Towards Funetional Complementation Cloning of the

Gene for the PiasnuIemmal Carnitine Transporter Defect:

Selective Media Development and Initial Transfections

Soott William Bukovac

A Thesis Submitted in Confdty with the Rquircmcnts

fa the Degee of Master of Science,

I)e-nt of QiiirPI Biochcniistry, at the

University of Toronto

8 Copyright by Scott W. Bukovac 1996

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TABLE of CONTENTS i

Page

ABSTRAa'

ACKNOWLEDGEMENTS

LIST of FIGURES

LIST of TABLES

PUBUCATIONS

GENERAL INTRODUCTION - CARNITINE:

Historical Information

Biosynthesis and Nutritional Rquircmtnts

Characterhtion of Plasma Membrane Camitine Transport in Various Tissues and Species

kxidation

Pathophysiology of Fauy Acid Oxidation Defects

Camitine Deficiencies - Rmiary and Secondary

EXPERIMENTAL APPROACH

CHAPTER 1: SELECTIVE MEDIA DEVELOPMENT

MateriaisandMcthais (a) CellLines/Matcrials (b) Ce11 Cultue Mcthodology (mcûia, rcçhniques, etc.) (c) Cell Counting (d) Media Famukition

ibj ~ani'puhian of Gilp*ose and Pbosphafc Corictntratiias (c) Maniputstion of Aniino Acid Conparneion (d) "CaniitUie Rcscue" - Effcct of Caniieinc Supplcmcntation (e) Effi~xt of Hygnnnych B pnd odia Antibiotics (9 Finel SeIcctivt Medium

vii

Discussion

CHAPTER 2: FUNCTIONAL COMPLEMENTATION CLONING

Experimental Approach

Matenais and Methads (a) m h y (b) Transfec tion Optimization (c) Transfection with Library (d) PassageISelec tion of Cells (e) Isolation of CellslDNA (f) PCR for Selection (g) Transformation of E.coli and Isolation of individual colonies (h) Sequencing of individuai clones (i) Computerized BLAST-malysis of sequences

Results (a) M'IT' Analysis (b) Transfection Optimization (c) PCD Ce11 Transfection with cDNA Library and Growth

in Selective Medium (d) Isolation of DNA and Confirmation of "Selection" (e) Typical Screening Gel Results (f) Summary of ScreenindSequencing Results (g) Results of BLAST searches

Discussion

FUTURE DIRECTIONS

a) Specificity of Hygromycin B in Selective Medium - Carnitine Rescue Experiment

b) Mechanism of Hygromycin B effect on Selection C) Further Analysis of Clones d) Constniction of a new cDNA lihrary in pREPR vector

APPENDICES:

Table 1: Literature Review of L-Camitine Uptiike Studies in Various Tissues and Species

Table 2: Clone Summary - Round 1

Table 3: Clone Summary - Round 3

Table 4: Clone Summary - Round 5

BIBLIOGRAPHY

ABSTRACT

Patients exhibiting a camitine deficient state in tissue a bloai can have a number of

underlying etiologies. The primary systemic camitint deficiency syndnmie is characte* by early

infantile onset wîth cardiomyopathy, hypotonia, rcclimnt hypokttotic hypoglycemic encephalopathy,

weakness, very low semm and tissue caniitine concentrations and a dnimatic clinical response to high

dose oral camitine supplcmentatim. Biochemical and uptalce snidies suggest chat the defect present

in these patients is a dcfcctive plasma m m b r ~ a e camitine tmspmt protein* A functional

wmplementation based protoc01 was o~empted to try and obtah the cDNA fa the transporter. A

selective p w t h medium (RPMI-1640 minus aspamgine, with 15% fed calf serum, 5 m M

galactose, 100 p M BSA / 100 jM paimitate and 0 8 0 Wrnl Hyprnycin B) was developcd which

eflectively suppressed the p w t h of primary camitine &fiCient patient ali lines in contrast to ~ ~ ) r m a l

conad ce11 lines. As lymphoblasts express the transporter defcct, a cDNA library, originally

prepamâ frmi lymphoblast mRNA, was useû to üansfiit mutant lymphoblast ceii lines. Following

p w t h in the selective mdium for various pcriais of the, the cDNAs h m thc suMving cells wae

shunled hm Ecoli, isolatal individuaiiy, and characterkd by so~utncing. The sc~ucllces weie

BLAST searched againa the NIWNCBI Gabanlr Aatabase and those clones containing sm@y

homologous scqucnccs were eliminated h m fitrthtr analysis. Sncening of 700 clones revded 467

with inserts, 367 of which were sequcnced and 92 of which w a e retained for future analysis.

ACKNOWLEDGEMENTS J'

I wish to acknowledge, with tbe completion of this Thesis. the following people for their many and varied types of assistance:

To Dr. Ingrid Teh, my thanks for h a help, guidance, encouragement, financiai support and advice on bah my scientific work and otha mattem.

To Dr. Brian H. Robinson and Dr. Don Mahiiran for swing on my thesis Committtt and for th& helpfui wmments and advia durhg the course of my thesis prr,ges.

To Dr. Zhong-Wei Xie and MIS. Wendy Chow, for invaluable technicai assistance throughout the course of my research work.

To my many fricnds throughout the city, meny fiom the Massey College and Metropolitan Community aiurch of Tmnto coamiunities, whose support has kai invaluable to m during the course of my saidKs, in particular Chris EL, Ann B., Glen A., and Alena S.

Fiiaiiy, 1 &dicatc this thesis to my parents. Peter and Bctty, without whose ullconditionai lwe, constant support, encouragement and advice, none of this would have been possible.

LIST of FIGURES: Page

GENERAL INTRODUCTION - CARNITINE EXPERIMENTAL APPROACH

Figure 1 : Carnithe Biosynthesis 17

Figure 2: Ovewiew of Camitine Cycle and Mitocbondrial Fatty Acid Oxidation 18

Fïgutt 3: Cycle of Intramimhondrial bxidation and its Ass0ciatd Defats 19

CHAPTER 1: SELECTIVE MEDIA DEVELOPMENT

Figure 1 : Fctai Calf Saum-96 Rcduction Study without BSA-palmitate Supplementation for Normal Conml (UIûû6) and PCD (Loo1 1) Cells (RPMI with 5 mM Gd.) 38

4

Figure 2: Fetal Calf Serum-% Reduction Study with 100 phd BSA-paimitatc Supplementation for Nomial Connd 06) and X D (Loo1 1) Cas (RPMI with 5 mM Gal.) 38

Figure 3: Comparison of Growth of Nonnai Conad 0 and PCD (LOO1 1) Celi Lines in Diffmnt Aniino acid DiogOut Media Conditions (RPMI with 15% saun 5 mM Gala and 100 phi BSA-palmitate) 39

Figure 4: Cornparison of Growth Characteristics of N d Conml (Lo and PCD (Loo1 1) Ce11 Lines in Dinaent Amino acid h o p a i t Media Conditions (RPMI with 15% saun, 5 mM Gal. and 100 pM BSA-palaitate) 39

Fi- 5: Cornparison of Griowth Chara~tqistics of Nonnal Conml and PCD (Loo1 1) Cell Lines in Serine, Cystcinc, and Glutamine Dmp-Out Media conditions (RPMI with 15% saum, 5 rnM Gd. and 100 BSA-~dmiate) 40

Fi- 6: Camitinc Rescue - Growth of~onnal Contn,l Ce11 LUie 0 in- Asn hop(nit Medium (RPMI 4th 15% scrum, 5 mM GaL and 1 0 plld BSA-palmitate) fœ inaewing camitinc carentrahims 41

Figure 7: Carnithe Rescue - Growth of- Celi Line (Loo1 1) in Asn hop-Out Medium (RPM with 15% scrum, 5 mM Gd. iind 100 phd BSA-palmitate) fa increasing camitint oo~ntiatims. 41

Figure 8: Hygriomycin B Sensitivity of PCD o2, UK)11) and Nomial Conad (LûûM, Looos) Cd Lines in N d Medium @PM with 2Wb senun) foUowing 14 doys of Hygmmycin B txposure 42

Figure 9: Hygriomycin B Sensitivity of PCD 02, UK)11) Md Namnl C a i a o i ~ L 0 0 0 9 ) W L i n e s i n S c 1 ~ v C M e d i i m i @PMI, Am Dmp-ûut, 15% Saum. 5 mM Gd. ancl 100 pM BSA-Ppimitatt) foliowing 14 days of Hygmmycin B Exposm 42

Figure 10: PCD CeLl Lines (iAMl2, UNI) and Nonnal Coriml Ccll LUies (Lo6, LûUl9. L O S , U1017): Growth in S e W v t Medium (RPMI, Asn hopûit , 15% serum, 5 m M Gai.. 100 pM BSA-Palmitate) with O p @ d Hygmmycin B 43

Figure 11: PCD Cd Lines oûZ, Ulû11) and Normal Ccmml Ccll Lims (LAXM. L0009. L0005. L0017): Growth in Selective Medium (RPMI, Am DmpIOut, 15% seruxn, 5 mM Gd.. 100 pM BSA-Palmitatc)with 40 pg/M Hygromycin B 43

CHAPTER 2: FU'IVCTIONAL COMPLEMENTATION CLONING:

Figure 1: Map of pREP4 vcctœ with Multiple Cloning Site and Insm 66

Figure 2: MTi' Dye CeIl Vinbility Assay Standard Curve - Live Ce11 Numba 67 versus MTI' dyc Absorbanœ (A57GA630)

Figure 3: Typical Scrteriing Gd Result 68

LIST of TABLES Page

CHAPTER 1: SELECTIVE MEDIA DEVELOPMENT

Table 1: Characteristics of Amino Acids

Table 2: ResultslObsewations of Amino Acid Drop-Ou t S tudies

CIIAPTER 2: FUNCTIONAL COMPLEMENTATION CLONING:

Table 1: Electroporation Cd Killing Efficiency for PCD (LOO1 1) Ce11 Line - % Viability determined by M T ï Ce11 Viability Assay

Table 2: Summary of Clones Obtained

APPENDICES:

Table 1 : Litenture Review of L-Camitine Uptake Studies in Di fferent Tissues and Species

Table 2: Clone Summary - Round 1

Table 3: Clone Summary - Round 3

Table 4: Clone Summary - Round 5

PUBLICATIONS

Published in Refereed Journals

Ingrid Teh, Scott W. Bukovac, and Zbong-Wei Xie. Characteization of the Human P l d e m m a l Camitine Ttansparer in Culturd S b Fibrobiasts. Archives d Biochtmistry and Biophysics. (L9%), 329(2), pp. 145-155.

Manuscript in Preparation for Refereed Journai

Scott W. Bukovac, Zhong-Wei Xie, and Ingrid Teh. Characterization of the Human Plasmalemmal Camitine Transporter in Lymphoblasîs: Na and pH dependence, Kinetics. Manuscript in Prepatation (1996).

GENERAL INTRODUCTION a CARNITINE: i

Historical Information

L-carnitine was discovered in 1906 as a component of meat and in

1927, the chernical structure of this component was established (1,2):

It was not until sorne 30 years later that the biological importance of L-

camitine was discovered. when it was determined to be an essential

growth factor of the yellow mealworrn. Tenebrio molitor. In honor of this

discovery, it was called vitamin BT. In 1952, vitamin BT was confirmed

to be L-carnitine (3). In 1959, Fritz showed that carnitine increased long-

chain fatty acid oxidation (FAO) in liver and heart muscle (4). Since then a

large number of studies have detailed its biosynthesis, nutritional sources,

metabolism. and role(s) in metabolism. as well as the syndromes that

affect the concentrations of carnitine in tissue or blood (5).

Biosynthesis and Nutritional Requirements

Carnitine stores found in man corne from two main sources: diet and

endogenous bios yn thesis. In non-vegetarians, approximately 75% of .--

camitine cornes from the diet, in the principal dietary sources of red meat

and dairy products and the remainder from biosynthesis (6.7). Carnitine

appears in food sources as three forms: free carnitine, short-chain acyl-

carnitine esters and long chain acyl-carnitine esters. Like most of the

water soluble vitamins, it is absorbed efficiently in the small intestine and

very little of the consumed carnitine is found in stool (8).

