o o

MB m

p-

c\ <h to ©

<M

O O O <M in in if) H <N ro *r in (N CM CM CM

U

<5 r< H Q H H 13 O O

U CJ3 h u h

S 3 y

3 U

n o

S

i i i i i i i i u £ H O * 2 U H O

n s n s n

« K g O

o o " H g *

^ S s

£ J S p § El K f > 8 O O < & <

P ft s M a

i * n « g ° §

in u (Jv (5 O t"*

§ « S H S °

8 * S

H > S o I ^ U . S u «1 Q g > B o § g O H l S O O H u

I * 1 * I - i ° l w 1

g « s « * . g o g > § a g * g w | p ^ _

| B g r i g * g o g > i « i - 1 - | | |

a 0 H O E* b* h 5 5 a S « g s d tj *« g o g o < * **

g ii O < *•

K § S H Q H

a s a

S 8 5

2 h S

g 8 8

B « 8

« 8 8

< 9 H

«e o 8

I - 8

8 - S

§

ID fC O E-» H H

g - g > g * E - K - 8 - g g g

- 8 - % » I * I - 3 - S * 3 - g H

* § * 1 ° t* E f a M ' 1 ' J n

o § M g j g > g o |

1 » M « i

W *2 £ Jj

B s-» •• < f* •*

3 ° %« B

0 6H 0

g * g ° s >

H yj u>

g * g w

8 « M d 1 5

; *

i **

8 ° i * g « [ j * 1 ° 8 * 8

2 « 2 - § a E * l M 8 - a - 3 u

1 ° l u S ° I 5 4 ^ h I 1 4 I " I u 1 § §

O O fr« H © f* H

a * g f c s z r g n

O 0 fr* H © H <•

r r s ° | n

B O P 64 £ > § " ' £ *

u g ' ' S ° S u H " | u j

^ S S ^ 5 H ^ ^ ®

^ H * ° < H *<

o M § i s

§H E-i H <5 ^ O

" S * 3 " R ° § g g O C> O C» H « S ^ < U < < fi(4 < 0 «-» Du H hi H I " J H >

^ u U H t i ^ O g j g

1 « 1 ^ 3 " I s " § M E * ' 1 " 3 ® 1 | |

i » S H S J 1 " § h s g s

S P5 S 6- S a y r„ S ( 5 B h | m g p < a « e ? H e »

^ 0 . y p p

H H H U> C/ U H S ^ t j ^

* 8 * g 5 5 § > 3 * 3 * S S S

8

r> E* (J y> < g « 5

8 '

§ « § f a § ® i 1 *

£• g o e

^ "

a -

S H

95

Figure 10. The complete nucleotide sequence of the genomic clone pB2.5.

The 2.5 kb Bam Hl-Bam HI genomic fragment was cloned into pUC19 and

sequenced to completion. ORFs for Tb-24 and gene A (the first 18 nucleotides

of a calflagin coding sequence) are indicated by the single-letter amino acid

code. The upstream ORF that encodes a putative non-calflagin protein (bases

1-321) has not been marked.

96

assembled using DNAsis® software to obtain complete sequences of pB1.7

and pB2.5 (Figs. 9 and 10).

3.4. Comparisons of coding and flanking sequences to other calflagin clones.

A schematic diagram of the relationship between conserved coding and

flanking regions of different calflagin clones is shown (Fig. 11). Prominant

features of the genomic clones include an incomplete open reading frame of

321 nucleotides (1-321; Figs. 10,11,12 A, B) at the 5' end of pB2.5. A

hydropathicity plot (Fig. 12 B) indicate that the sequence may code for a

membrane associated protein. The putative translation product of this ORF

represents the carboxyl portion of a protein that is unrelated to the calflagins but

shows significant homology of 10/28 amino acid residues (35%) to a cDNA

sequence reported from Brugia malayi (EMBL acc. no. BM7041). Whether this

ORF is actually transcribed has not yet been determined. Downstream from the

5' ORF is a second ORF (Tb-24) that shares nearly identical 5' and 3' non-

coding flanking regions with TB-44A. It encodes a 24 kDa protein with

extensive homology to the carboxyl half of Tb-44A (Figs. 10,11,13). The 3'

flanking sequences of Tb-24 are identical to the 3' flanking sequences of Tb-

44A (Fig. 11). This conserved downstream segment is followed by 131

nucleotides that are repeats of a region found at the 51 end of Tb-24, and

includes the putative mini-exon acceptor site (Fig. 11). Thirty-six nucleotides

downstream from the putative mini-exon acceptor site, the first 18 nucleotides of

another calflagin gene was observed (gene A, Fig. 11). This calflagin member

97

differs from Tb-24, Tb-17, and Tb-44A in that it has a BamHI site at the 51 end of

the gene.

The sequence of the 1.7 kb BamHI genomic clone begins at precisely the

same location within the calflagin sequence where the 2.5 kb genomic clone

ends (Fig. 9 and 11). This is suggestive that the 2.5 kb and the 1.7 kb genomic

fragments may be linked however, Southern blots (51) and restriction analysis

suggest that more than one calflagin member might contain a 5' BamHI site.

The partial sequence for the calflagin on the 1.7 kb genomic clone was called

Tb-1.7g (Fig. 9,11, and 13). The 3' non-coding flanking sequences of Tb-1.7g

and the cDNA clone Tb-17 were related (Fig.11).

Proteins with homology to the T. brucei calflagins were sought in the

available databases of the NCBI (National Center for Biotechnology

Information). No other organisms were found top have related sequences other

than those within the genus Trypanosoma.. Calflagin sequences from T. brucei,

T. lewisi, and T. cruzi are compared in Fig. 13. Substitutions among the T.

brucei calflagins were most prevalent at the N-terminus. Variability occurred at

the C-terminus, where variations on the hexapeptide sequence GANEGD were

represented once in Tb-24 and Tb-44A and twice in Tb-17 and Tb1.7g. The

most extensive difference occurred between Tb-44A and the remainder of the T.

brucei group as a consequence of an internal repeat in Tb-44A. The T. cruzi

group (1F8, FCaBP, 24 kDa antigen, and FCaBP-PBOL), in contrast, differed

very little (Fig. 13) and the few differences that did occur appeared to be strain

specific. T. cruzi has been shown to produce a single calflagin band from

immunoblots (7,47) and Southern blots show that T. cruzi is more

homogeneous that T. brucei (127). Comparisons of EF-hand

9 8

pB25

pB1.7

B E E B

pTB44A r E

UXSE3 I I E E

I E E E

pTB17 ! EEttE f s I 1

E Bft l S

400 hp

99

Figure 11. Common features of calflagin clones pB2.5, pB1.7, pBTB44A, and

pTB17 are compared. Coding sequences are indicated with a black box.

Common elements in the 5' and 3' non-coding regions are indicated by boxes

that share a common design. The position of restriction endonuclease sites for

EcoRI (R), BamHI (B), Sa/I (S), and Bgl\ (BG) are shown.

100

9

5 1 ATC COT CTC

lie Leu Leu

63

GGC CCG AAA

Gly Pro Lys

117

GTC ATG GTT

Val Met Val

171

ATG AAT CCC

Met. Asn Pro

225

GCA GCA CCA

Ala Ala Pro

279

GGT OTA ATT

Gly Leu lie

18 TTT ATT AftC

27 CTG GOT

36 ATT GTT AST

45 ATT GAA CTC

54 GCT GTT CGG TTG

Arg Leu Phe lie Asn Leu Val H e Val Ser lie Glu Leu Ala Val

72 CAC GCT GTC

81 GTG AGC

90 CTG ATT GGT

99 GCT OTA CCG

108 GCA GCT GCG GTG

Ala Val His Ala Val Val Ser Leu lie Gly Ala Leu Pro Ala Ala

126 CTA CCA GCG

135 TTG CTC

144 ACT ATG CAG

153 GTT GAC CAT

162 GCT GTA TOT ATT

Phe lie Leu Pro Ala Leu Leu Tbx Met Gin Val Asp His Ala Val

180 GAT CGT GTG

189 ACG TTG

198 AAG TAC TOG

207 GGA AGC ATG

216 TTT ACG GAA GAG

Glu Glu Asp Arg Val Thr Leu Lys Tyr Trp Gly Ser Met Phe Thr

234 TCT TGG ACA

243 CGC ATT

252 CGT TGT TAC

261 TTC TAC ATT

270 ATC TTT GTG TTT

Val Phe Ser Trp Thr Arg lie Arg Cys Tyr Phe Tyr lie H e Phe

288 GTA ATG GGC

297 ACC TAC

306 AGC GTC ATT

315 GCT GAA CTC GTC ATG

Val Met Val Met Gly Thr Tyr Ser Val lie Ala Glu Leu

TGA 3'

B Kyte and Doolittle Hydropathicity Plot

Hydrophobic

5 Hydrophilic m

100

101

Figure 12. Panel A shows the sequence and the translation of the carboxyl end

of an ORF in the genomic clone pB2.5. The ORF is located at the 5' end of the

clone (Figs. 10 and 11). Amino acid sequence is given in three letter code

below the sequence. Numbers are in nucleotides. Panel B shows a Kyte and

Doolittle hydropathicity plot revealing the hydrophobic nature of the sequence.

The bottom numbers are in amino acid residues. The hydropathicity is shown

along the left side.

102

_ _ _ _ _ _ _ 60

Tb-44A MGCSASKDTTNSKDGAASKGGKDGKTTADRKVAWERIRCAIPRDKDAESKSRRIELFKRF

lb-17 ***AS**NAS*P**********************************************C*

tb-24 **•***.************...*****«****.*..*..*.**.************•**« Tb-1.7g G**NAS*P*******************************.**************Q* 57

FCaBP/lF8 MOACGSKG*TSDKGIASD****KAK***E******Q****E*T**A*Q* K* 24 kDa Aff MGACGSKG*TSDKGLASD****NAK***E******Q****E*T**A*Q*******K*

FCaBP-PBOL MGACGSKG *TSDKGLASD** * *NAK***E******Q****E*T**A*Q* * * * * * *K*

TL-17 RRSKNS*SSK* * * *AAKTARLRGR3JL*QT* * *ERTD*ARQ* * *D* **K* 49

EF-hand fl

Tb-44A DTNGTGKLSFREVLDGCYSILKLDEFTTHLPDIVORAFDKAKDLGNKVKGVGEEDLVEFL 120

jb.j.7 ********Q***************** ************ ************* *********

Tb-24 ********q*********o***************************************** Tb-1.7g ****** **g*********G*****************************************

FCaBP/lF8 *K*E**.**CYD**HS**LEV*******PRVR**TK******RA**S*LENK*S**F**** 117 24 kDa Ag *K*E****CYD**HS**LEV*******FRVR**TK******RA**S*LENK*S**F**** FCaBP-PBOL *K*E****CYD**YS**l,EV*******SRVR**TK*****SRT**S*LENK*S**F****

Tl-17 *K*NS****YD**YR**IDE********RVR**TK***N****M****EKT*S**F**** 109

EF-hand #2

Tb-44A EFRLMLCYiyDIFELTVMFDTMDKDGSLLIjELQEFKEALPKLKEWGVDITDATTVFNEID 180

Ws-17 *****************i**************j}*************************** ^,.24 ********************************************q***************

Tb-1.7g ************************************************************ FCaBP/lF8 ***********F********EI*AS*NM*VDEE*L*R*V***EA**AKVE*PAAL*K*L* 177 24 kDa Ag ********«**E********EI*AS*NM*VDEE*L*R*V***EA**AKVE*PAAL*K*L* FCaBP-PBOL ***********F********EI*AS*NM*VDEE*F*R*V*«*EA**AKVE*PAAL*K*L*