Camitine is a small water soluble quaternary amine that ;contains 7

carbon atoms. Formally, it is described chemically as P- h ydrox y-y-

trimethylaminobutyric acid and has a molecular formula of C7H 15NO3.

Carnitine can be synthesized endogenously in mammalian tissues via the

pathway shown below in Fig. 1 (9). This pathway requires protein-bound

lysine, S-adenosylmethionine to act as a methyl group donor and 5

enzymes with appropriate CO-factors to achieve the full de novo synthesis.

Most animal tissues contain the necessary enzymes to sequentially convert

protein-bound lysine to 6-N-trimethyl-lysine, 3-hydroxy-6-N-trimethyl-

lysine, y-butyrobetaine aldehyde and finally y-butyrobetaine. The final

step of hydroxylation, catalyzed by the y-butyrobetaine, 2-oxogiutarate

dioxygenase is present. in man, only in the liver. kidney and brain (10).

The plasma concentration of carnitine is largely maintained at a

constant level by the renal threshold (approximately 40pM) for this

important quaternary amine ( 1 1). Skeletal muscle is found to contain up

to 70-times the concentration fourrd' in serum, and therefore it is not

suprising to find that approximately 90% of the total body carnitine store

is found in the skeletal muscle. Tissues, in general. are found to contain

carnitine concentrations which closely parallel the dependence and

capacity of the tissue to metabolize fatty acids. Stanley (1987)

summarized the literature for human tissue concentrations (nmollg) as

follows (12): heart (3500-6000); skeletal muscle (2000-4600); liver

(1 000- 1900); and brain (200-500). Based on a normal serum carnitine

concentration of 40 - 60 FM, this would suggest that a large concentration

gradient (20-50-fold) exists between the serum and the tissues and that

some sort of plasma membrane-based active transport process must exist.

Characterization of Plasma Membrane Carnitine Transport in

Various Tissues and Species

Carnitine transport has been investigated by a number of

researchers in various tissues, cells, membrane preparations and in a

number of species. A summary of those studies performed and published

in the literature to date is shown in Appendix Table 1 (13-44). The data

to-date suggest that several functionally different transporters exist in

man. Most of these transporters appear to operate via sodium gradient-

dependent, active transport mechanisms. The specific, saturable high-

affinity transport system found in kidney, skeletal muscle, cardiac muscle,

skin fibroblasts, and lymphoblasts appear to share similar kinetic

properties with a Km of 2-5 pM and a Vmax of 1-3 pmol/min/mg protein.

This is distinctly differentiated from the transporter kinetics observed in

liver where the Km is 500 pM and in brain where the Km is >1000 PM.

Finally, human proximal small intestine uptake of carnitine is observed to

have a Km of approximately 974 PM.

In the absence of a primary plasmalemmal carnitine uptake defect,

orally administered carnitine is, observed to have a relative1 y low systemic - 7 .

bioavailability (45). This is likely contributed to by the intermediate Km

values for the intestine as well as by the large Km value in the liver. In

addition, the liver can markedly increase its carnitine content if supplied

with exogenous carnitine and likely "scavenges" the carnitine from the

portal circulation pnor to release to the systemic circulation (46). Finally,

the transport system found in skeletal and cardiac muscle as well as

kidney, is almost saturated by normal serum carnitine concentrations.

In the presence of a primary carnitine uptake defect, high, oral dose

carnitine supplementation partially restores the serum carnitine

concentration, but only slightly increases the skeletal muscle carnitine

concentration (40). In true primary systemic carnitine deficiency

patients, the response to high dose oral carnitine supplementation is very

dramatic, with almost al1 clinical sequelae resolving within months after

initiation of supplementation (40.47). Even though muscle carnitine

concentrations are not restored to normal, the amount of carnitine

provided is sufficient for efficient long-chah fatty acid oxidation (40).

Liver carnitine is observed to increase dramatically after oral carnitine

supplementation, which would suggest that the depletion observed prior to

treatment is due to low serum concentrations (48). Even though the

uptake defect is not corrected, the clinical response to high oral dose

carnitine supplementation is believed to occur because of the resultant

"flooding" of cells with carnitine which leads to a non-specific, low affinity,

diffusion of carnitine into the carnitine transporter deficient tissues by

mass effect, thereby bypassing the ' defec tive plasmalemmal carnitine

transporter (49).

Role(s) in Metabolism

Carnitine has been shown to have a number of important roles in

metabolism (50). One of its primary functions is to facilitate the

shuttling of long-chain fatty acids across the permeability barrier of the

inner mi tochondrial membrane. Another of its "shuttling" roles is in the

shuttling of the end products of peroxisomal fatty acid oxidation and the

shuttling of a-ketoacids derived from the metabolism of branched chain

arnino acids. As an esterifiable compound within the cytosol. it also

plays a role in the esterification of potentially toxic acyl-CoA metabolites.

These metabolites can impair the function of the citnc acid cycle.

gluconeogenesis. urea cycle and fatty acid oxidation. Perhaps its most

important role is in the modulation of the intramitochondrial acyl-

CoAIfree CoA ratio. This is done by exchange esterfication of acyl-CoA

and carnitine to form acylcmitine and free CoA. This reaction is freely

reversible and will allow the shift of CoA rnoieties from their acylated form

to free form. thereby creating a buffer system for not only the acyl groups,

but also for the free CoA used in the ce11 in a large number of biochemical

reactions, These roles are summarized in Fig. 2 (51).

The majority of the actions of carnitine are mediated through the

action of the camitine acyl transferases. These enzymes catal yze the

reaction:

Acyl-CoA + carni tine <---------- > acylcarnitine + free CoA.

The acyltransferases can be divided into three major groups depending on

their substrate specifici ty: carnitine acetyltransferase (CAT) for short

chah acyl groups; camitine octanoyltransferase (COT) for medium-chah

acyl groups; and carnitine palrnitoyltransferase (CPT) for long-chain acyl

groups. These enzymes appear to be mainly localized to the

mitochondria. peroxisomes and microsornes (52). Su bstrate specificity of

each group of enzymes varies between different species and tissues. but

can be generally designated as (53): CAT - C2-C4; COT - CS-CIO; and

CPT - C11-C20.

One of the more critical roles of camitine is in promotion ,of efficient

oxidation of long-chain fatty acids (4). Long-chah fatty acids are unable

to cross the permeability barrier of the inner mitochondrial membrane

without the process of esterification, translocation, and de-esterification

which is facilitated by carnitine. Al1 fatty acids, regardless of chain

length must be converted to fatty-acyl-CoA thioesters by the fatty-acyl-

CoA synthetases prior to participation in P-oxidation. The long chain

form (Cl0 - C20). which plays the role of initiating long chain fatty acid

oxidation, is membrane bound. either in the endoplasmic reticulum or the

outer mitochondrial membrane (54). The medium- and short-chain

synthetases are found primarily in the mitochondrial matrix (54). The

medium chain synthetase selects for C4 to Cl2 length precursors, while the

short-chain synthetase selects for acetate (C2) and propionate (C3). The

reaction and mechanism of this activation is shown below (55):

fatty-acid + ATP -------- > fatty-acyl-adenosine

fatty-acyl-adenosine + CoASH -------- > fatty-acyl-CoA + AMP

Using palmitate as an example of a typical long-chah fatty acid. the . --

process of long-chain fatty acid metabolism can be described as follows

(see Fig. 3 (12.56)). Initial activation of the fatty-acyl moiety accurs via

the fatty-acyl-CoA synthetase on the endoplasmic reticulum or outer

aspect of the outer mitochondrial membrane. The palmitoyl-CoA thus

formed is transported across the outer rnitochondrial membrane by an as

yet to be delineated system. Camitine palmitoyltransferase 1, on the

inner side of the outer mitochondrial membrane. converts the palmitoyl-

CoA and carnitine to palmitoyl-carnitine and free CoA via a

transesterification reaction. Translocation of palmitoylcarnitine~ across the

inner mitochondrial membrane occurs via the carnitine-acylcarnitine

translocase antiport system. Carnitine palmitoyltransferase II, on the

inner side of the inner mitochondrial membrane, converts the

palmitoylcarnitine into palmitoyl-CoA and carnitine via another

transesterification reaction. Finally palmitoyl-CoA enters the B-oxid ation

"spiral" via the long-chain acyl-CoA dehydrogenase, and carnitine returns

to the cytosol via the mitochondrial membrane based carnitine-

acylcarnitine translocase system.

P-ox ida t ion

P-oxidation occurs via a number of sequential reactions that take

place within the mitochondriai matrix. and is summarized below in Fig. 3

(1 2.56). The first reaction is catalyzed by the fatty acyl-CoA

dehydrogenases. This class of enzymes. like many belonging to the fatty-

acid metabolism family, is chain length specific (ahort. medium and Long)

and deficiencies of these enzymes- can lead to biochemically detectable

deficiency syndromes (short-chah acyl-CoA dehydrogenase deficiency

(SCAD), medium-chah acyl-CoA dehydrogenase deficiency (MCAD), and . -

long-chain acyl-CoA dehydrogenase deficiency (LCAD) (57). Deficiency in

the electron-transferring flavoprotein (ETF), which is used as an acceptor

of the electrons generated via the flavin adenine dinucleotide (FAD) linked

dehydrogenation. leads to a multiple acyl-CoA dehydrogenase deficiency

(MAD) (58). The acyl-CoA dehydrogenases catalyze the formation of a

double bond between the a- and P- carbons of the acyl-chain to form 2-

trans-enoyl-CoA. Following this, the double bond is hydrated (addition

of "water" across the double bond) via the enoyl-CoA hydratase enzyme, to

form L-3-hydroxyacyl-CoA which is then deh ydrogenated via the L-3-

hydroxyacyl-CoA deh ydrogenases. Deficiencies in these h ydroxyacyl-CoA

dehydrogenases can lead to definable clinical sydromes (short-chain L-3-

hydroxyacyl-CoA dehydrogenase deficiency (SCHAD), medium-chain L-3-

hydroxyacyl-CoA dehydrogenase deficiency (MCHAD), and long-chain L-3-

hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) or trifunctional

enzyme deficiency) (57). Finally, thiolytic cleavage of the 3-ketoacyl-CoA

occurs via thiolase. cleaving an acetyl-CoA group and creating an acyl chain

that is 2 carbon atoms shorter. Deficiency of the thiolase enzyme leads to

another clinically definable syndrome. One of the key sites of regulation

of long-chain fatty acid oxidation is through the specific and reversible

inhibition of CPT 1 by malonyl-CoA (59). Malonyl-CoA. the first

committed intermediate in fatty acyl biosynthesis, therefore. plays the

dual role of activating fatty acyl biosynthesis (as the product of the rate-

limiting step), and inhibition of fatty acid oxidation by preventing uptake

of palmitate in the mitochondrion.

One of the other very important roles for carnitine is in the buffering

of the short-chah acyl-CoA / CoA ratio (60). Initial studies . . .. by Pearson

and Tu bbs (1 967) suggested that the CAT (carnitine acetyl transferase)

system is near equilibrium and buffers acetyl-CoA / free-CoA, because

changes in metabolic state result in compensatory changes in carnitine

metabolism (61). Studies have shown that short-chain acylcarnitines

increase with fasting while free carnitine decreases (62). These effects

are further enhanced in diabetic ketosis where t h e increase in plasma

acylcarnitines and the decrease in free carnitine has been attributed to

insulin deficiency and glucagon excess (63). This change is likely

attributable to the increase in fatty acid oxidation, increased production of

acyl-CoA intermediates. increased esterification of carnitine (resulting in

decreased free carnitine), increased release of acylcarnitines from cells

and, eventually. increased urinary free carnitine excretion in exchange for

acylcarnitine reabsorption at the renal tubular site.

Pathophysiology of Fatty Acid Oxidation Defects

The most important and efficient fuel for oxidative metabolism is

fat. Within a few hours of fasting. the liver glycogen stores begin to be

depleted, and the predominant substrate for oxidation becomes fatty acids.

Fatty acids serve three major roles during fasting: 1) the partial

oxidation in liver of fatty acids to ketones produces an important auxiliary

fuel for al1 tissues. particularly brain; 2) fatty acids serve as a major fuel

source for cardiac and skeletal muscle; and 3) the high rates of hepatic

gluconeogenesis and ureagenesis used to maintain homeostasis are

sustained by the production of ATP. reducing equivalents and metabolic

intermediates derived €rom fatty acid oxidation (64). In children.

particularly infants. there is an earlier activation of fatty acid oxidation

(FAO) because: 1) there is an increased brain to body mass ratio

cornpared to adults, thereby causing a switch to F A 0 for ketogenesis much

earlier in fasting; 2) the surface-area to mass ratio is very high, resulting

in a much higher basal metabolic rate in order to maintain body

temperature (shivering thermogenesis is highly F A 0 dependent) (65); and

3) the activity of several key enzymes involved in energy production is

lower in infants, compared to older children or adults (66). In the

aggregate, these factors help to explain the particular metabolic

vulnerability of the newborn and young infant and therefore the increased

likelihood of the clinical presentation of fatty acid oxidation de@cts in the

early years of life.

Carnitine Deficiencies - Primary and Secondary

Carnitine deficiency disorders can be divided into two major

categories: primary and secondary (67). In the primary disorders, the

affected tissues show a profound reduction in carnitine concentration. In

the systemic form of Primary Carnitine Deficiency or the plasmalemmal

carnitine transporter defec t (hereafter referred to as PCD). serurn

concentrations of carnitine are also dramatically decreased.

The primary carni tine deficiency syndromes have been divided in to

myopathic and systemic forms (68). The myopathie form would likely be

characterized clinically by progressive muscle weakness. lipid storage

myopathy, normal serum carnitine concentrations and low muscle

carnitine concentrations. A specific defect in the carnitine transporter in

skeletal muscle would explain the i weakness. lipid storage in muscle.

normal serum carnitine concentration and low muscle concentration.

However, a recent study of a patient with this particular syndrome ..-

demonstrated a deficiency of short-chain acyl-CoA dehydrogenase (SCAD)

activity in muscle (69). Therefore a primary isolated muscle carnitine

transporter defect has not been documented to date.

The systemic fonn of primary carnitine deficiency (PCD) is currently

defined and characterized clinically by early infantile-onset with:

cardiomyopathy; hypotonia; recurren t h ypoketotic hypoglycemic

encephalopathy; weakness; very low serum carnitine concentrations and

low tissue carnitine concentrations, in which there is a demonstrated

defect in plasmalemmal carnitine uptake. and a dramatic response to high

dose oral carnitine supplementation (49). The transporter defect appears

to be expressed in skeletal muscle, heart. kidney, cultured skin fibroblasts.

and cultured lymphoblasts (40,70). A number of cases previously

described in the literature were not true cases of primary systemic

carnitine deficiency and were later shown to have other defects of fatty

acid oxidation such as MCAD deficiency (71). These cases have now been

re-classified as secondary carnitine deficiency sydromes. The

nomenclature of "systemic carnitine deficiency" is therefore reserved for

those cases which fulfil l the criteria outlined above and which. most

importantly , are characterized by carnitine-responsive cardiomyopathy.

The secondary carnitine deficiency States can be the result of

genetically-determined metabolic errors, acquired medical conditions, and

iatrogenic factorddrug therapy (72). These conditions may be

characterized biochemically as having either decreased tissue or serum

carnitine concentrations or an increased ratio of esterified to free carnitine

or both. The genetic conditions which affect carnitine are quite diverse,

but predominantly are involved with the metabolism of fatty acids or

amino acids. Historically, the conditions of isovaleric acidemia. propionic

acidemia, methylmalonic aciduria. and thiolase deficiency were the initial

disorders that were associated with disturbances in tissue or serum

carnitine concentrations. Today, the rnost distinctive organic aciduria

associated with secondary carnitine deficiency is medium-chain acyl-CoA

dehydrogenase (MCAD) deficiency (73).

The mechanisms of many of these disorders causing sec~ndary

carnitine deficiency have not yet been conclusively determined. Many of

these conditions have low plasma concentrations of carnitine, an increased

esterified carnitine to free carnitine ratio. and low tissue carnitine

concentrations (64). In a number of these conditions, there is an

excessive accumulation of acyl-CoAs in the mitochondria at the "expense"

of free CoA. By mass-action, the reaction of acyl-CoAs with carnitine is

pushed to form more acylcarnitines. This often leads to the finding of an

increased esterified fraction of carnitine in either serum or urine. The

patterns of chain length and type of carnitine esters found, which occur

proximal to the metabolic block in the in trami tochondrial F A 0 disorders,

can greatly aid the clinician i n making a specific enzymatic diagnosis (74).

An attractive and simple explanation for the carnitine deficiency

would be that because of the increased acylcarnitine formation a large

amount of acylcarnitines are being excreted in the urine and therefore the

body is constantly being depleted of its carnitine stores. However. there

have been no definitive

data would suggest that

decreased and therefore

quantitative studies to support this theory. Other

the renal threshold for free carnitine is greatly . -

free carnitine is not reabsorbed (75). A

mechanism which might explain this observation is that the increased

acylcarnitines in the renal filtrate act as cornpetitive inhibitors of free

carnitine uptake at the plasmalemmal carnitine transporter in the renal

tubular reabsorption site, ultimately leading to free carnitine excretion.

In-vitro studies of the carni tine transporter in fi broblasts support this

observation, by showing that acylcarnitines do in fact inhibit carnitine

uptake. where the KilICso concentrations for acetylcarnitine,

octanoylcarnitine and palmitoylcarnitine giving half-maximal ighibition of

L-(methyi-3H)-carnitine uptake are 4.6 t 0.5 PM. 2.9 t 0.4 PM, and 0.37 t

0.06 pM respcctively versus 3.05 + 0.31 pM with L-camitine (76).

In order to more fully understand the plasmalemmal carnitine

transporter defect and to be able to make more timely diagnoses,

characterization of the transporter at a basic biochemical and rnolecular

level is very important. To date. attempts to clone the transporter have

been unsuccessful. Knowledge of the transporter at the DNA level may aid

clinicians in making a more timely diagnosis (through easily performed

DNA testing), facilitate prenatal diagnosis and genetic counselling, as well

as aid in the understanding of the basic molecular defects responsible for

this disorder. As patients with the plasmalemmal carnitine transporter

defect are "exquisitely" treatable with high dose oral carnitine

supplementation, early diagnosis would lead to a dramatic reduction in

overall morbidity and mortality.

EXPERIMENTAL APPROACH I

The approach chosen to clone the plasmalemmal carnitine

transporter is one based on a "functional complernentation" strategy.

Functional complementation has a long history of use in bacterial and yeast

genetic systems. It is based on the premise that a diseased or mutant ce11

can be "corrected" if DNA from normal cells expressing the rnissing or

defective gene product is transfected into them. This premise is

sufficient if and only if the DNA source contains the desired DNA clone in

an intact and functional form. If the source is genomic DNA. the process is

limited primarily by the handling of DNA prior to and during library

construction (to prevent unnecessary random shearing). If the source is

isolated mRNA converted to cDNA, the process is limited by choice of

tissue/cells from which the mRNA is isolated and whether or not the

expression system chosen expresses the protein of interest properly

(location, amount. etc.). Without a "probe" for the desired DNA sequence,

this is a difficult premise to prove prior to actually doing the work of

isolating and sequencing a number of clones.

The use of functional complementation as a tool for genetic analysis

of human genetic disorders is relatively rare. For many years a form of

functional complementation has been used to determine the functional and

possibly genetic heterogeneity or homogeneity of certain diseases. In this

method, ce11 lines from a patient with a particular disorder are fused with

a ce11 line from a patient with the same chical and biochemical disorder

but whose specific molecular defect is known. After functional

complernentation. the disrppearance of the disease phenotype indicates

that functional complementation has occurred. A recent example of this

type of "complementation" cornes from the characterization of the genetic

subtypes of Fanconi's anemia, where €ive functional compiem~tation

groups have been established (77).

In the experimental protocol outlined here, the source of DNA is a

cDNA library constructed from normal lymphoblastoid ce11 mRNA and

incorporated into the pREP4 (Invitrogen) eukaryotic expression vector

(78). The pREP type vectors are based on an Epstein-Barr virus-based

system (79) and are maintained in the cell episomally (non-nuclear DNA

replicons) (80). The library used in this protocol has been previously

used to isolate the cDNA for the C-type of Fanconi's anemia (FA-C) (78).

The mRNA originally used ta construct the library was isolated from a

"normal" lymphoblastoid ceIl line. Lymphoblasts have been shown to

share the transporter defect (44). and therefore, at a minimum, express

the transporter protein. Use of functional complementation has allowed

the isolation of candidate cDNAs/genes for ataxia telangectasia (81). other

Fanconi's anemia subtypes (82) and xeroderma pigmentosum (83).

A search of the literature has shown that this approach has yet to be

used in diseases which affect intermediary metabolism. In the examples . . ..

discussed above, the selection process and the diseases themselves are

based on DNA synthesis andlor repair mechanisms (81,82,83). The

difficulty in using this method for intermediary metabolic diseases may be

due to the difficulty in isolating the desired clone from the "background"

clones obtained. An efficient selection rnethod is required to isolate the

clones which have had the defect corrected versus those that have not. in

order to minimize the nature and number of clones obtained. In many

cases, intermediary cellular rnetabolism has many redundant systems to

scavenge or alternately provide the necessary metabolic interpediates.

If it is possible to block those altemate pathways and force the ce11 to use

the affected pathway, then an efficient selection process can take place.

An example of this is the HAT selection medium (normal medium with

hypoxanthine, aminopterine, and thymidine) system used in cloning

hydrid cells. whereby aminopterin inhibits dihydrofolate reductase,

blocking de novo purine and th ymidylate synthesis. Cells which survive

have obtained both the TK (by-passes thymidylate block) and HGPRT (by-

passes purine synthesis block) genes through cell fusion (84).

The initial aim of this project was to develop an efficient selective

medium system whereby normal cells could be distinguished efficiently

from patient cells. After optimization of the selective medium conditions,

mutant carnitine transporter deficient cells would be transfected under

optimal conditions with the cDNA-library-vector construct and then

passaged through selective medium for varying lengths of time. After

isolation of surviving cells, the "selected" plasrnid DNA would be isolated

and a small portion transformed into competent E.coli cells. Individual

clones would be characterized and then partially sequenced. The . - ..

sequences would be analyzed for similarity and homology with the large

international DNA and protein databases and with the sequences of other

sodium-dependent membrane transporters and the catnitine

acyltransferase family of proteins. Elimination of those clones with high

sequence similarity andlor homology to known sequences in the databases

would leave a number of clones which would be re-transfected into patient

ce11 lines for the purpose of determining whether carnitine uptake is

res tored,

Protein-&und Lysine

(CH3)3N(CH2)3COOH y-butyrobetaine

If- NADH. H+

Figure 1 :

y-butyrobetaine aldehyde

Carnitine Biosynthesis

Enzymes: 1) protein (iysine) meîhyltransferase 2) 64-tmiethyliysine, 2-oxoglutarate dioxygenase 3) 3hydroxy-6-N-tMiethy Ilyshe aldolase 4) butyrobetaine aldehyde dehydrogenase 5) y.butyrobetaine, Z-oxoglutarate dioxygenase

1 acyl-CoA dehydrogenase (AD)

3-hydroxyacyl-CoA dehydrogenase (HADI

l- NADH, H+

SCAD MCAD LCAD VLCAD Multiple AD

crotonase

CoASH

thiolase

\ RCH~CH~COSCOA shortened fatty-acyl CoA

SCHAD MCHD LCHAD Trifunctional enzyme

de f iciency

thiolase

Figure 3: Cycle of lntramitoc hondrial f3sxidation and its Associated Oefects

Chapter 1:

SELECTIVE MEDIA DEVELOPMENT - - -

- - - - - - - - - - - - - - - -

7

Experimental Approach 5

Cell lines from patients with prirnary carnitine deficiency do not

metabolize long chain fatty acids well in the absence of high concentrations

of camitine supplementation (Tein and Xie, unpublished observations).