Tl-17 ***********Y********EI*TS*NM**DDK*****V***EK**LK*EHPEK**KQL* 169

EF-hand *3

Tb-44A

Tb-17g Tb-24

Tb-1.7 FCaBP/lFB 24 KDa Ag

FCaBP-PBOL Tl-17

TNGSGWTFDEFSCWAVTKKLQVCGDPDGEGAAKTTADRKVAWERIRCAIPRDKDAESKS 240

***********************G****D*ENGANEGDGANAODGVPAAEGSA

*************************** *G*ENGANEGN

* * * * * * * * * * * * * * * * * * * * * * * * * * * * 0 * E N G A N E G D < 3 A N A G D G V P A A E G S A

K**T*S******AA**SAV**DAD****NVPES*

K * *T*S * * * * **AA**SAV**ADD** **NVPES*

K**T*S«*****AA * * SAV* * EPTATRTTCRRAREARGRRVTCPRRRQCKHRVRMRRGTC 229

KK**********AA**SA***DAE****K 198

233 218

211

EF-hand #4

Tb-44A RRIELFKQFDTNGTGKLGFREVLDGCYSILKLDEFTTKLPDIVQRAFDKAKDLGNKVKGV 300

FCaBP-PBOL FCWAEHILHFM 240

EF-hand #5

TB-44A GEEDLVEFLEFRLMLCYIYDIFELTVMFDTMDKDGSLLLELQEFKEALKKLKEWGVDITD 360

EF-hand #6

Tb-44A ATTVFNEIDTOGSGWTFDEFSCWAVTKKLQVCGDPDGEENGANEGN 407

103

Figure 13. Comparisons of calflagin protein sequences from T. brucei, T. cruzi,

and T. lewisi. The sequences used are from T. brucei, Tb-44A (accession no.

U06463), Tb-17 (accession no. X53464), Tb-24 (accession no. U06644), Tb-

1.7g (accession no. U05882); from T. cruzi FCaBP/1 F8 (accession no.X02838),

24 kDa antigen (accession no. S43664), FCaBP-PBOL (accession no. L26971);

and from T. lewisi (Lee et al.,1990). Numbers in the right hand margin indicate

the position of amino acids. The amino acids in bold letters indicate the position

of sequences related to the hexapeptide GANEGD. Identical residues are

indicated with an *. Bars indicate the position of EF-hands.

104

Calcium-binding motifs in T. brucei calflagins (Fig. 13) show thatTb-24, Tb-1.7g,

and Tb-17 all have three EF-hand calcium -binding sites while Tb-44A has six

due to the duplication. Most known EF-hand proteins contain multiple copies

(two to eight) of helix loop helix calcium-binding motifs. The EF-hands are

generally arranged as interacting pairs (128). It was shown (51) that in Tb-44A

EF-hands 1 and 4 are unpaired. Instead, the adjacent regions (residues 99-139

and 288-318) have degenerated so that no evidence of calcium co-ordination

could be found. The degenerate calcium co-ordination sites have retained

considerable hydrophobicity, however and may still function to bind calcium. In

that case they may have evolved to interact with target proteins.

3.5. Analysis of a cloned T. brucei repeated element

Degenerate oligo probes acquired from Dr. Larry Ruben at the Southern

Methodist University were end-labeled with 32P and probed against restriction

digests of T. brucei. A weak band was detected from oligo probe #4 on a Hind

III digest at approximately 1 kb. The detected genomic fragment was

electroeluted from the gel and cloned into the Hind III site of pUC 19. Positive

clones were detected by dot-blot analysis with the original probe #4. Plasmids

were isolated by rapid prep procedures and 32P labeled by random priming.

Plasmid probes were then probed to a genomic Southern digested with Hind III

and various other enzymes (Fig. 14 A). Several genomic fragments were

detected in lanes digested with Hind III and double digested with Hind III and

Xho I. Plasmid preparations of the Hind III cut genomic fragment were used in

double stranded sequencing reactions using the Sequenase® version 2.0

105

c o o Q. JC 4Z g 2 g

G A T C

22.6

- %** Wmm

:>Mu

- < ^ '.vv , Sf •\* EmM&m

*>&.> i\:in Kt' •?- rvx ,w .v*

§111

f' WTOi

106

Figure 14. Analysis of a Trypanosoma brucei repeated element. Panel A

shows a Southern blot of T.brucei restriction digests probed with a 32P labeled

Hind III genomic fragment containing a 28 bp repeated element. Approximate

size in kilobases are shown to the left. Panel B shows an autoradiograph of a

portion of the repeated element. The sequence was generated from 35S

labeled sequencing reactions terminating in G.A.T, and C shown at the top. The

gel is read from the bottom to the top.

107

sequencing kit obtained from U S Biochemical. A sample of the sequence

showing the repeated segment is shown (Fig. 14 B). Blast analysis reveal

significant homology of 27/38 bases (71%) with a cDNA clone from Brugia

malayi (EMBL: BAA32121). There were no other matches reported in the

BLAST analysis using standard BLASTN parameters.

3.6. Detection of novel T.brucei sequences using M13 shotgun sequencing.

Genomic DNA was cut with XhoU and Xba\ and fragments from 10-14 kb

were obtained from the two digestions. The DNA from both digestions was then

nebulized together to generate small fragments in the range of 1.5-3 kb,

suitable for cloning into the bacteriophage vector M13. Of the 5,856 M13 clones

stored in 96 well plates, 433 were sequenced from the -21 M13 forward primer

by a single pass through an ABI 377 DNA sequencer. Some sequences were

interrupted by large regions (greater than 100 bases) of unresolved nucleotides

and were treated as two sequences. Therefore, a total of 455 separate

sequences are reported and submitted to the GSS database of the NCBI. An

average read length of 740 nucleotides was obtained for each sequence,

representing approximately 0.84% of the total T. brucei genome. Because M13

sequencing proceeds only in one direction, sequence information was not

obtained from both ends of the insert. The sequences were analyzed using the

BLAST family of software (NCBI) for regions that encoded putative proteins,

structural RNAs, or regulatory elements. 84 sequences with homologies to

sequences already in the database are reported (Table 4).

108

Si r-

H £**«

H-3 <

Pi p*

55 w

2 i—i C/3

Q

>> 0> > v« 5* C/5 <P U d a; 5* o1

a; 0T5

s o ti a; O

£ w> <M o XI CO <D U » ti x> 3 o 3 £>

B o o CJ c«

B

C/2 P4 O H eu, h-H 05 U c/3 m Q Z O M 5 U l~*4 U* HH E-55 W Q w > h-M

S D Pu

•8

i£> N oo t in in (Nt m in vD tJ-

£ ^ a\ in on ooh in vd in in r-

^ ^ ^ QV Q\ Cr* I/) H h» fOfO co ^ co in

SiHin rH vO in

£ &*CM v0 oo r- in OOOM 00 Vp CO

Si £

K 2 00 S

in h in h oo co I*- in

4 2 4 Of in % m CM

^ fM TJ- xr CO in (M

CO co r> in rH T|"

SS r*

104

457

292

64%

64

%

56%

2 V£J

u §

49%

38

%

37%

CM i ON CO

* <M ON

£ ON 00

1 * 3 1 5 £ | s ' t w i l l 'Qwrt o a trf 2 ti ^ t« o 9 § 5 o i i s > § «

&« vT

i I i I f •s t i

3 8* t=.

a> S?<y 9) tti

U"Bk

f S f f i l

qim i S f o - i

S S S g g l . r1 55 <1> 55 n o rrr t ^ c si ^ ^ S 00 ^ ? 'S *-• ?} <5 £» 00 m E in ^ S ON 00 o 8 VD fo T3 <M 00 r9 8 vH CM 3 :s? 5 53 OiCn W 6J) V5 W) Dfl CC v-i

<y

If &3 3 1

2*3

I Qi w V} CB

•3

^ H ^ g f l i - a s

f 9 s

§ 5 1 ^ O K cm fO ON Is- 2 Ji if 00

n J S 2 ^ ^ j t q oooo on 22 K in r-. rH T" *-H in rH U ' R ^ ' a b U

O^G-

S S g

3 * 8 w(M C-in *-J ON 00 £2 00 ^ 3 <*> fH *-H frj N ^ S *-j *n ^

o a • u u as

K 00 CO H ON *h m co o in Is* m h* ij5 «n CO CO CO CO co rH H r-i iH H W W M M W

in vo cm vo ^ m m in «sD CO CO CO

CM Tf VD N h CM • CO COON r~tTf 00 Vp CO VJD i-\D h h K ^ in co coco coco rococo

QJ

d S § -

c £ § ,§

S ^ 3 - C • . *•' «!

0QCQQQ CQ CQ CQ CQ P3 CQ M CO PQ PQ

CO ^ <M

1 0 9

hJ <

& Ph

55 w hJ

S )—( 00

Q

c/5 CtJ O E-" CM •—< es u C/5 W Q

2 O HH

5 U H~4

N"4 H £ W Q h«« m > HHI

5 J1<H tD P i

00 o oo ti a • 2 u si* ^ TJ «*

CM CO CO CM CM no H H ^ -

CM O h- I/) H <N rH CN h- CTN (N Tf rH VJD

N V) i * H H Irt H CM H Irt

g g 00 VJD CM nO 0" tH O rH Z Z v O f N O O ^ ^ ^ h h

•P "sP sP sP nP Sp nP sp M? £5** d * C 1 Cl 3

OMAVDI^VOI^.1^. rH no in v o o o m m r ^ T f m no v£ in

53*3 u o

e e •Q «Q

I S l S f f f

1 - 1 1 &

S . f S

f Cu 0)

£ 3 ^ < : ° w

<A Ch /ii 2i a. c* 3J t [p § § S '•S 3 er> " S g g g S k k | 1 B = to LO A ^ o «0 2 2 8 3 ° 3 r ! r i vo r r (n cm >£ j*5 VD Jt? <N r i r j H S ^ fO « cj 7T ~

i

£ CM

£ rH i n

£ i n "fr

no cm no -"t cm CM

no csl no Tf- nsi CM

38

64 h-

cn VD TH CM rH CO

rH CM

71%

75

%

79%

58

%

64%

62%

48%

72

%

59%

43

%

58%

43%

oo § r,„o S

• o f K | . S ? g S

35 & S i t e ?

to O N a n

g g e g

§ § i > N O ^ V ^ H O N 3" *n *h i n Tt* v£>

r t h- m h- i n no no no no no no no rH rH rH rH rH rH rH jjp jjp gQ CQ PQ PQ CQ

^ CM N i n v£> r^ M3 co m ro f o c o

OJOt ?

no no

3 50 - ^ V •*t W

H 0 0 g x £ ' 2

i w O > w ^ £ & i *n S? u ' £ ; nJ "M ' #5b'Sb.S

BQ GQ CQ QQ

rH 00 vO h* no no rH rH W M

h s &

S3 I

*2 o u *d >

ffi

5 o

i >i j j yW r-i

*~j CT» <j s"" ^ S <N ' 3 Q\ C i g O « N o ^ v o g,fM

aPfM q? 0 0 »CI <-i

^ Ss (N -ci> ®< 3 . ^ *ct DO V> OOuw 00 H ON H r - 5\

i n r f ro no ro H H H CQ OQ OQ

00 CM m no rH CQ

1 1 1 ^ s 6 a H t fo N H (N

rH ^ no M f \ | vO

no P no no v > i n oo 2 i n i n ^ xh

^ ^ ^ OS 0v Qk QS QS»

N i n l - o h i n vO 00 no no <N no

no .9 vO XJ cr a

o c g £

22 K » i 2 -O ?| U ^ J ^ g a b s & s & . E § s ^3

q j S

N l 1 S | S g tH w 00

- - , 3 8 3 2 s i M rs| fs| 00 f f j vD E S J g i i l

& M -5b "B,

no no h- r}* i n no no no no no no rH rH rH rH rH rH jjp gp OQ CQ CQ QQ

rH

1 ,

110

hJ < i (N4 % ro %• ro r o N rO(N M f O H

a x j

•a & & i n (N| ro rH

P<

2 5 w hJ

8 v £ > , _ H i n m H

N f O N fC CO CO <N CO

co H O t H

r o H TT oo

00 t H COI/) T H f O f O

N C 0 H

vo i n csi rH O V0 rH l/> CO

CN CO "t

£ ^ ^ ^ i N O P N O ^ ^ r o O ^ N

C/5 Tfr vO h- ,Zj N i / ) i n W N V0 vO i n

& ^ ^ ^ ^ CN 00 00 00 <N VD ^ CO i n _ _ __ _ c o x t ^ t v D i n c o i n co i n (N Tf Tf

: y y

i n h n v D o o h *

£ S 00 H N i n i n i n i n £ * ^ O (N O 0 0 ^

CO Pi! O H Ph *—« p s u 0 0 t u Q

5 5 O HH

U I—I

£ H z w Q M

w >

*•*<

g

0m

8 I

% *$& Q

1 J £ }

bO

crt ^

2 ^ * i * t k " h i *

CO r !

• S

0) $

Q« «0 O

§ £ g j

fisl

C/D C/0 O X i * 0 *

f n ^ ^ c "» fN| £^rH 53 oo ®2 o P o CO ° 0 -ro [*r CN

V C >«**

rt«0 0 0

00 CN ^ " -'t m ^t CM (N _ v £ > O S £ > 2 S n j zzf n i •m *w4 <*H *• oo oo bo w>m k co oo i n O M n oo cv m vO VD vD CO CO CO CO rH rH rH tH

F F S P P

2

^ o d ^ g ' S 0 0 s * £ 1 5 o 2 J

U v£>

00 oo & v O

? « 2

o i n

22 S - o o ^ *h t * £ c o i n d J o H (N (N o o ' j j S r o Q g j O ^ v O N 3 Ch i n O H i ; 2 oo

*5b {S^Tab'Sh "Sb'Sb

Si n o i n i n

00 CO • CM

c o ^ - c o ^ c n v D v O r ^ m CO CO CO CO CO " ~ pq cq pq pq pq

c o c o CO CO H H H H PQ PQ PQ PQ

j £ > , Q 0 tO 50

fM (M <M

CO CO CO H H H CO CO CQ * * *

h - 00 T* H 00 O

. . . . Q w V w x W ( S | v£> w r>. <r»

. m H ^ ^ d vo vo i n ^

H 00 h H h N ON ^ g

PI VP rH rjr r 4 J? U U ^ ' S c ^

i n vd h 0s* co ^ vp i n vD N ^ VJD CO CO CO CO H H H H PQ PQ PQ PQ

111

- 4 <

& Um

z w J

S M GO

Q

0 0 C4 O H

HH «

U 00 uu Q

Z O §

U HH PH NH H 2 IX| Q HH

m > \mmi

H <

D (X

•S £

I I f s | rH

^ xr

<M h-rH ro vO ro

$ & (N CM i/3 m

^ £ vD

ro ro

i

on o on fl O 2 w

a s « ns

•3 *3 w ,w ~ a § §

S*' S>* »mm* U~4

a a Si s o o k fe & A

1 3

. s . a (A (A

8 3

s s

m m rH rH tt-in in c/> cn

I S

h- ^ jsp Ch

ro ro ro

i co CM ro ro A ro

ro ^0 ro H M Tf ro

i n ? h- ro ro i n <n Is- ro o> crs

in

i n

sP *P s& sO sO ^ ON ON ON ON ON ON ^ ON N N r o i n H v o O h -h- -3* N i r j i n n vo

^ ^ ^ ^ ^ ^ nP ^ ON ON ON qV ON 0^ O v O V 0 N 00 00 N ON v£> ro in ro ro rf CM

i f f 8 °

© s « » 2 w

rt ^ N . 2 N1

cn r» m

prj en N CO Tf o> vd r>

00 00

<y

i — 8 * 1

- 3

s £ £ £ s §

rs CM £t W o o ^

°o KJ <ys CM )g r s <y ^

S 5 § fM Pi !Z?

a i t .

cr> i n h* i n in o ro ro

i . & <N Cq rO U WTO vtj

N N i n H ^ N r o v D o O v O

M "iL

r M r o ^ m r o f N f o ^ r o c M ^ r H

CO Th N* t-H 1-4 vM h« N N fO i n i n N 00 N

• — H in in 1 - 00 {N (M S

t t H f O H N ^ H H i n n t

ON <J\ ON ON ON ON ON ON ON r O V ^ m r O ^ O O r H C M C f t f O -c r s t t t n ^ t m v D i n i n i n T t - i n

ON ®N ON 0^ ©N ON ON { N N rO N 00 VO 00 vJD rH rH

r o r o r o i n r o ^ r o ^ ^ O N

SP

« $ & . i q «

4 i f i

a

I S l

0 —k ^ *•«immmt

§ Vp "•>. " Q w « H

» q § 2 S ?

g y f i g s S 5 ^ 5 ; j s

S CO KJprjCM 2 CTi <n » O N ^ Q fO CJ rsi ^ } S v o w

c\ <y% d r-i <p CM CN • a n a a f t %

a i |

s M l

£ i l l O ro

'T? f/) O «f 5g 00 fM 5 » h h E

§ 3 1 5 - 8 * . - X ^ (M ^ ^ 2 H 00 H S h \ £ > 0 \ S ^ C% <M 5? 00 ON (M ?m h» in —1

'5b "3b *5

(NoOOCMCfrONCMVg - - w w ro CM ro G M ^ i o r o r o < N v 0 v 5 i ^ c o i ^ . < T N m v ^ i ^ i n ^ m r ^ v i > r ^ v 0 w 0 i n ro r o r o r o r o r o r o r o r o r o r o

PQ S W W w w w w w fq w w w w w w w w w w w w

112

Table 4. illustrates the variety of sequences identified. Internally repeated

motifs were found primarily in structural proteins and proline rich proteins. The

repeats were evident because one or more segments of the query sequence

aligned with multiple regions of the subject sequence (superscript a).

Alternatively, two or more segments of the query sequence overlapped one

another when aligned with the subject sequence (superscript b). Finally, one or

more non-overlapping segments of the query aligned with overlapping regions

of the subject (superscript c).

113

3.7. M13 Sequences of particular interest

Several protein coding regions previously unidentified in T, brucei were

of particular interest. Pyruvate: ferridoxin oxidoreductase (PFO) is one such

structure. The partial amino acid sequence of a trypanosome PFO (dbGSS no.

B13566) is shown with high homology to the PFO from E. histolytica (Fig. 15 A).

A PFO has not yet been described in 7. brucei.

A dismutase capable of destroying superoxide radicals has not been

described previously for T. brucei, although an Fe-superoxide dismutase has

been identified in Crithidia fasciculata and T. cruzi (129,130). Evidence that T.

brucei may contains a Fe-superoxide dismutase (dbGSS no. B13733) with

homology to the enzyme from T. cruzi is shown (Fig. 15 B).

Apoptosis-like events have been described for T. cruzi (131) and

Leishmania (132). Evidence that those events may occur in T. brucei are found

in a clone (dbGSS no. B13593) that contains an ORF and a downstream

untranslated region of 402 bases. The untranslated region has a 95% identity

to human DNA sequences found immediately downstream of the stop codon for

apoptosis inducer Nbk (U49730) (Fig. 16). Two other descriptors contained

similar homology, including a human STS (G26842) and a human Bcl-2

interacting killer (BIK) (U34584) [50,51]. No homology was found between the

coding region for any of the apoptosis genes and the trypanosome sequence.

114

3.8. Cosmid library analysis.

In the pursuance of other novel targets for therapy and of ultimately

sequencing the entire Trypanosome genome a 1X deep cosmid library from T.

brucei strain M110 was analyzed by DNA fingerprinting and cataloged (Fig.17).

1276 random cosmid clones representing approximately 51 million bases were

digested with EcoR I and electrophoresed on 0.8% agarose gels with Hind III

cut lambda size markers. The gels were stained with EtBr and photographed.

Four copies of the library were prepared and stored at -80° C as well as one set

of filters for other analysis and probing.

115

pyruvatesferredoxin oxidoreductase

T.brucei

E.histolytica

3 SNTGGHASKASNLGQVAQFAAAGKNIKKKSLAEIAMSYGYV SNTGG SKA+NLG V +FA++G KK L IAM+YG V

993 SNTGGQKSKATNLGAWKPAS SGCKRPKKDLGAIAMAYGDV

T.brucei

E.histolytica

YVAQVAMGANPAQTLKAIHE 185 YVA +A+GANPAQ KA E YVASIALGANPAQAFKAFKE 1049

B

Fe-superoxide dismutase

T. brucei

T.cruzi

145 GYDGRAAQGLSTVHGRIHYEQDN*GYAN*AIAVAXWGS 32 GYDG AA+GLS +HY++ + GY A A S

11 GYDGLAAKGLSKQQVTLHYDKHHQGYVTKLNAAAQTNS 48

116

Figure 15. Identification of genes involved in regulation of oxidative stress in 7".

brucei. Panel A shows a comparison between a translated region of T. brucei

sequence B13566 and pyruvate: ferredoxin oxidoreductase from E. histolytica

(U30149). Panel B shows a comparison between a translated region of T.

brucei sequence B13733 and Fe-superoxide dismutase from T. cruzi (U39401).

The single letter amino acid code is shown. Identities between the sequences

are indicated by listing the amino acid between the alignments while similarities

are indicated with a plus sign. The asterisk indicates the position of stop

codons. The residue numbers refer to the nucleotide position from the 5' end of

the insert or start methionine of the subject. The region of overlap is short for

the pyruvate: ferredoxin oxidoreductase because the 5' end of clone B13566

encodes the carboxyl terminus of the protein. Conversely, the region of overlap

for the Fe-superoxide dismutase is short because the 3' end of clone B13733

encodes the amino terminus of the protein.