This basic biochemical observation provides a unique way for cells having

the genetic defect for primary carnitine deficiency (PCD) to be selected

against on the basis of growth response to long-chain fatty acids (LCFAs).

Prior work in Our lab (Tein and Xie, unpublished observations) have

shown that fibroblasts containing the PCD defect do not grow well in a-

MEM medium containing no serum supplementation, with 5 mM galactose

replacing glucose and supplemented with 100 pM BS A-palmitate. Using

this basic medium composition as a starting point, the nutritional

conditions of the media were further manipulated to obtain a final

selective medium for lymphoblasts. As a basic principal, the cells could

obtain their "dietary" metabolic fuel requiremen ts from three main sources

- fatty acids, carbohydrates and protein. In addition. manipulation of CO-

factor concentrations or inhibition of other basic biochemical processes

such as protein synthesis would also allow access to selection between

normal and PCD patient ce11 lines. Ideally, in this selective medium,

forcing the PCD ce11 lines to use only LCFAs as their prirnary fuel source

should cause either cessation of cell growth or ce11 death. Galactose was

added to the growth medium in order to provide a potential "slow-release"

rate-limiting source of glucose- 1 -phosphate as well as to maintain normal

glycoprotein structure and function within the cellular membranes. Use of

lower concentrations of galactose could provide further selection. Addition

of the BSA-palmitate supplement provides a source of relatively non-toxic

LCFAs. Experiments done in previous work demonstrated thgt at higher

concentrations of the BSA-palmitate supplement or when the palmitate

concentration exceeded the BSA concentration, the supplement proved to

be very toxic to al1 ce11 lines tested.

Further nutritional manipulation can be achieved by changing the

supply of amino acids. Amino acids are divided into 4 categories (85):

essential; non-essential; lipogenic; and glucogenic. Each arnino acid can

be classified as essential or non-essential, based on whether the organism

(in this case human) can synthesize it endogenously. and as lipogenic or

gluconeogenic (or both) based on whether it can supply substrates for

lipogenesis or gluconeogenesis. The table below shows the distribution of

the amino acids according to these characteristics.

TABLE 1: Characteristics of Amino Acids --

C h a r a c t e r i s t i c s Non-Essential - Gluconeogenic

Amino Acids asparagine, aspartate, glutamine,

Essential - Gluconeogenic

Those amino acids which have been determined to be essential cannot be

glu tamate. proline valine, methionine, histidine.

- - -- -

Essential - Lipogenic Non-Essential - Lipogenic and .;

Gluconeogenic Essential - Lipogenic and

Gluconeoeenic

manipulated in the growth medium. as lowering of the concentration or

deletion from the medium can or will cause ce11 death. In developing the

selective medium, the best amino acids to manipulate will be those that

are either non-essential gluconeogenic or non-essential lipogenic amino

tryptophan, leucine, lysine alanine. glycine. cysteine, - serine, t y r O s i n e threonine, isoleucine. ~henvlaianine

acids. With those that are non-essential giuconeogenic, ideally, dropping

them out of the medium (combined with the existing galactose and BSA-

palmitate conditions) will cause further dependence on LCFA oxidation for

oxidative fuel. Manipulation of the lipogenic amino acids, by increasing

the concentration will provide increased endogenously synthesized fatty

acids for oxidative metabolism.

Materials and Methods

(a) Ce11 Lines / Mriteriols:

Lymphoblastoid cell lines were obtained from the Human Genetic Ce11

Repository at the Hospital for Sick Children (Toronto, Canada). Al1 ce11

lines were assayed for carnitine uptake prior to use in experiments. The

results of the uptake experiments are published (44). Those cell lines

(L0002, L0011) demonstrating no measurable carnitine uptake were

designated as Primary Carnitine Deficient Cell (PCD) ce11 lines. Normal ce11

lines were those that demonstrated normal carnitine uptake (L0006,

L0009, L0005, L0017). Al1 othet materials. drugs and compounds were of

the highest grade available and were purchased from Sigma unless

otherwise stated.

(b) Cell Culture Methodology (media, techniques. etc.):

All ce11 lines were cultured from previously frozen (-800C) stock vials

of cells. After thawing on dry-ice for 15-30 min.. the cells were quickly

thawed by gently agitating tightly closed vials in a 370C water-bath. As

soon as the ice crystals had melted. the ceIl suspension was transferred

into a filter-top tissue culture flask (T25) which contained 10 ml of

medium. Normal culture medium for all lymphoblast cultures was RPMI-

1640 (University of Toronto Media Preparation Service) supplemented

with 20% Fetal Calf Serum (Cansera). The ce11 suspension was allowed to

grow to confluence (highly saturated cell suspension - approximately

2x106 cells/ml) prior to the addition of more medium or splitting to new

flasks.

(c) Cell Counting:

Cells were resuspended by repeated pipetting with 5 ml pipettes.

After adequate resuspension, the ceIl suspension was sampled by using a

flame sterilized pasteur pipette. The ce11 suspension was allowed to fil1 the

chambers of a haemocytometer ce11 counting slide by capillary action.

Typically, the 4 corner segments of each grid were counted in sequence

with the total number of cells being recorded. Actual ce11 concentration

was determined as: ( Total niimber counted ) x (10000 cells/ml)

4

(d ) Media Formulation: t

"Normal" medium was formulated by using a base of RPMI-1640 as

provided from the University of ,Toronto Tissue Culture Service. Fully

supplemented medium was made by using approximately 400 ml of base

RPMI-1640 medium and then adding 100 ml of fetal calf serum to bring

the supplementation to 20% fetal calf serum.

"Drop-Out" media (those lacking one or more of the amino acids

normally found in RPMI-1640) were formulated using the RPMI-1640

Select-Amine Kit (Gibco-BRL). Instructions given with the kit were

followed, except that the glucose and the specified amino acid(s) were

omitted. Galactose ( 5 0 mM) solution instead was used to give the media a

final galactose concentration of 5 mM. Individual "drop-out" media were

formulated and then filter sterilized (0.2 pm) prior to the addition of

previously sterilized BS A/palmitate supplement (see below), galactose,

serum and drugs. Addition of drugs or other compounds was done by

dissolving the drug in an appropriate solvent at as high a concentration as

possible. This solution was then diluted with PBS and added to ce11

cultures, or added directly (at al1 times keeping any solvent concentration

at <OS%).

The final selective medium formulation was as follows: base medium

- RPMI-1640 minus asparagine, with 5 mM galactose, 15% fetal calf serum,

100 pM BSA / 100 pM palmitate and Hygromycin B 40-80 pglml.

BSAlpalmitate was made as a 10X concentration stock solution by

dissolving 14.74 g fatty acid-free BSA in approx 90 ml sterile PBS. The

mixture was allowed to slowly dissolve overnight at 40C. After cornplete

dissolution had taken place. the palmitate was added (2.2 ml, 50 m M

palmitic acid in ethanol). After absorption of palmitate ont0 the BSA

(ovemight at 4W). the volume was brought to 110 ml, and the solution was

filter stedized. Galactose was made as a lOOX concentration (500 mM)

stock solution in PBS. Hygromycin B (Calbiochem) was made in stock

concentrations of 1 0 . or 1 .O m g h l in PBS, filter sterilized and then added

to the medium to give the desired final concentration.

Results

The development of the selective medium for distinguishing PCD

from normal control ce11 lines was done through progressive manipulation

of the nutritional conditions of the medium. Initial experiments were

perfomed using either one or two PCD ce11 lines and one or two normal

control ce11 lines. Final experiments were done, in triplicate, using both

available PCD ce11 lines and four normal control ce11 lines.

(a) Manipulation of Fetd Caif Serurn Concentration:

Manipulation of the serum requirements for ceIl growth was studied

first. Initial experiments (Figs. 1 and 2) determined the growth

characteristics of the patient and normal control ce11 lines in different

serum concentrations (20%. 10%. and 5%) in galactose media conditions

(RPMI with galactose replacement), both in the absense and presence of

the BSA-palmitate supplement (final BSA/palmitate concentration =

100 pM1100 PM). In the absense of the BSA-palmitate supplement

(Fig. I), the PCD ce11 line (LW1 13. grew at a slower rate than the normal

control ce11 line (L0006). At the lowest semm concentration tested (5%)

the patient cells had an approximately 3-4 fold slower growth rate than

controls. In the presence of the BSA-palmitate supplement. the PCD and

normal control ce11 lines declined rapidly in the lowest serum

concentration (5%) tested (data not shown). With 10% fetal calf serum

added (Fig. 2), both .ce11 lines grew minimally, but did not decline as in the

5% fetal calf serum added conditions. At a serum concentration of 20%. a

significant differentiation in proliferation rate was observed (normal

control : patient ratio = approximately 29). As a compromise between

the differentiation observed in 20% fetal calf serum and the lac& of growth

in 10%. a final value of 15% serum was used for further expetiments.

(b) Manipulation of Galactose and Phosphate Concentrations:

A set of experiments were performed where the carbohydrate

component concentration and the phosphate concentration in the medium

were manipulated individually. The carbohydrate was manipulated by

reducing the amount of galactose added to the medium (5.0, 2.5. 1 .O,

0.5 mM). The phosphate concentration (and secondarily ATP) of the ceIl

was manipulated by increasing or decreasing the amount of phosphate

added to the medium formulation (2.0~. 1.0~. 0 . 5 ~ ~ 0 . 1 ~ normal

concentration). Results of these experiments showed that manipulation of

either of these two quanitities did not result in an increased degree of

selection between the PCD and normal control ce11 lines (data not shown).

(c) Manipulation of Amino Acid Composition:

The next set of experiments:determined the effect that exclusion of

particular amino acids from the growth medium would have on

differentiation of the PCD from the normal control cell lines. Ali amino

acids which were considered essential were added to al1 media prepared.

Drop-out media excluding each of the non-essential gluconeogenic and

lipogenic amino acids alone andlor in combination (asparagine. aspartate,

glutamine, glutamate. proline, alanine, glycine, cysteine. serine and

tyrosine) were prepared, as described above, with galactose replacement,

15% fetal calf serum serum and BSA-palmitate (100pM1100pM) added.

The cells were plated into the "drop-out" selective media and the results

are shown in Table 2 below.

This initial screen suggested that selection was best

and normal control ce11 lines with drop-out of asparagine

between the PCD

only, aspartate

only, asparagine plus aspartate, and glutamate only. Results of

experiments with proline, alanine, glycine and tyrosine showed no increase

in the selection between PCD and normal control ce11 lines. Repetition of

the experiments and cornparison of results in different cell lines led to the

identification of the "asparagine-only" drop-out medium as the most

consistent and reproducible in terms of growth characteristics and growth

differentiation (Figs. 3. 4. and 5). Figs. 3 and 4 show the results of two of

the studies done to confirm the reproducibility and consistency of the

different amino acid drop out conditions. From these two studies, and

other duplicated studies. it was concluded that the best and most

consistent growth differentiation between control and PCD ceIl lines was

TABLE 2: Results/Observations Amino Acid /

Combination Droo-Out Glutamine Glutamic Acid

Asparagine

AsDartic Acid Cysteine Serine Asparagine and Glu tamine

Aspartic Acid and Glutamine

Glutamine and Glutamic Acid As~aranine and AsDartic Acid

of Amino Acid Drop-Out Stuc

R e s u l t -neither ce11 line grew well -growth rate of normal vs. PCD is

approximately 4X higher -normal vs. PCD rate = 4X -more consistent results in

multiple studies -normal vs. PCD rate = 3.5X -normal vs. PCD rate = 4.OX -normal vs. PCD rate = 2.5X -bath lines grew marginally, then

declined rapidly -both lines grew marginally, then

declined rapidly -minimal growth observed -normal vs. PCD rate = 3SX

achieved with the exclusion of asparagine from the medium. Tfie results

presented in Fig. 5, for the serine and glutamine drop-out media,

demonstrate that the differentiation in growth rates between the control

and PCD ce11 lines is less than that observed in the asparagine drop-out

medium.