117

T.brucei MTKRSKTKGKKDARKRAEKELQCAEKTEEGTSRGAEKPTSEQPQRRRRR 147 M L E

Human MSEVKPLSRDILMETLLYEQLLEPPTMEVLGMTDSEEDLDPMEDFDSLE 147

T.brucei XRTKRRGKPSEDCGQDLRPPRVREWRPLPRKAHRPILDGPTDPEQGEGR 294 L

Human CMEGSDALALRLACIGDEMDVSLRAPRLAQLSEVAMHSLGLAFIYDQTE 294

T.brucei SNQRVNPS* gaggggctga 331 V S

Human DIRDVLRSFMDGFTTLKENIMRFWRSPNPGSWVSCEQVLLALLLLLALL 441

T.brucei cctgaggccaagggaGCCCCGGCGGTTCAGNGGGGGGNNGGCCCCACCC 380 llllllllll Mil I Mil E 1 I I I I I II I

Human LPLLSGGLHLLLK *gGCCCCGGCGGCTCAGGGCGGGGCTGGCCCCACCC 518

T.brucei CCATGACCATTGCCCTGGAGGTGGCGGCCTGNTGCTGTTATCTTNTTAA 429 11IIII tl I 1111II111111111111111 M I N I M U M Mil

Human CCATGACCACTGCCCTGGAGGTGGCGGCCTGCTGCTGTTATCTTTTTAA 567

T.brucei CTGTTTTCTCATGATGCTTTTTANTATTTAAACCCCGAGATAGTGCTGG 478 lllllllllllllllil Mil 1111111111111111111111111

Human CTGTTTTCTCATGATGCCTTTTTATATTTAAACCCCGAGATAGTGCTGG 616

T.brucei AACACTGCTGAGGTTTAATACTCAGGTTTTTTGTTTTTTTTTTATTCCA 527 I I I II I I I I I I I I I I I M M I I I I I I I I I I I I I I I I I I I I I I I I I II

Human AACACTGCTGAGGTTTTATACCCAGGTTTTTTGTTTTTTTTTTATTCCA 665

T.brucei GTTTTCGTTTTTTCTAAAAGATGAATTCATATGGCTCTGCAATTGTCAC 576 II I I I I I I I I I I I I I I I I II I I I I IiI I I I I I II I I I I I I I II I II I

Human GTTTTCGTTTTTGCTAAAAGATGAATTCCTATGGCTCTGCAATTGTCAC 714

T.brucei CGGTTAACTGTGGCCTGTGCCCAGGAAGAGCCATTCACTCCTGCCCGTG 625 I I I I I II I I I II I IMI I II I I I I I I I I I I I I I I I I I I I I I I II I I II

Human CGGTTAACTGTGGCCTGTGCCCAGGAAGAGCCATTCACTCCTGCCCCTG 763

T.brucei CCCACACGGCAGGTAGCAGGGGGAGTGCTGGTCACACCCATGTGTGATA 674 I I I I I I I I I I II I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I II I

Human CCCACACGGCAGGTAGCAGGGGGAGTGCTGGTCACACCCCTGTGTGATA 812

T.brucei TGTGATGCCCTCGGCAAAGAATCTACTGGAATAGATTCCGAGGAGCAGG 723 I I I I I I II I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I II

Human TGTGATGCCCTCGGCAAAGAATCTACTGGAATAGATTCCGAGGAGCAGG 861

T.brucei AGTGCTCAATAAAATGTTGGTTTCC 748 I I M I I I I M I M I M M I M M M

Human AGTGCTCAATAAAATGTTGGTTTCC 886

118

Figure 16. Comparison between coding and non-coding regions of T. brucei

sequence B13593 and a human apoptosis gene sequence (U49730). Coding

regions are represented with the single letter amino acid code and are

italicized. Shared amino acid residues are shown between the alignments

while identical nucleotides are connected with a line. Stop codons are indicated

by an asterisk. Intragenic non-homologous sequence are in lower case letters.

N's in the DNA sequence are unresolved nucleotides.

119

- 1 -12 -

GEL#1|

-13-36—

J s ' • > ' i * «•- i n

37-48- 49-62

GEL#2|

78-95— GEL#3|

™96*108——

M m ® ^

w i g » * • *

w *

1 2 0

-109-120- -121-132- -133-144—

GEL#4| ^ m i | H If p ^ If m • m ' i I ^ y f If# *m

-145-156-- 157-168- 169-180—

GEL#5|

GEL#6|

181-192- 193-205

-206-217- -218-229- 230-241-

GEL#7

i —0~—242-253- -254-265- X" -266-277-

GEL#8

- «t|

H & m m •

tp m mm m

GEL#9|

121

X

290-301 302-305* j.

1 2 2

-306-317- -318-329— -330-341-

GEL#10

354-365 366-377-

Gel#11

——342-353———

GEL# 12

1 2 3

-414-425- -426-437-

GEL#13l

GEL#14

450-461 -v -462-473- 474-485

486-497- 1 -498-509 i —- 510-520

GEL#15

GEL#16

545-556— 533-544 521-532

te W

-557-568— -569-580- -581-592

GEL#17

GEL#18

-605-616 617-628-593-604

1 2 5

-629-640- -641-652-

GEL#19 "~X~

-653-664-

—665-676

GEL#20!

725-736-

GEL#21

1 2 6

-737-748 749-760-

GEL#22

-773-784

GEU23I

. 809-820- 821-832- - 8 3 3 - 8 4 4 -

GEU24

127

845-856 857-868 -869-880-

GEL#25

—881-892 1 -893-904 -905-916

GEL#26

917-928- 929-940 1 941-952-

GEL#27

1 2 8

-953-964- -965-976- -977-988-

GELJ28

-989-1000 1001-1012- 1013-1024 1

GEL#29

—1025-1036- 1037-1048 ^ 1049-1058 ^

GEL#30

1 2 9

1059-1070 1071-1082- -1083-1094-

GEL#3'

1095-1106™

GEL#32 * " X

1107-1118-

•i r ||||

-1119-1130-

m mrn W m . m * W Ua *e+W '

i i i " ? «* W

-1131-1142-

GEL#332 .. C M fiK*S,

'IB m I I w j

W ** ** v '

————1143-1154-- '

w • ^ | p

H m' buwt

130

•1167-1180 — i ————"1181-1192' 1193-1204 GEL#34

—1217-1228 1229-1240-GEL#35|

1205-1216

-1241-1252 ..—1253-1264——^———1265-1276— GEL#36

131

Figure 17. Analysis of random cosmid clones from T. brucei strain M110. Clones are digested with EcoR I and electrophoresed on 0.8% agarose gels with Hind III digested lambda DNA. The gels were stained with EtBr to reveal banding patterns. Clone numbers are shown between lambda markers at the top of the gels.

4. Discussion

This work is a continuation of work begun by Wu et al.,1992. The

sequences, transcription and genomic organization of EF-hand proteins were

obtained using several different trypanosome strains. The original partial

protein sequence (data not shown) of Tb-44 was obtained from strain M110,

while the Tb-44A cDNA was cloned from a library of IsTAFM. Tb-17, Tb-24, and

Tb-1.7g were all obtained from strain 427-60. The M13 library and the cosmid

librarys were constructed from strain M110. Some sequence variability that

was observed may therefore reflect differences between strains.

Several lines of evidence indicate that multiple calflagin family members

occur within a single trypanosome. These include: the detection of multiple

bands by immunoblot analysis of strain M110 (47); the detection of Tb-44-

related sequences in both the IsTAFM cDNA library and M110 cell lines; the

detection of identical restriction fragments when genomic DNA of strains M110

or 427-60 were hybridized with coding regions of calflagins (Fig. 4); and the

cloning of Tb-17, Tb-24 and Tb-1.7g from the same strain. These data are in

contrast to T. cruzi where genomic Southern blots identified a 900 bp direct

repeat upon digestion with BamH\, Hind\\\, Pst\ or Sail (127), and only a single

band on immunoblots (7,47). Overall, the data suggest that multiple functions of

the T. cruzi calflagins are limited.

The different patterns of 3' flanking sequences for the calflagins may help

indicate the evolutionary origins of the calflagin variants. For example, Tb-17

and Tb-1.7g are very similar in their coding sequences. They share the same

133

amino terminus and carboxyl terminal extensions as well as the same 3' non-

coding sequences. Therefore they are probably variants of a common

progenitor (Fig. 11,13). Tb-24 and Tb-44A also share similar coding sequences

including amino and carboxyl termini that are distinct from Tb-17 and Tb-1.7.

They share the same distinct 3' non-coding region, suggesting that Tb-44A

arose from a progenitor to Tb-24. In addition, Tb-44A contained a near perfect

internal repeat of 559 bp, which indicated that further heterogeneity in the

calflagin family arose from gene duplication of internal sequences. The

presence of only 12 nucleotide substitutions between the two halves of Tb-44A

indicates that the duplication event occurred relatively recently in the evolution

of trypanosomes. This suggestion was strongly supported by the observation

that T. cruzi contained a 23 kDa calflagin, but not the 44 kDa calflagin

(133,134). These data indicate that the duplication event occurred after the

lineage for T. brucei and T. cruzi diverged. Two different mRNA species of 1.2

and 1.6 kb were identified that encoded the calflagins. The larger mRNA likely

results from the transcription products of Tb-44A, Tb-17, and Tb-1.7g. Despite

the large size differences between the coding regions for Tb-44A and Tb-17,

each cDNA was approximately 1.5 kb. Tb-1.7g has downstream sequences

that are related to Tb-17, suggesting the use of a common polyadenylation site

and common sized transcript. By contrast, the short 3' non-coding intergenic

region of Tb-24 suggests that the smaller 1.2 kb mRNA is encoded by this gene.

The presence of multiple EF-hand calcium-binding proteins in cilia and

flagella may be a general phenomenon. The flagellum of Chlamydomonas

contains calmodulin, and a separate EF-hand protein (135,136,137). Cilia from

Tetrahymena contain at least 3 other EF-hand proteins besides calmodulin

134

(138). All of the EF-hand proteins of cilia and flagella are structurally divergent,

and may have evolved to perform special functions. The function of

trypanosome calflagins is under investigation. The flagellar location of these

calcium-binding proteins suggests a role in the motility of trypanosomatids.

However, the requirement of multiple calcium-binding proteins including

calflagins and calmodulin for motility is not known. The situation in T. cruzi

provides evidence that multiple calflagins, especially the larger Tb-44A is not

an absolute requirement for motility. Both immunoblot analyses (7,47), and

molecular cloning (127) demonstrate that the T. cruzi flagellum functions as a

motile apparatus in the absence of Tb-44A. An alternative possibility is that

some members of the calflagin family mediate signal/recognition functions of

the trypanosome flagellum. Calcium-sensitive adenylate cyclase (139), host-

parasite junctional complexes (140), or the cellular interactions required for

genetic exchange (141) are potential targets for calflagin regulation. Gene

cloning of the trypanosome calflagins will facilitate these functional analyses.

The repeated nature of the calflagins reflects the organization of the

trypanosome genome. Approximately 15% of the trypanosome genome is of a

repeated nature. Tandemly arranged 177 bp repeats of the minichromosomes,

telomeric repeats, and several families of retroposon repeated elements make

up the bulk of non-coding repeated DNA. Coding sequences lack introns,

occur at high density and are often duplicated in tandem arrays. This level of

repetition presents special problems in applying classical molecular techniques

to gene analysis. Restriction analysis of the calflagin gene locus using

Southern blotting for example was inconclusive due to similarities between

135

coding and non-coding sequences. The current hypothesis is that at least one

and possibly two other calflagin genes may exist at the locus (data not shown).

Strategies to resolve the calflagin locus are currently being considered for

investigation. In highly repeated genomes, false bands may be observed when

labeled probes bind non-specifically to DNA dense areas within a Southern

blot with certain restriction digests. False bands may also occur when probes

bind weakly to repeated elements resulting in a signal that can be amplified

many times over. Such was the case where a degenerate oligo probe for a T.

brucei VSG was used to probe a genomic Hind III digest (data not shown). A

weak band was observed and a positive Hind III fragment was cloned into a

pUC plasmid vector and sequenced (Fig. 14A). The sequence revealed a 28

bp repeated element. Reevaluation of a heavily EtBr stained gel revealed an

abnormal distribution of Hind III fragments. A dense area of fragments was

observed in the area from which the positive fragment was cloned suggesting

non-specific binding of the probe to that area. The clone was then probed back

to a Hind III digest of genomic DNA where several bands were observed

(FIG.14B). The 1.5 kb band (Fig.14A) may be the same band observed with the

degenerate oligo probe (data not shown).