(d) "Carnitine Rescue" - Effect of Carnitine Supplementation:

To determine whether or not the growth inhibition observed in the

asparagine drop-ou t medium could be restored with carnitine

supplementation, carnitine was added to the asparagine drop-out selective

medium and the gtowth rate was observed in one normal control (L0006)

and one PCD ce11 line (L0011). Figs. 6 and 7 show that an increase in the

initial growth rate is observed in the PCD ceIl line (LOO1 1). and not in the

normal control ce11 line (L0006). The slightly higher growth rate

observed for the PCD cells in these experirnents comprred to the previous

results (Fig. 4) might be due to the stage of growth of the cells pnor to

plating (as has been observed previously) or due to slight variations in

medium composition. In previous experiments where growth was

monitored for long periods of tirne (14 days or more), eventually a plateau . ,

of ce11 population was reached, followed by a fairly rapid decrease in ce11

nurnber (likely due to toxic metabolite accumulation andlor cytolysis).

Therefore, the sharp downward deflection of the curves in Fig. 6 is likely

due a large increase .in growth rate, followed by a growth plateau and

subsequent cytolytic loss of cells. It appears that carnitine, at least in the

control lines, exhibits a dose dependent effect on the growth rates

observed, whereby the higher carnitine concentrations may shorten the

time for the growth plateau to be reached. Therefore more cytolysis (and

fewer ce11 numbers) would be observed at the final time point.

(e) Effect of Hygromycin B and orher Antibiotics:

In preparation for transfections, killing curves for the addition of

dmgs to the medium were perforrned. Because of the need to add

Hygromycin B (Hyg B) to either normal medium or selective medium for

selection of cells containing the pREP4-cDNA constructs. killing curves were

deveioped for both media conditions. In the process. a serendipitous

discovery was made. The PCD patient ce11 lines appeared to have a rnuch

higher sensitivity to the drug than the normal control ce11 lines at both 7 or

14 days when assayed in normal medium (7 day data not shown). This

effect was even more pronounced in the selective medium developed to

this point in time. Figs. 8 and 9 show the results of the addition of

Hygromycin B to growing ceIl cultures. Exposure was allowed to occur for

14 days, and 9b ce11 survival was assayed by the MTT method (see Method

- Chapter 2, p. 49). In normal -.growth medium, the ICso (concentration

at which 50% survival is observed) of the normal control ceIl lines (L0006,

L0009) is approximately 100 and 140 pg/ml respectively. and for the PCD

ce11 lines (L0002, L0011) is approxirnately 40 pg/rnl. In thé selective

medium (RPMI minus asparagine, with 5 mM galactose, 15% fetal calf

serum. 100 PM BSA-palmitate). both normal control ceIl lines have an ICso

of 140 pg/ml and the PCD ce11 lines (L0002, Lûû11) have an ICSO of 30 and

50 pg/ml respectively. Comparing the results in normal and selective

medium, very slight differences are obsewed for the ICso values of the

two PCD cell lines. A significant decrease in sensitivity (from 100 to 140

pg/rnL) is observed for one of the control ceIl lines (L0006) when assayed

in selective medium. Since the cells would be transferred for long term

culture into the selective medium, and since this is where "selection" would

take place, the Hygromycin B effect on cell survival in the selective

medium is the more important of the results presented.

After the identification of Hygromycin B as an agent which further

increases the growth selection between the normal control cells and the

PCD ce11 lines, a search for other drugs having a similar mechanism of

action was undertaken. Hygromycin B is an aminocyclitol that acts on

cells through inhibition of protein synthesis by binding to ribosomes and

by disrupting translocation and promoting mistranslation (86.87). S ince

the vector which holds out library, namely pREP4, contains a hygromycin

resistance gene, it would be useful to find another drug with a sirnilar

mode of action to replace the Hygromycin B in the selective medium.

Inhibitors of protein synthesis (tetracycline, ch loramphenicol, kanamycin,

geneticinlG418) and one ATP depleting agent (oligomycin) were screened

at different concentrations. AI1 of the drugs tested. except Hygromycin B,

gave similar

lines tested

against PCD

ICso concentrations in both the normal control and PCD ce11

(graphs not shown), and thereby did not increase the selection

ce11 lines.

(f) Final Selective Medium:

The final selective medium formulation therefore was varied slightly

according to the ce11 line k i n g testedhsed. The basic formulation was:

RPMI-1640 without asparagine; with 5 m M galactose; 15% fetal calf

serum; 100 pM BSA - 100 pM palmitate; and Hygromycin B - 40 pg/ml

(for L0011) and 80 pglml (for L0002). Final experiments to show the

ability of the selective medium to adequately differentiate between the

normal control and PCD ce11 lines is shown in Figs. 10. 11. and 12. In Fig.

10 (without Hygromycin B). the PCD ce11 lines appear to be able to continue

to grow at a very slow rate. In Fig. 11, the LOO1 1 PCD ce11 line shows little

or no growth, while the LOO02 PCD ce11 line grows at a very slow rate in 40

pg /d of Hygromycin B. With the addition of 80 pg/ml of Hygromycin B to

the medium (Fig. 12), both PCD cell lines showed little or no growth, while

the normal control ce11 lines appeared to grow reasonably well. One line

(L0006) was observed to grow very rapidly. then "plateau". followed by a

decrease in cell number.

Discussion

In order to develop an effective cloning strategy that is based on

expression of a particular phenotype, one musc develop a method to

differentiate between those clones which are and are not expressing the

phenotype. In the case of functional complementation based cloning

strategies this is absolutely essentid, otherwise an unacceptably high

number of "background" clones will be selected and assayed. The assay

process is time consuming and xostly and therefore the fewer the number

of clones which are selected and screened in full, the better. Therefore,

an effective selective growth medium, which allows for the "enrichment"

(increased growth) of those cells which have the defect corrected over

those that do not (decreased or no growth), is absolutely essential.

The final selective medium developed in these experiments (Figs. 10

- 12) exploits the biochemical abnormality of the PCD ce11 lines. PCD ce11

lines do not grow well in a medium where the availability of the preferred

bioenergetic substrate, namely glucose, is limited due to replacement with

galactose, and where the primary bioenergetic substrate is thellong-chah

fatty acid palmitate, which they cannot efficiently metabolize. This

observation could be logically predicted based on the minimal intracellular

cmitine contained in PCD cells. With minimal intracellular carnitine, the

ce11 would be unable to translocate palmitate as palmitoylcarnitine across

the rnitochondrial membrane for ensuant intramitochondrial j3-oxidation

for adequate ATP production. These PCD cell lines are unable to

accumulate carnitine inside the cytosol. This was directly demonstrated

by assays where 3~-carnitine uptake across the plasma membrane of the

affected lymphoblasts was found to be severel y reduced (44).

As shown in Figs. 3. 4, and 5. a basic selection is observed between

PCD ce11 lines and normal control ce11 lines. Presurnably, those ceIl lines

that have other fatty acid oxidation defects would also have slow growth

rates in this selec tive medium. This selective medium should

preferentially select against long-chain fatty acid oxidation defects (eg.

CPT 1, CPT 2, LCAD, LCHAD deficiences as well as the carnitine transporter

defect). Wi th the addition of palmi ta te (a representative LCFA). the

severity of the effect would likely be related to the proximity of the defect

to the fatty acid entry point into:; p-oxidation, which in this case would be: .-.

the primary carnitine transporter defect = long chain defects > medium

chain defects > short chain defects. In addition those cells unable to

convert galactose into glucose-1-phosphate (inherited disorders of

galactose metabolism) via the reactions catalyzed by: galactokinase;

galactose- 1 -phosphate uridyl transferase; and uridine di phosphate-

galactose-4-epimerase, would also have impaired growth rates. The most

common of the galactosemir defects is the uridyltransferase reaction step

(88). The slow conversion of galactose to glucose-1-phosphate would

significantly lower the availability of glucose for glycolytic flow, and

therefore would force the cells to become metabolically dependent upon

long chain fatty acid oxidation.

The removal of asparagine from the medium caused an increased

differentiation in the growth velocity of PCD versus normal control ce11

lines. This increased differentiation might be explained by several

mechanisms. Asparagine is a gluconeogenic amino acid, and therefore

removal of asparagine from the medium would result in a decrease in the

availability of gluconeogenic substrates. One example of growth inhibition

due to an asparagine-deficient medium has been provided by the use of L-

asparaginase in certain leukemias (89). L-asparaginase is an enzyme

which hydrolyzes asparagine. Asparagine is considered to be a non-

essential amino acid but certain leukemias and other malignancies are

unable to synthesize asparagine due to a lack of asparagine synthetase

activity (89). Therefore, asparagine becomes an essen tial amino acid for

these cells which are then dependent upon extracellular sources of

asparagine to complete protein syothesis. When L-asparaginase activity

depletes asparagine in plasma, these leukemic cells are unable to derive

asparagine from extracellular sources to maintain cell viability, thereby

providing the basis for the selectivity of L-asparaginase against malignant

cells. Asparagine also has a role in providing the cell with oxaloacetate.

Asparagine is hydrolyzed to aspartic acid via the asparaginase reaction.

Aspartate then donates its amino group to a-ketoglutarate in a

transmination reaction to yield glutamate and oxaloacetate. Oxaloacetate

is also generated in the citric acid cycle and anapleurotically from

pyruvate via pyruvate carboxylase (PC). However, lymphocytes have

been shown to have very limited PC activity (90). This reduced PC

activity in lymphocytes combined with the removal of asparagine €rom the

medium, as well as the reduced glycolytic flux secondary to galactose

replacement of glucose in the medium, in the aggregate, would

significantly reduce the oxaloacetate levels in the cells. In normal cells

this is likely compensated by an increase in P-oxidation which provides

acetyl-CoA to the citric acid cycle for the generation of ATP and reducing

equivalents. Growth of PCD ce11 lines would therefore be inhibited

because of insufficient compensatory fboxidation for the generation of

ATP, in the setting of rate-limiting glycolysis and gluconeogenesis and a

substrate-limited citric acid cycle.

In order to test whether or not the selection process i s based on the

carnitine deficiency , a carni ti ne "rescue" experimen t was performed. Fig . 6 shows that the growth rate of the normal control cells is basically

unaffected by the addition of carnitine to the selective medium. However.

when camitine is added to the PCD ce11 line growing in the asparagine

drop-out selective medium, an approximately 40-5056 increase in growth

rate is observed. This would suggest that the growth medium developed

to this point is selective against the intracellular carnitine deficiency which

develops in the PCD ce11 lines. A further experirnent which should have

been performed to test the specificity of the selective medium would have

been to perform a carnitine rescue experiment once the Hygromycin had

been added to the medium.

With the discovery chat the PCD ce11 line is more sensitive to

Hygromycin B than the normal control cell line. a new way to further

"select" against PCD cells was discovered. The mechanism for this further

selection is unclear. Because of the inability of the PCD ce11 lines to

metabolize long-chain fatty acids efficiently, these fatty acids or theit

metabolites (fatty-acyl carnitine esters or fatty-acyl glycine esjers) may

accumulate and may eventually become toxic to the cells. High levels of

long-chain acylcarnitines may predispose the ce11 to lipid membrane

peroxidative injury (91). The ce11 would respond by initially turning on

the cellular detoxification mac hinery followed by the reaction pathway

which initiates ce11 death by apoptosis or necrosis. Hygrornycin B is an

aminocyclitoi that causes cell toxicity by binding to ribosomes and

inhibiting ptotein synthesis (86,87). At concentrations below the IC50, the

ce11 is able to either detoxify the Hygromycin B. or is able to bypass the

protein synthesis inhibition by increasing the rate of protein synthesis.