The repeated sequence was, however, interesting in its own right in that

it consisted of an unusual size (28 bp) repeated element. It was considered to

be to small for a minichromosome repeat and to large for a telomeric repeat.

The size was closest to a type of 50 bp repeat associated with some VSG

promoters and antigenic variation. BLAST analysis revealed that it had

significant homology to a single element found in a B. malayi suggesting that

the sequence may be conserved. This repeated element is an example of an

136

interesting sequence that was randomly extracted from a size selected

trypanosome genome, a strategy that has proven to be fruitful in the discovery

of novel trypanosome sequences (105,106).

A seemingly unrelated discovery of the 3' end of an ORF found at the 5'

end of Tb-24 was also found to have significant homology to B. malayi.

It's proximity to the 5' end of the calflagin locus suggested that it may be co-

transcribed with the calflagin genes perhaps as a house keeping gene or a

control/regulatory element. Strategies to PCR amplify the sequence between

the ORF and the calflagins from total mRNA were not with out concern due to

the conserved sequences 5' and 3' to the genes. In addition to understanding

the ORF's transcriptional status, was the desire to ultimately sequence the

entire calflagin locus as well as the 5' ORF sequence. A strategy was therefore

undertaken to isolate one or more fragments that would contain the entire

calflagin locus as well as the 5' ORF. Since the calflagin gene locus is

contained within Xba I-Xba I and Xho I-Xho I restriction fragments (Fig. 4) size

selected fragments would be taken from one of the digest. A consideration to

creating and screening such a library was that a significant number of

sequenceable clones would be generated. To sequence the clones from one

end (500 bp) would significantly contribute to trypanosome sequencing efforts.

The prospects of identifying novel genes that might be exploited for therapy was

also a motivating factor to sequence as many of the clones as possible.

Detection of novel T.brucei sequences using shotgun cloning of genomic DNA

has previously been reported using pCR-Script™ as a vector followed by

double stranded sequencing of the insert DNA (106). This approach worked

well due to the high density and close spacing of genes in T. brucei. The

137

decision to use the M13 bacteriophage as a shotgun vector was in part due to

the logistical situation at that moment, but was also based on several

advantages including: high copy numbers of the recombinant double stranded

replicative form (RF) per cell; no strict constraints on insert size due to the

filamentous nature of the phage; Isolation of single stranded templates resulting

in high quality sequencing reactions and clean sequence reads. It was further

considered that since the focus was extended to include a sequencing project

as well as an effort to isolate one or more calflagin containing fragments, a

further step could be taken to increase the number of different non-calflagin

containing fragments. By cloning a mixture of both Xba I-Xba I and Xho I-Xho I

fragments the variety of non-calflagin containing fragments would be increased.

While not completely random the use of two size selected restriction digests

increases the number of different non-calflagin fragments while keeping the

relative number of calflagin fragments the same. This could be accomplished

because Xba \-Xba I and Xho \-Xho I fragments containing the calflagin locus

are basically the same size (Fig.4). The DNA was size select between 10 and

14kilobases. For example:

Xho I fragments + calflagin fragments

Xba I fragments + calflagin fragments

2X DNA ss 2 fragment families + 2X calflagin fragments

Initially, all 455 sequences were analyzed by the TBLASTX algorithm

with the filters off. Each sequence was then submitted to the GSS database

and "Nearest Neighbor" analysis performed. Those sequences for which the

TBLASTX and Nearest Neighbor analysis gave different results were blasted

again using BLASTP and BLASTN searches with SEG (120,121) and DUST

138

(125) filters on, respectively. The differences between searches were largely

due to the parameter options of the blast source. All of the highest ranked

descriptors were considered while only one was reported for each sequence.

Those sequences chosen for inclusion in Table 1 would typically have low p

values (p»0.05), high complexity, and length of homology typically greater

than 30 amino acids. Some sequences with high ranking descriptors were

eliminated due to a highly repeated nature or low complexity of the homologous

region (i.e. proline rich, histidine rich, or glycine rich repeats). Some

sequences were included that only appeared in non-filtered BLAST formatting

but were of high enough homology over a long enough region to warrant

reporting. These sequences demonstrate that filtering can delete some useful

data. The initial BLAST analysis was done with filtering off to prevent loss of

these coding sequences and repeated elements in the genome. All of the

sequences were investigated for any mammalian contamination in the form of

repeated elements by the NCBI under the direction of Dr. David Litman. Only

one sequence was found to be remotely related to a mammalian repeated

element (mir), but the findings were not considered to be significant and the

sequence was left in the database.

In this report several protein coding regions previously unidentified in T.

brucei were of particular interest. Pyruvate: ferredoxin oxidoreductase (PFO),

Fe-superoxide dismutase, and a descriptor for the apoptosis inducer Nbk

warrant further discussion.

Pyruvate: ferridoxin oxidoreductase. In mammalian cells, oxidative

decarboxylation of pyruvate to form acetyl-CoA is accomplished by the enzyme

pyruvate dehydrogenase. However, a variety of anaerobic pathogens,

139

including Entamoeba histolytica, Trichomona vaginalis and Giardia lamblia

utilize PFO to conduct the same function (142,143,144). The biochemical

distinction between PFO and pyruvate dehydrogenase has been exploited in

the design of therapies against anaerobic pathogens since PFO can donate

electrons to nitroheterocyclic compounds (145). The resulting nitro-free radical

and subsequently formed superoxide anion are toxic to the cell (145).

Interestingly, nitroheterocylcic compounds such as Metronidazole are also

effective against T. brucei (146,147,148,149). In this case, it has been

assumed that trypanothione reductase is responsible for transfer of electrons to

the inactive drug (150). The trypanosome PFO might be used to generate free

radicals from nitroheterocyclic compounds, or might be targeted directly as a

critical component of metabolism. The absence of a functional mitochondrion in

bloodstream forms would suggest that the trypanosome PFO is likely to be

present during this stage and be developmental^ regulated.

Fe-superoxide dismutase. The efficacy of Metronidazole and other

nitroheterocyclic compounds depends in part on the rate of nitro free radical

production, and the rate of dismutation. Trypanosomes are lacking in some

enzymes which protect against oxidative stress, including catalase and

glutathione peroxidase (151). Defense against hydrogen peroxide relies

instead on the activity of a trypanothione dependent peroxidase (152,153,154).

A dismutase capable of destroying superoxide radicals has not been described

previously for T. brucei, although a Fe-superoxide dismutase has been

identified in Crithidia fasciculata and T. cruzi (129,130). The sensitivity of T.

brucei to free radicals is the basis for trypanotolerance in Cape buffalo (155)

and N'Dama cattle (156), where each of these organisms contain hydrogen

140

peroxide generating enzymes in their blood. An understanding of the oxidative

defense mechanisms of T. brucei is a first step towards exploiting these

pathways in therapy design.

A descriptor for apoptosis inducer Nbk. Xanthine oxidase is the enzyme

responsible for hydrogen peroxide production in Cape Buffalo blood (155). It

has been recently shown that exogenous xanthine oxidase/xanthine can kill

procyclic form trypanosomes in a manner which is blocked by catalase, and

involves fragmentation of the nuclear DNA and loss of mitochondrial function

(Dr. Larry Ruben, Southern Methodist University personal communication).

These data suggest that hydrogen peroxide can initiate an apoptosis-like event

in T. brucei. Apoptosis has already been described following treatment with the

lectin, concanavalin A (157,158). Vacuolization of the cytosol and

fragmentation of the nuclear DNA precedes cell death (157). Additionally, a

programmed pattern of RNA synthesis also precedes cell death (158).

Apoptosis-like events have also been described for T. cruzi (131) and

Leishmania (132).

The highly conserved nature of this DNA between humans and

trypanosomes suggests that it contains a critical regulatory motif or that it was

recently acquired by the trypanosome from the mammalian host. Activation of

apoptotic pathways may form the basis of interesting new therapies in which the

cell is essentially induced to commit suicide during the infection cycle.

Overall, this sequencing study reflects a continuation of work begun by

others (105,106). The goal has been to describe the calflagin gene locus and

sequence large quantities of the trypanosome genome in an effort to identify

new targets for diagnosis and treatment of trypanosomiasis. This is an ongoing

141

project that hopes to contribute to the elucidation of the entire trypanosome

genome. Sequencing of the entire genome might be accelerated by using

existing sequence data in conjunction with a cosmid (40 kb insert) or PAC

(140kb insert) library and constructing a minimal tiling path based on PCR

amplified sequence-tagged sites (STSs) and/or cosmid or PAC finger printing,

as described for Giardia lamblia (159). Select cosmids or PACs could then be

shotgun cloned into the M13 vector for sequencing and assembly. The 1X

deep cosmid library for T. brucei (Fig. 17) was created with this long term goal

in mind. Powerful new software is currently being developed to assemble

restriction fragments such as those shown in figure 17. Recombinant cosmid

DNA has been prepared to extend the depth of the library many fold if the

opportunity arises to apply this type of software or to create a working library for

sequencing. A set of filters for the cosmid library has also been stamped using

a Biomeck® robot for the purpose of hybridization experiments.

References

[1] Katz, M., Despommier, D.D., Gwadz, R. (1984) Parasitic Diseases

Springer-Verlag: New York, Heidelberg, Berlin.

[2] Caphern, A., Giroud, C., and Baltz, T. (1977) Trypanosoma equiperdum:

Etude au Cours de la Trypanosomose du lapin. Experimental

Parasitology 42,6-13.

[3] Hajduk, S. L., K. Hager, and J. D. Esko. (1992) High-density

lipoproteins-mediated lysis of trypanosomes. Parasitol. Today 8: 95-97.

[4] Markell,E.K.,Voge,M.,andJohn,D.T. (1992) Medical Parasitology,

7th Edition, 137-147.

[5] Brener, Z. (1979) Present status of chemotherapy and

chemoprophylaxis of human trypanosomiasis in the western

hemisphere. Pharmacol Ther. 7, 71-90.

[6] Skolnic A. (1989) Does influx from endemic areas mean more

transfusion-associated Chagas disease JAMA 262, (11) 1433.

[7] Engman, D., Heinz-Krause, K., Blumin, J., Kim, K.,, Kirchhoff, L. and

143

Donelson, J. (1989) A novel flagellar Ca2+-binding protein

trypanosomes. J. Biol. Chem. 264,18627-18631.

[8] Boutilier. B., Cheek, T. (1993) Calcium regulation and signaling. The J.

Exp. Biol. 184, introductory remarks.

[9] Chafouleas, J., Dedman, J., Munjaal, R. and Means, A. (1979)

Calmodulin: development and application of a sensitive radioimmuno

assay. J. Biol. Chem. 254,10262-10267.

[10] Klee, C. B., Crouch, T. H. and Richman, P. G. (1980) Calmodulin. Ann.

Rev. Biochem. 49, 496.

[11] Klee, C. B. and Vanaman, T. C. (1982) Calmodulin. J. Adv. Protein

Chem. 35, 213-321.

[12] Marshak, D., Clarke, M., Roberts, D. and Watterson, D. (1984) Structure

and functional properties of calmodulin from the eukaryotic

microorganismDictystelium discoidem. J. Biochem. 23, 2891-2899.