Once the IC5o is reached or exceeded. a higher percentage of proteins are

either not sythesized or sythesized incorrectly. This drug-induced effect,

coupled with the asparagine "drop-out". could be sufficient. in the PCD ce11

lines, to begin the signal transduction process which eventually leads to

ce11 death. Whether or not the two processes of fiitty acid mediated

toxicity and Hygromycin B mediated toxicity act by necrotic means or by

initiating a similar part of the apoptosis pathway remains

deterrnined.

In the next phase of the .experiments. the cells will

with a cDNA library which is borne in the pREP4 vector.

to be

be transfected . - -

This vector is an

episomally maintained vector which contains a segment of the Epstein-

Barr Virus (EBV) parental genome (oriP - origin of plasmid replication) and

the EBV encoded nuclear antigen-l (EBNA-1). The vector also contains

the "strong" Rous Sarcoma Virus (RSV) promoter which drives the

expression of the inserted cDNA and the SV40 promoter which drives the

expression of the Hygrornycin resistance gene. In addition to the

Hygromycin resistance gene, used for selection in eukaryotic cells, the

vector also contains the p-lactamase gene which confers ampicillin

resistance in E.coli. The presence of the Hygromycin resistance gene in

this vector poses a problem for the selection process designed. The

degree to which the resistance marker will affect the specificity of the

selection process has not been determined. Once the cDNA library is

vansfected into the PCD ceIl line(s), the cells will be selected for the

presence of the plasmid by growing the cells in normal medium with

Hygromycin B present, followed by passage into selective medium. If a

ratio of the increase in ce11 number of normal control versus PCD cells can

be used as a rnarker for the rate of selection that i s occurring i n the ce11

population, then a marked difference in growth rates is seen between the

O and 80 pg/ml Hygromycin B medium conditions. In O pglml

Hygrornycin B, the average increase in ce11 nurnber for control and PCD

cells is 2 . 5 ~ and l . l x per day respectively, giving a ratio of approximately

2.3:l. At 80 pg/ml of Hygromycin B. the average increase in ce11 number

for control and PCD cells is 2 . 4 ~ and 0.06~ per day. respectively, giving a

selection ratio of 40: 1. Therefore. the 80 pglml Hygromycin B

concentration will produce a more npid and "cleaner" selection (i.e. those

clones correcting the defect will be visible above background sooner) than

the O pg/ml Hygromycin B concentration. However, even if acquisition of

the Hygrornycin resistance gene decreases the effect of Hygromycin B on

the selection. a selection will still continue to occur, albeit at a slower rate.

Figure 1: Fetal Calf Serum-% Reduction Study without BSA- 3 8 palmitate Supplementation for Normal Control (L0006) and PCD (L0011) Cells (RPMI with 5 m M Gal.)

D a y s

Figure 2: Fetal Calf Serum-% Reduction Study with 100 pM BSA-palmitate Supplementation tor Normal Control (L0006) and PCD (L0011) Cells (RPMI with 5 m M Gal.)

0 2 4 6 8

D a y s

Figure 3: ~omparison of Growth of Normal Control (LOO061 and PCD 3 9 (~00i1) CeIl Lines in Different Amino acid ~ r o ~ - ~ u t Media Conditions (RPMI with 1596 serum, S m M Gal. and 100 p M

D a y s

Figure 4: Cornparison of Growth Characteristics (L0006) and PCD (LOOf1) Cell Llnes in acid Drop-Out Media Conditions (RPMI 5 m M Cal. and 100 pM BSA-palmitate)

of Normal Control Diffetent Amino with 15% serum,

Glu-Control

Asp-Control

Asn-Conttol

AsplAsn-Conuol

Glu-PQ)

4-PQ)

Asn-PCD

Asp/Asn-PCD

D a y s

Figure 5: Cornparison of Growth Characteristics of Normal Control (L0006) and PCD (L0011) Cell Lines in Serine, Cystelne, and Glutamine Drop-Out Media conditions (RPMI with 15% serum, 5 m M Cal. and 100 CM BSA-palmitate)

2 4 6

D a y s

Figure 6: Carnitine Rescue - Growth of Normal Control Cell Line (L0006) in Asn Drop-Out Medium (RPMI with 15% serum, 4 1 5 m M Gai. and 100 pM BSA-palmitate) for increasing carni t ine concentrations

OuMCamadded - 1ouMCamadded -1 SOUMCarnadded - 500 uM Cam added

O 2 4 6 8

D a y s

Figure 7: Carnitine Rescue - Growth of PCD Cell Line (L0011) in Asn D ~ o D - O U ~ Medium (RPMI with 15% serum, 5 m M Cal. and 100'pM ~ ~ ~ - ~ a l m Ï t a t e ) for increasing carnitine c o n c e n t r a t i o n s

O uM Cam added

10 uM C m added 50 uM C m added 500 uM C m a d M

O 2 4 6 8

D a y s

Figure 8: Hygromycin B Sensitivity of PCD (L0002, L0011) and Normal Control (L0006, L0009) Ce11 Lines in Normal Medium (RPMI with 2096 serua) following 14 days of Aygromycin B exposure

120

œ-*-. m - m 2

O-+ - . PCD-LOO 1 1

Control-LOO06

Conuol-LBOO9

. O . I . I . # 1 r I

O 100 200 300 400 500 600

Aygromycin B Conc. (uglml)

Figure 9: Hygromycin B Sensitivitj of PCD (L0002, LOOll) and Normal Control (L0006, L0009) Cell Lines in Seltctlve Medium (RPMI, Asa Drop-Out, 15% Serum, 5 mM Gai. and 100 pM BSA-palmitate) following 14 days of Hygromycin B e x p o s u r e

140 . .

..-

.-*o. PCD-Loo02

-,*W. PCD-Loo11

--c-. Conuol-Lod - Conuol-Loo09

O I I I I 1 . \

O 1 O0 200 300 400 500 600

Hygromyein B Conc. (uglml)

Figure 10: PCD Cell Lines (M002, L0011) and Normal Control Cell Lines (L0006, L0009, L0005, LOO17): Growth in Selective

4 3

Medium (RPMI, Asn Drop-Out, 15% serum, 5 mM Gal., - 100 pM BSA-Palmitate)-with O pglml Hygromycin i B

Days in Selective Medium

Figure 11: PCD Cell Lines (LOO02, L0011) and Normal Control Cell Lines (L0006, L0009, LOOOS, L0017): Growth in Selective Medium (RPMI, Asn Drop-Out, 15% serum, 5 m M Gai., 100 pM BSA-Palmitate) witb 40 pglml Hggrompcin B

O 2 4 6 8 1 O 1 2

Days in Selective Medium

Figure 12: PCD Cell Lines (LOOOZ, UOll) and Normal Control Cell Lines (LOOO6, L0009, L0005, L0017): Growth in Selective Medium (RPMI, Asa Drop-Out, 15% serum, 5 mM Cal., 100 p M BSA-Palmitate) with 80 pglmi Hygromycin B

Days in Selective Medium

Chapter 2: -

FUNCTIONAL COMPLEMENTATION CLONING

Experimental Approach i

The use of functional complementation as a cloning strategy has been

part of the standard repertoire of microbiologists and microhial geneticists

for a number of years because of the ease of manipulation of the system.

As detailed in the Experimental Approach section above (pp.14-16), the

use of functional complementation as a cloning strategy in eukaryotic

systems is relatively new, and has been limited to diseases which affect

DNA or RNA synthesis or repair.

i

After development of a selective medium (Chapter 1). the general

approach for the cloning project is as follows: obtaining and

characterizing a suitable cDNA library; choosing and optimizing

transfection conditions; transfection of cell line with cDNA library ;

selection (growth of cells in selective medium); isolation of remaining

cells/DNA; isolation and characterization of individual colonies;

sequencing of individual clones; characterization of sequence

information; elimination of "close~match" clones with no "interesting"

homologies; large-scale prep of remaining clones; re-transfection of cells

with "sets" of DNA; functional carnitine uptake assay; and final

characterization of remaining "lead" clones.

The cDNA library used for this project was obtained as a gift from Dr.

F. Merante €rom the .laboratory of Dr. B. H. Robinson and was originally

prepared by the laboratory of Dr. M. Buchwald (78). The library was

prepared using a vector primed synthesis strategy and isolated mRNA, in

order to enhance the yield of full length inserts oriented 5'-to-3' with

respect to the RSV-LTR promoter and the SV40 polyadenylation signal

respectively. The mRNA was obtained from a normal lymphoblastoid ce11

line and purified through two rounds of oligo-dT chromatography. The

vector used. pREP4. is an Epstein-Barr virus-based expression shuttle

vector. These vectors are maintained episomally (80). and the plasmids

obtained after selection can be easily shuttled into E.coli. One drawback

of this type of vector is that plasmids can be maintained in the

lymphoblast cells in the absence of direct selection. because the EBV

replicon contained in the vector is so efficient (79). A map of the vector,

including the restriction sites introduced by means of the vector-primed

synthesis strategy as well as the location and orientation of the insert, is

presented as Fig. 1.

The cDNA library construct used for this project contains a

Hygromycin B resistance gene (see Fig. 1). The selective medium

developed also contains Hygromycin B (see Chapter 1). In the

development of the selective medium. a number of other drugs were

tested (with a similar rnechanism bf action) and none provided the same

degree of selection provided by Hygromycin B. In the absence of

Hygromycin B. a selection between normal control and PCD ce11 lines was

still observed (see Chapter 1 - Figs. 10-12). The approach which would

provide the "cleanest" selection would be to have a vector with another

resistance gene on it (eg. histidinol or geneticin). In this case, some

selection will still occur in the selective growth medium, regardless of the

presence of the Hygromycin B resistance gene on the vector, because the

PCD cell lines are still basically defective in long chain fatty acid oxidation

and do not survive well in the glucose-free, palmitate supplemented,

asparagine drop-ou t medium.

After growth in the selective medium, isolation of cells and DNA, and

shuttling of plasmid DNA into E.coli cells, the individual clones are

characterized by restriction digest analysis with BamH1. From the map

shown in Fig. 1, it can be seen that the full length and therefore size of the

clone would be obtained by digestion with BamH1, except when a BarnHl

site is contained internally in the insert. Those clones which were 0.8 kb

and larger were sequenced. Initially, sequencing was attempted by using

the standard technique of dideoxynucleotide-based labelling using the kits

available through Pharmacia (T7 polymerase) and United States

Biochemical (Sequenase), but neither produced satisfactory results.

Finally, the sequencing was performed using a thermal cycling protocol

(Thermo Sequenase), which was commercially available through

Amersham. This protocol uses two thermal cycling runs to generate the

final labelled product. The first run is done in dNTP lirnited reaction

conditions in order to generate labelled "primers" which have been

lengthened slightly by the process. For the second cycling run, the dNTP

concentration is markedly increased, ' and the final labelled DNA is

produced.

Following gel electrophoresis, x-ray film exposure and reading of the

sequence. the sequence is inputted into a DNA sequence handling software

package. The sequence thus obtained is compared with al1 "known" DNA

sequences by using the e-mail accessible BLAST search engine (92) and the

Genbank database available at the National Institute of Health, USA.

From the output of the search, a number of key pieces of information can

be obtained. Firstly, the identity of a particular clone/DNA sequence can

be identified if the "P" value is very low. The "P" value estimates the

probability that the "query" sequence and the database sequenw are "not"

identical. If the "P" value is very low (40-5). then there is a strong

likelihood that the sequences are identical. If this "identity" is only for a

small number of the total bases compared, then the sequence obtained

from the database may correspond to a functional motif (such as a

membrane spanning domain, an ion translocation channel, or a binding

site). Those sequences which had strong identity over a large portion of

the queried sequence were discarded. The remaining clones were, and

are continuing to be. characterized by further sequencing , database

searching and functional analysis.

Materials and Methods

(a) M ï T Assay: a

Ce11 survival was assayed by using a modified MTT cytoproliferation

assay (modified from Mossman (93)). Btiefly, cells were resuspended

and 100 p1 of ceIl suspension was transferred to a 96-well plate (Costar).