[13] Putkey, J., Ts'ui§, K., Tanaka, T., Lagase, L., Stein, P., Lai, E., and

Means, A. (1983) Chicken calmodulin genes: a species comparison of

cDNA sequences and isolation of a genomic clone. J. Biol. Chem. 258,

11864-11870.

144

[14] Marx, J. L. (1980) Calmodulin: A protein for all seasons. Science

208, 274-276.

[15] Kooter, J., Winter, A., d'Oliveira, C., Wagter, R. and Borst., P. (1988) 5'

Boundaries of telomere conversions map at different positions in a VSG

expression site of T. brucei and may disrupt an expression site

associated gene. Gene 69,1-11.

[16] Szebenyi, D., Obenforf, S. and Moffat, K. (1981) Structure of vitamin D-

dependent calcium binding protein from bovine intestine. Nature 294,

327-332.

[17] Herzberg, O. and James, M. (1985) Structure of the calcium regulatory

muscle protein troponin-C at 2.8 angstrom resolution. Nature 313, 653-

659.

[18] Sundaralingam, M., Bergstrom, R., Sraburg, G., Rao, S., Roychowdhury,

P., Greaser, M., and Wang, B. (1985) Molecular structure of troponin C

from chicken skeletal muscle at the 3-angstrom resolution. Science 227,

945-948.

[19] Anderson, J. M., Charbonneau, H. J., Jones, H. P., McCann, R. O., and

Cormier, M. J. (1980) Characterization of the plant NAD kinase activator

protein and its identification as calmodulin. J. Biochem. 19, 3113-3120.

145

[20] Dedman, J. R., Potter, J. D.p Jackson, R. L., Johnson, J. D. and Means, A.

R. (1977) Physicochemical properties of rat testis Ca2+-dependent

regulator protein of cyclic nucleotide phosphodiesterase. J. of

Biol.Chem. 252: 8415-8422.

[21] Kilhoffers, M-C., Demaille, J. G. and Gerard, D. (1981) Tyrosine

fluorescence of ram testis and octopus calmodulins : effects of calcium,

magnesium and ionic strength. J. Biochem. 20, 4407-4414.

[22] Richman, P. andKlee, C. (1978) Conformation-dependent nitration of

the protein activator of cyclic adenosine 3',5'-monophosphate

phosphodiesterase. J. Biochem. 17, 928-935.

[23] Kretsinger, R., and Barry, C. (1975) The predicted structure of the

calcium-binding component of troponin. Biochim. Biophy. Acta 405,

40-52.

[24] Kretsinger, R. H. and Nockold, C. E. (1973) Carp muscle calcium-

binding protein II. Structural determinationand general desription. J.

Biol. Chem. 248, 3313-3326.

[25] Babu, U., Sack, J., Greenhough, T., Bugg, C., Means, A., and Cook, W.

(1985) Three-dimensional structure of calmodulin. Nature 315, 37-40.

[26] Da Silva, Ana Claudia R. & Reinach, Fernando C. (1991) Calcium

146

binding induces conformational changes in muscle regulatory proteins.

Trends in Biomedical Science 16, 53-57.

[27] Heizmann, C. and Hunziker, W. (1991) Intracellular calcium-binding

proteins: more sites than insights. Trends in Biomedical Sciences 16:

98-103.

[28] Zvelebil, M.( & Thornton, J. (1993) Peptide-protein interactions: an

overview. Quarterly Review of Biophysics 26, 333-363.

[29] Heidorn, D. B and Trewhella, J. (1988) Comparison of the crystal and

solution structures of calmodulin and troponin C. J. Biochem. 27, 909-

915.

[30] Ikura, M., Spera, S., Barbato, G., Kay, L E. Krinks, M. and Bax, A. (1991)

Secondary structure and side-chain 1H and 13C resonance assigments

of calmodulin in solution by heteronuclear multidimensional NMR

spectroscopy. J. Biochem. 30, 9216-9228.

[31] Torok, K., Lane, A., Martin, S., Janot, J. and Bayley, P. (1992) Effects of

calcium binding on the internal dynamic properties of bovine brain

calmodulin, studied by NMR and optical spectroscopy. Biochem. 31,

3452-3462.

147

[32] Torok, K. and Whitaker, M. (1994) Taking a long, hard look at

calmodulin's warm embrace. BioEssays 16, 221-224.

[33] Fairlamb, A. and Bowman, I. (1980) Maintenance of concentrated

suspensions of bloodstream trypomastigotes in vitro using continuous

dialysis for measurement of endocytosis. Exp. Parasitol. 49, 366-380.

[34] Langreth, S. and Balber, A. (1975) Protein uptake and digestion in

bloodstream and culture forms of Trypanosoma brucei. J. Protozoology

22, 40-53.

[35] Coppens, I., Baudhuin P., Opperdoes, F. and Courtoy, P. (1988)

Receptors for the host low density lipoproteins on the hemoflagellate

Trypanosoma brucei: Purification and involvement in the growth of the

parasite. Proc. Natl. Acad. Sci. USA. 85, 6753-6757.

[36] Coppens, I., F. R. Opperdoes, P. J. Courtoy, and P. Baudhuin. (1987)

Receptor-mediated endocytosis in the bloodstream form of Trypanosoma

brucei. J. Protozool. 34, 465-473.

[37] Dixon, H., Ginger., C. and Williamson, J. (1971) The lipid metabolism of

blood and cultural forms of Trypanosoma lewisi and Trypanosoma

rhodesiense. Comp. Biochem. Physiol. B 39, 247-266.

[38] Threlfall, D., Williams, B. and Goodwin, T. (1965) Excerpta Med. Sect.

148

Progr. Protozool. 91, 141.

[39] Carroll, M. and McCrorie, P. (1986) Lipid composition of bloodstream

forms of Trypanosoma brucei brucei. Comp. Biochem. Physiol. B 83,

647-651.

[40] Webster, P. and Shapiro, S. (1990) Trypanosoma brucei: A membrane-

associated protein in coated endocytic vesicles. Exp. Parasitol 70,154-

163.

[41] Webster, P. (1989) Endocytosis by African trypanosomes: 1. three

dimentional structure of the endocytic organelles in Trypanosoma brucei

and T. congolense. J. Cell Biol. 49, 295-302.

[42] Borst, P. (1986) Discontinuous transcription and antigenic variation in

trypanosomes. Ann. Rev. Biochem. 55, 701-732.

[43] Donelson, J. and Rice-Ficht, A. (1985) Molecular biology of

trypanosome antigenic variation. Microbiol. Rev. 49,107-125.

[44] Pays., E. (1986) Variability of antigen genes in African trypanosomes.

Trends Genet. 2, 21-26.

[45] Shapiro, S. and Pearson, T. (1986) African trypanosomiasis: antigens

149

and host-parasite interactions. In: T.M. Pearson (ed.): Parasite antigens,

towards new strategies for vaccines. 215-274. Marcel Dekker, Inc., New

York.

[46] Wong, S., Morales, T., Niegel, J. and Cambell, D. (1993) Genomic and

transcriptional linkage of the genes for calmodulin, EF-hand 5 protein,

and ubiquitin extension protein 52 in Trypanosoma brucei. Mol. and

Cell. Biol. 13, 207-216.

[47] Wu, Y., Haghighat, N. and Ruben, L. (1992) The predominant

calcimedins from Trypanosoma brucei comprise a family of flagellar EF-

hand calcium-binding proteins. J. Biochem. 287,1-7.

[48] Lizardi, P., Lerner, T., Gonzalez, A. and Nogueira, N. (1985) Expression

in Escherichia coli of a cDNA clone encoding a hypothetical calcium-

binding protein from trypanosoma cruzi epimastigotes. Vaccines 85, 67-

70.

[49] Ouaissi, A., Aguirre, T., Plumas-Marty, B., Piras, M., Schoneck, R., Gras-

Masse, H., Taibi, A., Loyens, M., Tartar, A., Capron, A. and Piras, R.

(1992) Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific

antigen released in association with membrane vesicles and defined by

a monoclonal antibody. Biol. Cell 75,11-17

150

[50] Lee, M., G-S., Chen, J., Ho, A., D'Alesandro, P. and Van der Ploeg, L.

(1990) A putative flagellar Ca2+-binding protein of the flagellum of

trypanosomatid protozoan parasites. Nucleic Acids Research 18,

4252.

[51] Wu, Y., DeFord, J., Benjamin, R., Lee, M. G-S. and Ruben, L. (1994) The

gene family of EF-hand calcium-binding proteins from the flagellum of

Trypanosoma brucei. J. Biochem. 304, 833-841.

[52] Ruben, L., Haghighat, N. and Campbell, A. (1990) Cyclical

differentiation of Trypanosoma brucei involves changes in the cellular

complement of calmodulin-binding proteins. Exp. Parasitol. 70, 144-153.

[53] Ruben, L., Hutchinson, A. and Moehlman, J. (1991) Calcium

homeostasis in Trypanosoma brucei. J. Biochem. Chem. 266, 24351-

2435.

[54] Ruben, L., Egwuagu, C. and Patton, C. (1983) African trypanosomes

contain calmodulin which is distinct from host calmodulin. Biochimica et

Biophysica Acta 758,104-113.

[55] Ruben, L. (1987) Identification of a developmental^ regulated calcium-

binding protein mTrypanosoma brucei. J. Protozool 34, 367-370.

151

[56] Ruben, L. and Patton, C. (1987) Calmodulin from Trypanosoma brucei:

immunological analysis and genomic organization methods in

Enzymology 139, 262-276.

[57] Paindavoine, P., Rolin, S., Van Assel, S., Geuskens, M., Jauniaux, J-C.,

Dinsart, C., Huet, G. and Pays, E. (1992) A gene from the variant surface

glycoprotein expression site encodes one of several transmembrane

adenylate cyclases located on the flagellum of Trypanosoma brucei.

Mol. and Cell. Biol. 12, 1218-1225.

[58] Alexandre, S., Paindavoine, P., Tebabi, P., Pays, A., Halleux, S., Steiner,

M., and Pays, E. (1990) Differential expression of a family of putative

adenylate/guanylate cyclase genes in T. brucei. Mol. Biochem. Parasitol.

43, 279-288.

[59] Alexandre, S., Guyaux, M., Murphy, N., Coquelet, H., Pays, A., Steinert,

M. and Pays, E. (1988) Putative genes of a variant specific antigen gene

transcription unit in Trypanosoma brucei. Mol. Cell. Biol. 8, 2367-2378.

[60] Cully, D., Ip H. and Cross G. (1985) Coordinate transcription of variant

surface glycoprotein genes and an expression site associated gene

family in Trypanosoma brucei. Cell 42,173-182.

[61] Pays, E., Tebabi, A. Pays, H. Coquelet, P. Revelard, D. Salmon, and M.

152

Steinert. (1989) The genes and transcripts of an antigen gene

expression site from T. brucei. Cell. 57, 835-845.

[62] Graham, S. and Barry J. (1991) Expression site-associated genes

transcribed independently of variant surface glycoprotein genes in

Trypanosoma brucei. J. Mol. and Biochem. Parasitol. 47, 31-42.

[63] Mathews, K., Shiels, P., Graham, S. and Barry, J. (1990) Structure and

expression of trypanosome metacyclic VSG expression sites. In

Parasites: Molecular Biology, Drug and Vaccine Design (Agabian, N.,

and Cerami, A., eds.): 73-86. Wiley and Liss, New York.

[64] Keith, K., Hide, G., .Tait, A. (1990) Characterisation of protein kinase C

like activities in Trypanosoma brucei. Mol. and Biochem. Parasitol. 43,

107-116.

[65] Parsons, M., Valentine, M. and Carter, V. (1993) Protein kinases in

divergent eukaryotes: Identification of protein kinase activities regulated

during trypanosome development. Cell Biol. 90, 2656-2660.

[66] Parsons, M., Valentine, M., Deans, J. Schieven, G. and Ledbetter, J.

(1991) Distinct Patterns of tyrosine phosphorylation during the life cycle

of Trypanosoma brucei. Mol. and Biochem. Parasitol. 45, 241-248

153

[67] Carafoli, E. (1987) Intracellular calcium homeostasis. Annu. Rev.

Biochem. 56, 395-433.

[68] Benaim, G., Bermudez, R., and Urbina, J. (1990) Ca super (2+) transport

in isolated mitochondrial vesicles from Leishmania braziliensis. Mol.

Biochem. Parasitol. 38, 61-68.

[69] Benaim, G., and Romero, P. (1990) A calcium pump in plasma

membrane vesicles from Leishmania braziliensis. Biochimica et

Biophysica Acta 1027, 79-84.

[70] Cohen, B., Benaim, G., Ruiz, M., and Michelangeli, F. (1990) Increased

calcium permeability is not responsible for the rapid lethal effects of

amphitericin B on Lieshmania sp.. FEBS Lett. 259, 286-288.

[71] Philosoph, H., and Zilberstein, D. (1989) Regulation of intracellular

calcium in promastigotes of the human protozoan parasite Leishmania

donovani. J. Biol. Chem. 264,10420-10424.

[72] Ghosh, J., Ray, M., Sarkar, S. and Bhaduri, A. (1990) A high affinity

Ca2+-ATPase on the surface membrane of Leishmania donovani

promastigote. J. Biol. Chem. 265,11345-11351.

[73] McLaughlin, J. (1985) A high affinity Ca2+-dependent ATPase in the

154

surface membrane of the bloodstream stage of Trypanosoma

rhodesiense. J. Mol. and Biochem. Parasitol. 15,189-2.

[74] Benaim, G., Losado, S., Gadelha, F. and Docampo, R. (1991) A

calmodulin-activated(Ca2+-Mg2+)-ATPase is involved in Ca2+-transport

by plasma membrane vesicles from Trypanosoma cruzi. J. Biochem.

280, 715-720.

[75] Laveran, A. (1902) De I' action du serum humain sur le Trypanosome

du Nagana (Tr. Brucei). C. r. hebd. Seanc. Acad. Sci. 134, 734-739.

[76] D'hondt, J., Van Meirvenne, N., Moens, L and Kondo, M. (1979) Ca2+

is essential cofactor for trypanocidal activity of normal human serum.

Nature 282, 613-615.

[77] Rifkin, M. (1978) Identification of the trypanocidal factor in normal

human serum: high density lipoprotein. Proc. Natl. Acad. Sci. USA 75:

3450-3454.

[78] Rifkin, M. (1991) Role of phospholipids i n the cytotoxic action of high

density lipoprotein on trypanosomes. J. Lipid Research 32, 639-647.

[79] Hager, K., Pierce, M., Moore, D., Tytler, E., Esko, J. and Hajduk, S.

(1994) Endocytosis of a cytotoxic human high density lipprotein results

in disruption of acidic intracellular vesicles and subequent killing of

155

African trypariosomes. J. Cell Biol. 126 (1) 155-167.

[80] Hajduk, S., Moore, D., Vasudevacharya, J., Siqueira H., Torri A.,

Tytler E. and Esko, J. (1989) Lysis of Trypanosoma brucei by a toxic

subspecies of human high density lipoprotein. J. Biol. Chem. 264: 5210-

5217.

[81] Hajduk, S. L., Hager, K. and Esko, J. D. (1992) High-density

lipoproteins-mediated lysis of trypanosomes. Parasitol. Today 8:95-97.

[82] Smith, A.B., Esko, J.D. and Hajduk, S.L. (1995) Killing of trypanosomes

by the human haptoglobin-related protein. Science 268, 284-286.

[83] Clarkson, A. and Amole, B. (1982) Role of trypanocidal drug action.

Science 216:1321-1323.

[84] Scheibel, L., Colombani, P., Hes, A., Aikawa, M., Atkinson, C. and

Milhous, W. (1987) Calcium and calmodulin antagonist inhibit human

malarial parasites (Plasmodium falciparum): implications for drug design.

Proc. Natl. Acad. Sci.USA. 84, 7310-7314.

[85] Pearson, R., Manian, A., Harcus, J., Hall, D. and Hewlett, E. (1982)

Lethal effects of phenothiazine neuroleptics on the pathogenic protozoan

Leishmania donovani. Science 217, 369-371.

156

[86] Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A., Kirkness, E.F.,

Kerlavage, A.R., Bult, C.J., Tomb, J.F., Dougherty, B.A., Merrick, J.M.,

McKenney, K., Sutton, G., GitzHugh, W., Fields, C., Gocayne, J.D., Scott,

J., Shirley, R., Liu, L.-I., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips,

C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Hanna,

M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L.D., Fritchman,

J.L., Fuhrmann, J.L., Geohagen, N.S.M., Gnehm, C.L., McDonald, L.A.,

Small, K.V., Fraser, C.M., Smith, H.O. and Venter, J.C. (1995). Whole-

genome random sequencing and assembly of Haemophilus influenzae

Rd. Science 269, 496-512.

[87] Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A.,

Fleischmann, R.D., Bult, C.J., Kerlavage, A.R., Sutton, G., Kelley, J.M.,

Weidman, J.F., Small, K.V., Sandusky, M., fuhrmann, J., Nguyen, D.,

Utterback, T.R., Saudek, D.M., Phillips, C.A., Merrick, J.M., Tomb, J.-F.,

Dougherty, B.A., Bott, K.F., Hu, P.-C., Lucier, T.S., Peterson, S.N., Smith,

H.O., Hutchison III, C.A. and Venter, J.C. (1995) The minimal gene

complement of Mycoplasma genitalium. Science 270,397-403.

[88] Bult, C.J., White, O., Olsen, G.J., Zhou, L., Fleischmann, R.D., Sutton,

G.G., Blake, J.A., FitzGerald, L.M., Clayton, R.A., Gocayne, J.D.,

Kerlavage, A.R., Dougherty, B.A., Tomb, J.F., Adams, M.D., Reich, C.I.,

Overbeek, R., Kirkness, E.F., Weinstock, K.G., Merrick, J.M., Glodek, A.,

Scott, J.L., Geoghagen, N.S.M., and Venter, J.C. (1996) Complete

157

genome sequence of the methanogenic archaeon, Methanococcus

jannaschii. Science 273, 1058-1073.

[89] Williams, N. (1996) Yeast genome sequence ferments new research.

Science 272, 481.

[90] Borst, P., van der Ploeg, M., van Hoek, J.F., Tas, J., and James, J.,

(1982) On the DNA content and ploidy of trypanosomes. Mol. Biochem.

Parasitol. 6,13-23.

[91] Gottesdiener, K., Garcia-Anoveros, J., Lee, M.G., and Van der Ploeg, L.H.

(1990) Chromosome organization of the protozoan Trypanosoma

brucei. Mol. Cell Biol. 10, 6079-6083.

[92] Van der Ploeg, L.H., Cornelissen, A.W., Michels, P.A., and Borst P.

(1984) Chromosome rearrangements in Trypanosoma brucei. Cell 39,

213-221.

[93] Henriksson, J., Aslund, L., Macina, R.A., Franke de Cazzulo, B.M.,

Cazzulo, J.J., Frasch, A.C. and Pettersson, U. (1990) Chromosomal

localization of seven cloned antigen genes provides evidence of diploidy

and further demonstration of karyotype variability in Trypanosoma cruzi.

Mol. Biochem. Parasitol. 42, 213-223.

158

[94] Wagner, W., arid So, M. (1990) Genomic variation of Trypanosoma

cruz'r. involvement of multicopy genes. Infect. Immun. 58, 3217-3224

[95] Lighthall, G.K., and Giannini, S. H. (1992) The chromosomes of

Leishmania. Parasitology Today 8,192-199.

[96] Bastien, P., Blaineau, C., and Pages, M. (1992) Molecular karyotype

analysis in Leishmania. Subcell Biochem. 18, 131-187.

[97] Weiden, M., Osheim, Y.N., Beyer, A.L., and Van der Ploeg, L.H. (1991)

Chromosome structure: DNA nucleotide sequence elements of a subset

of the minichromosomes of the protozoan Trypanosoma brucei. Mol.

Cell Biol. 11, 3823-3834.

[98] Turner, C.M., Sternberg, J., Buchanan, N., Smith, E., Hide, G., and Tait, A.

(1990) Evidence that the mechanism of gene exchange in Trypanosoma

brucei involves meiosis and syngamy. Parasitology 101, 377-386

[99] Gibson, W., Garside, L. and Bailey, M. (1992) Trisomy and chromosome

size changes in hybrid trypanosomes from a genetic cross between

Trypanosoma brucei rhodesiense and T. b. brucei. Mol. Biochem.

Parasitol. 51,189-199.

159

[100] Paindavoirie, P., Zampetti-Bosseler, F., Pays, E., Schweizer, J., Guyaux,

M., Jenni, L., and Steinert, M. (1986) Trypanosome hybrids generated in

tsetse flies by nuclear fusion. EMBOJ. 5,3631-3636.

[101] Benne, R. (1990) RNA editting in trypanosomes: is there a message?

Trends Genet. 6,177-181

[102] Johnson, P.J., Kooter, J.M. and Borst, P. (1987) Inactivation of

transcription by UV irradiation of Trypanosoma brucei provides evidence

for a multicistronic transcription unit that includes a variant surface

glycoprotein gene. Cell 51, 273-281.

[103] Walder, J.A., Eder, P.S., Engman, D.M., Brentano, S.T., Walder, R.Y.,

Knutzon, D.S.,Dorfman, D.M. and Donelson, J.E. (1986) The 35-

nucleotide splice leader sequence is common to all trypanosome

messenger RNA's. Science 233, 569-571

[104] Boothroyd, J.C. and Cross, G.A.M. (1982) Transcripts coding for variant

surface glycoproteins of Trypanosoma brucei have a short, identical

exon at their 5' end. Gene 20, 281-289.

[105] el-Sayed, N.M., Alarcon, C.M., Beck, J.C., Sheffield, V.C., and Donelson,

J.E. (1995) cDNA expressed sequence tags of Trypanosoma brucei

rhodesiense provide new insights into the biology of the parasite. Mol.

Biochem. Parasitol. 73, 75-90.

160

[106] el-Sayed, N.M., and Donelson, J.E., (1997) A survey of the

Trypanosoma brucei rhodesiense genome using shotgun sequencing.

Mol. Biochem. Parasitol. 84,167-178.

[107] Maniatis, T., Fritsch, E.F., and Sambrook, J. (1989) Molecular Cloning: A

laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY.

[108] Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction

procedure for screening recombinant plasmid DNA. Nucleic Acids Res.

7, 1513

[109] Ish-Horowicz, D. and Burke,J.F. (1981) Rapid and efficient cosmid

cloning. Nucleic Acids Res. 9, 2989

[110] Messing, J., Gronenborn, B., Muller-Hill, B. and Hofschneider, P.H.