MTT solution (1011 of 5 mglmlb. 3-(4.5-dimethylthiazol-2-yl)2.5-diphenyl

tetrazolium bromide in PBS) was added and the plates were incubated for

4 hr at 5% CO2 and 370C. Hydrochloric acid in isopropanol (100~1 of

0.04N HCl in isopropanol) was added and the resulting suspension was

pipetted vigorously to mix the contents well. . Absorbance at 570 and 630

nm was determined on an automated 96-well plate reading apparatus.

Transformation of the MTT dye from yellow to purple, with a subsequent

higher absorbance, was indicative of the presence of live cells. Cell

survival was calculated as the ratio of absorbance ( A s ~ o - A ~ ~ o ) for treated

cells versus untreated cells multiplied by 100%. To prepare a standard

curve. a dilution series of cells was plated into 96-weli plates and then the

full MTT assay was carried out.

(b) Transfection Optirnizution:

Transfection was optimized so that an electroporation apparatus

(BioRad GenePulser) could be used. Cells were isolated by centrifugation

(800 rpm / 10 min., Hettich) from cultures which were grown to a

population of 1 -2x 106 cells/ml and were resuspended in RPMI- 1640 with

20% FCS (fetal calf serum) supplernentation at approximately j9xl06

cellsld. Ce11 suspension (approximately 1 .O mllcuvette) was transferred

to electroporation cuvettes. stored on ice for 10 minutes. electroporated.

stored on ice for a further 10 minutes and then aseptically transferred to a

T25 flask containing 9.0 ml of RPMI-1640 (20% FCS). Electroporation was

camied out at various voltagelcapacitance combinations in order to achieve

a condition that kills between 20430% of the input cells (suggested

conditions by manufacturer of electroporation apparatus). Cells were

incubated at 5% CO2 1 370C for 2-3' days and then assayed for survival.

(c) Transfection with Library:

Using the optimal conditions determined above (500 pF / 250 V).

both patient lines (LOO02 and LOO1 1 ) were transfected with the library.

Bnefiy, 20 pg of the cDNA library construct was transferred into a sterile

plastic culture tube. . Ce11 suspension (1.0 ml as prepared above) was

added, mixed gently and transferred to an electroporation cuvette, stored

on ice for 10 minutes, electroporated, stored on ice for 10 minutes and

then transferred to a flask containing RPMI-1640 with 20% FCS. Initial

growthlproliferation was allowed to occur for 2-5 days in "normal"

medium prior to transfer into selective medium.

(d) Passagdsefection of Cells:

After 2-5 days in normal medium, transfected cells were transferred

to a sterile centrifuge tube and then centrifuged (1200 rpm, 7-10 min.).

After aspiration of the supernatant, the pellet was resuspended in

selective medium (usually 10 ml). Selection was allowed to proceed by

growth in this selective medium for varying pends of time. I Following

"selection", the cells were either centrifuged and resuspended in normal

medium (for a "rest"/recovery period) or passaged continuously in the

selective medium (slightly expanding medium volume each time).

r

(e) Isolation of CelldDNA :

Cells were isolated by centrifugation (800 rpm, 10 min.), followed by

aspiration of the supernatan t (ce11 debris, remaining DNA, etc.). Ce1 1

pellets were stored at -800C until prdcessed for DNA isolation. DNA

isolation was performed according to the alkaline lysis-precipitation

protocol generally used for bacterial cells (described in detail below). For

this application, 200 pl lysis buffer (50 mM glucpse, 10 mM EDTA, 5 mM

Tris, pH 8.0 and 2.0 mglml lysozyme), 400 pl NaOH/SDS (0.2N NaOH, 1%

SDS), and 400 11 3M sodium acetate neutralization buffer (pH 4.9) were

used, followed by isopropanol precipi tation, 70% ethanol wash,

lyophilization, and resuspension in TE buffer(l0 m M Tris pH 8.0, 1 mM

EDTA).

If) PCR for Selection: Z

To confim that selection did in fact occur. polymerization chain

reaction (PCR) was perfomed on a small sarnple of the plasmid DNA

obtained €rom the lymphoblast cells. Primers were used that amplified

from the RSV-LTR motif (S'-end) and from the SV40-pA motif (3'-end).

Reactions were set-up by preparing a cocktail of the reaction cornponents

as follows (quantity for each 50pl reaction): 10X reaction buffer (5 1 1 of

10X stock, 1X final concentration); MgCl2 (3 pl of 25 mM, 1.5 mM final);

dNTP nucleotide (4 pl of 25 mM. 2 mM final); primers (0.5 pliof 0.5 pg/ml.

0.005 pglml final); water (30.8 pl); Taq polymerase (0.2 pl of 1 U/ml. 0.2U

final). Reaction cocktail was distributed to individual reaction tubes

(48 pl each), followed by addition of DNA (2 pl). Minera1 oil was added.

and the reactions cycled as follows: 940 1 1 rnin,650 1 1 min, 720 / 1 min

for 30 cycles. Reac tion contents were transferred to individual

microfuge tubes. ethanol precipitated. washed. lyophilized. and

resuspended in 20 pl TE. Glycerol containing dye was added to the

solution (2 pl), which was then mix'ed and electrophoresed on a 1% agarose

gel. Results of the electrophoresis did not photograph well but were

observable under direct UV illumination and viewing. Results were

recorded as observations in note form.

9

(g) Transformation of Exoli and Isolation af individual colonies:

Transformation. of competent E.coli cells was done according to the

protocol provided by Gibco-BRL with their Library Efficiency Competent

DHSa E.coli cells. with modifications as follows. E.coli cells were thawed

on ice and then 50 pl aliquots were transferred to 1.7 ml eppendorf

centrifuge tubes (previously piaced on ice). DNA isolated from the

selection procedure (1-3 pl) was added, followed by gentle vortexing to

evenly distribute the DNA. After 30 minutes incubation on ice. the cells

were heat shocked at 370C for 45 seconds, followed by chilling on ice for 2

minutes. Luria-Bertani (LB) medium (950 PL) was added. The tube was

capped, and was then placed in the incubatorlshaker for 1.5 hours at 370C.

LB medium was prepared by dissolving 10 g NaCl, 5 g Yeast Extract, and 10

g tryptone in 1.0 L of double-distilled water. followed by pH adjustment to

pH 7.3 and then autoclaving. Aliquots of cells (20-200 pl) were plated on

LB-Amp plates (LB-medium plus 0.75% wlv agar and 100 pglml

Ampicillin) and grown overnight at 370C. Individual colonies were

either picked immediately or the plates were stored at 40C in sealed plastic

"zip-lock" bags. F

C

Individual colonies were picked (using a sterile pipette tip),

transferred to 4.0 mL LB-Amp medium (LB medium plus 50 pg/ml

Ampicillin) and grown overnight in a bacterial incubator-shaker at 370C.

Glycerinated stocks of individual tolonies were prepared by transferring

0.85 ml of the culture to a sterile microfuge tube, adding 0.15 ml sterile

glycerol, mixing well, freezing in a dry-ice/EtOH bath and followed by

storage at -800C. The remaining culture was centrifuged (5-7 min., 3500

rpm, Large Hettich centrifuge). Plasmid DNPI was prepared according to

the mini-al kalinelNaOH1detergen t method (94). Briefly, the cells were

resuspended in 200 pl lysis buffet (50 mM glucose, 10 mM EDTA, 5 rnM

Tris. 2 mg/ml lysozyme, pH 8.0) and were then incubated at room

temperature for 5 min and subsequently placed on ice for 5 minutes.

Alkaline-detergent solution (400 pl. 0.2N NaOH / 1% SDS) was added and

mixed well and then the solution was incubated on ice for 5 minutes.

Neutralization was achieved by addition of 400 pl of 3M sodium acetate

(pH 4.9). After incubation on ice for 15 minutes the samples were

centrifuged at 12 000 rpm for 10 minutes. Plasmid DNA was obtained by

decanting the supernatant into separate sterile microfuge tubes

(approximately 1.0 ml). Pure isopropanol (0.7 ml) was then added and

mixed well. The solution was allowed to sit at 40C for 30-60 minutes,

followed by centrifugation at 12 000 rpm for 15 - 30 minutes. The

supernatant was removed. The pellet was washed with 70% EtOH and then

dried under vacuum for 10 minutes, followed by dissolution i d 5 0 pl of TE.

Screening of individual colonies was done by digestion with BamHl

restriction enzyme. A cocktail of buffer concentrate. water. ribonuclease

(10 mg/ml solution) and restriction enzyme (2:2:03:0.5) was prepared.

This restriction cocktail (5 pl) was transferred to individual 0.7 ml

microfuge tubes. to which DNA (5 pl) was added. The mixture was

incubated at 370C for 1-4 hrs. followed by electrophoresis through a

1%w/v agarose in IxTBE gel. lxTBE contains 90 mM Tris. 90 m M Borate,

1 mM EDTA. Colonies showing no insert were discarded. Initial

sequencing was performed on those colonies containing inserts of 0.9 kb or

greater.

(h) Sequencing of individuat clones:

Sequencing reactions of individual colonies were performed by Dr.

Zhong-Wei Xie and Mrs. Wendy Chow. using a cycle-sequencing protocol.

as distributed by Amersham (Thermosequenase cycle sequencing kit) with

3%-labelled ATP (95). Primers used for the forward (5'-end of insert) and

reverse (3'-end of insert) reactions were as follows:

RSV-LTR (AACGCCATITGACCATTCACCAC) a d

SV40-PA (~AGITGTGGTGGTITGTCCAAACKATC).

Initially, 10 pl of DNA (prepared above) was mixed with buffer,

nucleotides (lnbelled and unlabelled). primers and thermosequenase, and

was layered with minera1 oil and cycled (950 / 15". 550 / 30". 50 cycles).

After cycling was completed. the reaction was split between four

termination reaction tubes. con taining hig her nucleotide concentrations

and dideoxynucleotidetriphosphates. and cycled again (950 / 30", 680 / 30".

720 1 90"). Reactions were transferred carefully. ensuring that no oil was

transferred, into large microfuge tubes containing stop solution and stored

at -200C until the sequencing gel was run. Following denaturation and

snap-cooiing on ice. the samples were run on ultra-thin sequencing gels

(7% acrylamide / OSxTBE), drkd and exposed to Xaray film.

( i ) Computerized BLAST-analysis of sequences:

Sequences were read from individual clones for 125-200 bases.

Individual sequences were inputted ' in to a DNA sequence handling

prograrn for the Apple Maclntosh (DNA Strider) and saved as a sequence

file. Sequences were "packaged" as e-mail messages and compared

against the GenBank Non-Rcdundant DNA Sequence Database at the

NCBINIH using the BLAST e-mail search engine. E-mail based replies

were edited and shortened reports were printed out for tabular

sumrnarization and anal ysis. Those clones closely matching sequences

already reported in the database for known proteins/DNA (closely

matching king defined as BLAST P value c 1x10-5). were eliminated from

further analysis, unless the matches were to cDNAs with no functional

infornation or if the homology was one that may be consistent with the

characteristics of the transporter (membrane protein, sodium gymporter,

etc.). Rernaining sequences were translated in 3 frames using

DNAstnder. BLAST searched against the Non-Redundant Protein Database,

and the results surnmarized in tabular form. Those clones yielding

"close-match" results were eliminated from further processing and

analysis. The remaining clones were denoted as being "active".

Active clones were analyzed by a number of methods. Firstly,

further DNA sequence information was generated by additional

sequencing . Further BLAST searching was perforrned by searching the

DNA sequence against the Expressed Sequence Tag Database (dbEST), and

comparing it to the newly completed (April 1996) genome database of

Saccharomyces cerevisiae (Brewer's Yeast).

Results:

Progress towards the functional cornplementation cloning of the

piasmalemmal carnitine transporter was achieved by confirmation of a

nurnber of steps in the process ,of transfection. DNA isolation and

experimental analysis. Initial experiments confirrned a number of

important pieces of information. PCR was used to confirm the presence of

a broad range of sizes of inserts in the library used. Further experiments

were used to optirnize the transfection conditions and to demonstrate that

some sort of selection was occumng. Final experiments led to the

isolation and screening of a large number of clones, followed by sequencing

and sequence analysis.