(1977) Filamentous coliphage M13 as a cloning vehicle: Insertion of a

H/ndlllfragment of the lac regulatory region in M13 replicative form in

vitro. Proc. natl. Acad. Sci. 74, 3642-3647

[111] Tabor, S. and Richardson, C.C. (1989) Effect of manganese ions on the

incorporation of dideoxynucleotides by bacteriophageT7 DNA

polymerase and Escherichia coli DNA polymerase I Proc. Nat. Acad.

Sci. USA 86, 4076-4080.

161

[112] Tabor, S. and Richardson, C.C. (1989) Selective inactivation of the

exonuclease activity of bacteriophage T7 DNA polymerase by invitro

mutagenesis J. Biol. Chem. 264, 6447-6448.

[113] Tabor, S. and Richardson, C.C. (1987) DNA sequence analysis with a

modified bacteriophage 17 DNA polymerase. Proc. Nat. Acad. Sci. USA

84, 4767-4771.

[114] Southern, E.M. (1975) Detection of specific sequences among DNA

fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503.

[115] Maxam, A.M. and Gilbert, W. (1977) A new method for sequencing DNA.

Proc. Natl. Acad. Sci. 74, 560.

[116] Balber, A.E. (1972) Trypanosoma brucei: fluxes of the morphological

variants in intact and X-irradiated mice. Exp. Parasitol. 31, 307-319.

[117] Medina-Acosta, E., and Cross, G.A. (1993) Rapid isolation of DNA from

trypanosomatid protozoa using a simple 'mini-prep' procedure. Mol.

Biochem. Parasitol. 59, 327-329.

[118] Horwitz, J. P., Chua.J., Tomson, R.J., Darooge, M.A., Fisher, B.E.,

Mauricio, J., and Klundt, I. (1964) Substrates for cytochemical

demonstration of enzyme activity. I. Some substituted 3-indole-l3-D-

glycopyranosides. J. Med. Chem. 7, 574

162

[119] Ulmann, A., Jacob, F., and Monod, J. (1967) Characterization by in vitro

complementation of a peptide corresponding to an operator-proximal

segment of the 3-galactosidase structural gene of Escherichia coli. J.

Mol. Biol. 24, 339.

[120] Wootton, J. C., and Federhen, S. (1993). Statistics of local complexity in

amino acid sequences and sequence databases. Computers in

Chemistry 17,149-163.

[121] Wootton, J. C., and Federhen, S. (1996). Analysis of compositionally

biased regions in sequence databases. Methods in Enzymology 266,

554-571.

[122] Altschul, S. F., Warren G., Miller W., Myers E. W., and Lipman D.J. (1990).

Basic local alignment search tool. J. Mol. Biol. 215, 403-10.

[123] Altschul S.F., Boguski M.S., Gish W., and Wootton, J.C. (1994) Issues in

Searching molecular sequence Databases. Nature Genetics 6, 119-129.

[124] Gish W., and States D.J. (1993). Identification of protein coding regions

by database similarity search. Nat. Genet. 3, 266-72.

[125] Hancock, J. M., and Armstrong, J.S. (1994) SIMPLE34: an improved and

enhanced implementation for VAX and Sun computers of the SIMPLE

163

algorithm for analysis of clustered repetitive motifs in nucleotide

sequences. Comput. Appl. Biosci. 10, 67-70.

[126] Lanham, S.M. (1968) Separation of trypanosomes from the blood of

infected rats and mice by anion-exchangers. Nature 218, 1273-1274.

[127] Gonzalez, A., Lerner, T., Huecas, M., Sosa-Pineda, S., Nogueira, N.,

and Lizardi, P. (1985) Apparent generation of segmented mRNA from

two separate tandem gene families in Trypanosoma cruzi. Nucleic Acids

Research 13 (16), 5789-5802.

[128] Strynadka, N.C., James, M.N. (1989) Crystal structures of the helix-

loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58, 951-998.

[129] LeTrang, N., Meshnick, S.R., Kitchener, K., Eaton, J.W., Cerami, A.

(1983) Iron-containing superoxide dismutase from Crithidia fasciculata.

Purification, characterization, and similarity to leishmanial and

trypanosomal enzymes. J. Biol. Chem. 258(1), 125-130

[130] Temperton, N.J., Wilkinson, S.R., and Kelly, J.M. (1996) Cloning of an Fe-

superoxide dismutase gene homologue from Trypanosoma cruzi. Mol.

Biochem. Parasitol. 76(1-2), 339-343

[131] Ameison, J.C., Idziorek, T., Billaut-Mulot, O., Loyens, M., Tissier, J.P.,

Potentier, A., and Ouaissi, A. (1995) Apoptosis in a unicellular eukaryote

164

(Trypanosoma cruzi): Implications for the evolutionary origin and role of

programmed cell death in the control of cell proliferation, differentiation

and survival. Cell Death Differ. 2, 285-300.

[132] Moreira, M.E.C., Del Portillo, H.A., Milder, R.V., Balanco, J.M.F., and

Barcinski, M.A. (1996) Heat shock induction of apoptosis in

promastigotes of the unicellular organism Leishmania (Leishmania)

amozonensis. J. Cell Physiol. 167, 305-313.

[133] Schneider, A., Lutz, H.U., Marugg, R., Gehr, P., and Seebeck, T.

(1988) Spectrin-like proteins in the paraflagellar rod structure of

Trypanosoma brucei. J. Cell Sci. 90(Pt 2), 307-315.

[134] Brostrom, M.A., Brostrom, C.O., and Wolff, D.J. (1978) Calcium-

dependent adenylate cyclase from rat cerebral cortex: activation by

guanine nucleotides. Arch. Biochem. Biophys. 191(1), 341-350.

[135] Gitelman, S.E., and Witman, G.B. (1980) Purification of calmodulin from

Chlamydomonas: calmodulin occurs in cell bodies and flagella. J. Cell

Biol. 87(3 Pt 1), 764-770

[136] Huang, B., Mengersen, A., and Lee, V.D. (1988) Molecular cloning of

cDNA for caltractin, a basal body-associated Ca2+-binding protein:

homology in its protein sequence with calmodulin and the yeast CDC31

gene product. J. Cell Biol. 107(1), 133-140

165

[137] Taillon, B.E., Adler, S.A., Suhan, J.P., and Jarvik, J.W. (1992) Mutational

analysis of centrin: an EF-hand protein associated with three distinct

contractile fibers in the basal body apparatus of Chlamydomonas. J. Cell

Biol. 119(6), 1613-1624

[138] Takemasa, T., Takagi, T., Kobayashi, T., Konishi, K., and Watanabe, Y.

(1990) The third calmodulin family protein in Tetrahymena. Cloning of

the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23).

J. Biol. Chem. 265(5), 2514-2517.

[139] Rolin, S., Halleux, S., Van Sande, J., Dumont ,J., Pays, E., and Steinert,

M. (1990) Stage-specific adenylate cyclase activity in Trypanosoma

brucei. Exp. Parasitol.71(3), 350-352

[140] Tetley, L., and Vickerman, K. (1985) Differentiation in Trypanosoma

brucei: host-parasite cell junctions and their persistence during

acquisition of the variable antigen coat.

J. Cell Sci. 74, 1-19

[141] Tait, A. (1990) Genetic exchange and evolutionary relationships in

protozoan and helminth parasites. Parasitologyl 00 Suppl, S75-S87

[142] Reeves, R.E., Warren, L.G., Susskind, B., and Lo, H.S. (1977) An energy-

conserving pyruvate-to-acetate pathway in Entamoeba histolytica.

166

Pyruvate synthase and a new acetate thiokinase. J. Biol. Chem. 252(2),

726-731

[143] Williams, K., Lowe, P.N., and Leadlay, P.F. (1987) Purification and

characterization of pyruvate: ferredoxin oxidoreductase from the

anaerobic protozoon Trichomonas vaginalis. Biochem. J. 246(2), 529-

536

[144] Ellis, J.E., Williams, R., Cole, D., Cammack, R., and Lloyd, D. (1993)

Electron transport components of the parasitic protozoon Giardia lamblia.

FEBS Lett. 325(3), 196-200

[145] Muller, M. (1986) Reductive activation of nitroimidazoles in anaerobic

microorganisms. Biochemical Pharmacology 35, 37-41

[146] Jennings, F.W. (1991) Chemotherapy of CNS-trypanosomiasis: the

combined use of the arsenicals and nitro-compounds. Trop. Med.

Parasitol. 42(2), 139-142

[147] Jennings, F.W. (1991) Chemotherapy of CNS-trypanosomiasis:

combination chemotherapy with a 5-nitroimidazole (MK 436), an

arsenical (Cymelarsan) and suramin. Trop. Med. Parasitol. 42(3),

157-160

167

[148] Otigbuo, I.N. and Woo, P.T. (1988) The in vitro and in vivo effects of

metronidazole and chloroquine on Trypanosoma brucei brucei. J.

Parasitol. 74, 201-206.

[149] Foulkes, J.R., (1996) Metronidazole and suramin combination in the

treatment of arsenical refractory rhodesian sleeping sickness-a case

study. Trans. R. Soc. Trap. Med. Hyg. 90(4), 422

[150] Kelly, J.M., Taylor, M.C., Smith, K., Hunter, K.J., and Fairlamb, A.H.

(1993) Phenotype of recombinant Leishmania donovani and

Trypanosoma cruzi which over-express trypanothione reductase.

Sensitivity towards agents that are thought to induce oxidative stress.

Eur. J. Biochem. 218, 29-37.

[151] Fairlamb, A.H., and Cerami, A. (1992) Metabolism and functions of

trypanothione in the Kinetoplastida. Annu. Rev. Microbiol. 46, 695-729.

[152] Henderson, G.B., Fairlamb, A.H., and Cerami, A. (1987) Trypanothione

dependent peroxide metabolism in Crithidia fasciculata and

Trypanosoma brucei. Mol. Biochem. Parasitol. 24, 39-45.

[153] Penketh, P.G., and Klein, R.A. (1986) Hydrogen peroxide metabolism in

Trypanosoma brucei. Mol. Biochem. Parasitol. 20,111-121.

168

[154] Penketh, P.G., Kennedy, W.P., Patton, C.L. and Sartorelli, A.C. (1987)

Trypanosomas hydrogen peroxide metabolism. FEBS Lett. 221,

427-431.

[155] Muranjan, M., Wang, Q., Li, Y.-L., Hamilton, E., Otieno-Omondi, F.P.,

Wang, J., Van Praagh, A., Grootenhuis, J.G., and Black, S. (1997) The

trypanocidal cape buffalo serum protein is xanthine oxidase. Infect.

Immun. (In Press).

[156] Traore-Leroux, T., Fumoux, F., Chaize, J., and Roelants, G.E. (1987)

Trypanosoma brucei: Polyamine oxidase mediated trypanolytic activity

in the serum of naturally resistant cattle. Expt. Parasitol. 64, 401-409.

[157] Welburn, S.C., Dale, C, Ellis, D, Beecroft, R., and Pearson, T.W. (1996)

Apoptosis in Trypanosoma brucei rhodesiense in vitro. Cell Death Differ.

3, 229-236.

[158] Murphy, N.B. and Welburn, S.C. (1997) Programmed cell death in

procyclic Trypanosoma brucei rhodesiense is associated with differential

epression of mRNAs. Cell Death Differ. 4, 365-370.

[159] Smith, M.W., Holmsen, A.L., Wei, Y.H., Peterson, M., and Evans, G.A.

(1994) Genomic sequence sampling: a strategy for high resolution

sequence-based physical mapping of complex genomes. Nat. Genet.

7(1), 40-47