(a) M ï T Analysis

The MTT cytotoxicity/cytoproliferation assay as initially descnbed

by Mossman (93) was used, after slight modification, to determine the

survival of cells after treatment under varying conditions. In order to

confirm that the method was suitable for use in the lymphoblastoid ce11

lines that we were using, a standard curve was constructed by sequential

dilution of the cells into the 96-well plate wells, followed by treatment as

described in the Materials and Methods section. The results of this

experiment are presented in Fig. 2. The results show a linear relationship

( ~ 2 = 0.984)

difference in

between the number of living cells in the well and the

absotbance A(A570-A630) observed.

(b) Transfecfion Optirnizndon:

Literature presented by the manufacturer of the electroporation

apparatus. and empirical results of other researchers have shown that

determining the vol tage-capaci tance ; combination used in electroporation,

which kills approximately 50% of the cells, is optimal for transfection

efficiency (96.97). Table 1 shows the results of two separate assays

where the cells were isolated and treated, as if DNA were going to be

added. and then electroporated under di,fferent voltage capaci tance

conditions. The results presented in Table 1 (p.69) show that the 500 pF

and 250 V combination provides the most consistent results, resulting in

approximately 50% ce11 survival after the electroporation "zap".

(c) PCD Cell Transfection with cDNA Library and Growth in Selective Medium:

Cells were transfected with the pREP4-cDNA library as described

above and allowed to grow in selective medium for varying lengths of

t h e , with or without recovery growth penods in normal medium. After

the desired length of time was reached, an aliquot of cells was removed

from the flask(s) and h a ~ e s t e d as described. followed by PCR and then

individual colony isolation.

i

(d ) Isolation of DNA and Confirmation of "Selection":

After isolation of DNA from the "selected" cells, an experiment using

PCR was performed to help confirm that selection was in fact occurring.

Unfortunatel y the " srnears" of DNA bands produced on eth idium bromide t-

C

stained agarose gels. in each reaction, were not readily visible by

photographic methods, and therefore observations were made directly

from the gel into the experirnental notebook. For the PCR. the following

were used as controls: no DNA; DNA from cells with no library added (in t

theory no plasmid DNA should be present); and a srna11 aliquot of the

original library. In both the "no DNA" and the DNA from cells with no

library added, no smear of DNA bands was observed. In the lane where

the original library was used, a "long" smear of bands was observed from 0

approximately 4.5 kb to 0.3 kb. In al1 of the "selected" DNA samples, the

smear obsewed had less intensity (fewer bands) in the small size range

and also in the very large size range. thereby limiting the range to

approximately 0.8 - 3.5 kb.

(e) Typical Screening Gel Results: i

After PCR of a small aliquot of the isolated DNA to confirm the

process of selection. the original "selected" DNA sample was used to

transform competent E.coli cells, followed by plating, isolation and growth

of individual colonies. and isolation of plasmid DNA. After digestion of

the plasmid obtained from each colony. the reaction mixture was

electrophoresed on an agarose gel and a photograph was taken. The

results in Fig. 3 (p.68) show the results of a typical "screening" agarose gel.

The large band at the top of the gel (nearest the wells) corresponds to the

vector. The bands below the vector bands in the individual lanes

correspond to the insert which can be sized approximately according to the

markers run simultaneously with the samples. Those colonies containing

inserts larger than 0.8 kb were marked for sequencing. In the initial

round (marked Round 1) and half of the next round (marked Round 3). al1

of the colonies, regardless of size, were sequenced.

Cf) Surnmary of Screening/Sequencing Results:

Sequencing of the S'-end of the cDNA inserts was performed

according to the thermal cycle sequencing protocol described in the

Material and Methods section. Attempts at using other commerially

available, and less expensive. kits (T7 polyme~ase. Sequenase, etc.) proved

unsuccessful. Typically. 125-250 bases could be easily read manually

directly from the autoradiography film. The length of the readable

sequence depended primarily on the purity of the DNA sample, percentage

of the sequencing gel used, and the length of the gel nin. A surnmary of

the number of clones grown. screened, sequenced and BLAST searched is

presented in Table 2 (p. 69).

(g) Results of BLAST searches: i

After inputting of the sequence of each clone into the cornputer, the

sequence was searched using the e-mail accesible BLAST search algorithm

at the National Centre for Biotechnology Information (NCBI) at the United

States' National Institutes of Health (NIH). Higher similarity scores on a

number of clones at the initial nucleotide search level led to elimination of

these clones. After three-frame translation of the nucleotide sequence

into amino acid sequence, each protein sequence frame was searched

against the protein database at NCBI. Further clones were eliminated at

this time when a high degree of similarity with amino acid sequences

already in the database was found. The identity of the sequence with

high sirnilarity (i.e. function andlor structural motif) and the number of

identical bases/amino acids was taken into account when eliminating

clones from further analysis. If in doubt. the clone was analyzed further,

then eliminated if necessary at that point. The results of this analysis are

presented in the Appendix in Tables 2, 3. and 4.

Discussion

With the development of ,a selective growth medium in the work

described in Chapter 1 of this thesis, a method has been developed to

select for those cells which have had the plasmalernrnal carnitine

transporter defect corrected. Paralleling the development of the

selective medium was the developrnent of techniques for: transfecting the

PCD lymphoblast ce11 lines; isolating the DNA back from the cells;

screening the clones for insert size; sequencing the S'-end of the insert;

and finally, detailed sequence analysis and homology searches.

The MTT ~yt~toxicity/cytoproliferation assay was based on the

ability of live cells to be able to convert the MTT dye from the initial

yellow colour to the purple water insoluble dye. After solubilization of

the dye in acidic isopropanol. the change in absorbance A(A570-A630) was

proportional to the nurnber of live cells remaining. The results shown in

Fig. 2 demonstrate that this relationship was linear and therefore the

mathematical manipulation of the data into ratios or percentages was

valid.

Table 1 shows the results of using the MTT assay to assess the

degree of ce11 killing when the isolated lymphoblast cells were subjected to

an electncal pulse during the process of electroporation. Electroporation is

based on the ability of discrete electrical pulses to open a hole in the ce11

membrane and allow molecules, which have been adsorbed on the outside

of the cells. to be taken inside. In the case of cells and DNA, whether

plasmid or linear DNA, this results in transfection of the cells. Literature

from the manufacturer of the electroporation apparatus and from

empirical evidence

voltage-capaci tance

approximatel y 50%

€rom other researchers, suggested that the optimal

combination conditions for transfection occurs when

of the cells are killed by the electroporation process

(96,97). Presumably at this level of ceIl killing. a balance is achieved

between the creation of membrane defects which allows entry of DNA. and

membrane disruption which leads to the disruption of intracellular

conditions (and therefore ce11 death). Table 1 shows that this "50%"-point

was reached at 500 pF and 250 V for the lymphoblast ce11 line tested.

The cDNA library used for this project was kindly providrd by the

laboratory of Dr. Brian Robinson, as originally prepared by Strathdee, et. al.

(78). Fig. 1 shows the basic map and essential restriction enzyme sites on

the pREP4 vector plus the cDNA insert construct. Based on the map of the

vector, primers for RSV-LTR and SV40-pA can be used for sequencing or

to amplify the cDNA inserts via PCR. Sequencing of the S'-end of the

inserts proved to be quite difficult. Attempts to use the standard T7

polymerase and Sequenase based protocols failed, due to either

inconsistent results or total failure to sequence successive clones. Finally,

an attempt was made to use a thermal-cycling based protocol. which

proved to be successful. The failure of the other methods may be due to

the large size of the template (approximately 10 kb base vector) or other

kitlmethod-vec tor interactions. The success of the thermal-cycling based

protocol may be due to the higher overall temperatures used throughout

the reactions to generate the sequences. thereby keeping the DNA strands

of template apart, while allowing primer-template annealing and

subsequent polyrnerization of sequence-specified length DNA pieces.

Evidence for selection occurring in the DNA pool during growth in

selective medium is supported by several independent observations. PCR-

based confirmation of the changes in the insert size of the DNA pool is one

source of evidence. PCR amplification of the insen pool of the original

library compared to a number of the "selected" DNA pools showed a

distinct decrease in the range of DNA sizes (from 0.3-4.5 kb to 0.8-3.5 kb,

and in the intensity of the banding prttern(s) at smalier sizes. In addition,

a summary of the results presented in Table 2 show that in Round 1

approximately 45% of the colonies picked contained inserts whereas in

Round 3 and 5. 66% and 73% contained inserts. In round 1. the selection

was carried out by 3 successive 10 day passes through selective medium

followed by 4 day recovery periods in normal medium. Rounds 3 and 5

were performed continuously in selective medium. The number of clones

sequenced in rounds 3 and 5 vs round 1 also increased. In addition, the

proportion of "active" clones in rounds 3 and 5 also increased marginally

(11% and 17% respectively vs. 10%). Together. these results may suggest

that selection within the DNA pool was occurring, the ultimate test of

which would be to obtain the actual transporter clone. A control

experiment for the selection process. which should have been performed

prior to transfection of the PCD cells with the cDNA library. would have

been to transfect the normal control and PCD cells line with an "empty"

vector followed by characterization of growth in the selective medium with

comparison of these results to those previously obtained.

After sequencing was performed, al1 of the sequence information was

entered into a computer and sent +via e-mail for sequence similarity 1

homology comparison via the BLAST algorithm at the NIHMCBI. This

algorithm is based on cornparison of the supplied sequence data with the

complete genetic repository database called Genbank. A list is then

returned of those sequences which most closely match it. Results obtained

by e-mail suggest that the "queried" sequences have either a complete lack

of, questionnable, or. strongly homologous regions when compared to al1 of

the known protein or DNA sequences (depending upon the specific

database used). Those clones with large, strongly homologous regions to

DNA sequences encoding known proteins were noted and usually

eliminated from funher analysis unless significant structural or functional

features which were compatible with our proposed mode1 for !he camitine

transporter were present (suggestive domains for eg. membrane

transporter, channel characteristics, p-oxidation capabilities, etc.). The

remaining clones were searched via 3-frame translation followed by

protein database BLAST searchs. Again those clones containing

significant homologies were eliminated from further anatysis. This

process of search and removal of clones with significant homologies to

known DNA sequences will hopefully promote the eventual enrichment of

the "pool" for the carnitine transporter clone. Finally each of the

remaining approximately 100 clones was compared against the human EST

(Expressed Sequence Tag) databnse. This database catalogues the effort of

groups trying to sequence short pieces of cDNA (via mRNA to cDNA) as a

way of looking at al1 of the mRNA/cDNA that is expressed in a particular

tissue, organism, or developmental point in time. Further analysis of the

sequence against the newly completed Yeast genome database was not

performed due to problems with availability of cornputer resources.

One important problem that could be encountered in this process of

clona1 selection and analysis is the possibility of isolation of a clone which

"complements" the defect (in this case restores growth of the PCD lines in

selective medium) but which does not correct the actual biochemical defect

in carnitine uptake. In other words. the restoration of growth could be

the result of an alternative compensatory metabolic mechanism conferred

by the isolated clone. The precedent for complementation. without

correction of the specific genetic defect. has been demonstrated in the

attempts of other investigators to isolate and clone the gene(s) for ataxia

telangiectasia (98). For this reason, it will be critically important in future

studies, to assay carnitine uptake in the transfected lymphoblasts that

have survived in the selective growth medium, in order to determine

whether the restoration of growth is due to successful transfection of the

cDNA encoding the plasmalemmal carnitine transporter.

The remaining approximately 100 clones, plus further clones to be

generated by efforts using the selective medium based system. provide an

initiai methodology for trying to obtain a cDNA clone for the plasma

membrane carnitine transporter. Techniques developed during the

course of the thesis project will facilitate the search for the elusive

carnitine transporter cDNA clone. Identification and characterization of

the transporter cDNA will eventually allow identification of the gene

encoding the plasma membrane carnitine transporter and will lead to the

examination of important structure-function relationships of this

transporter.

Figure 1: Map of pREP4 vector with Multiple Cloning Site and tnsert Legend: hph = Hygromycin resistance gene

EBV (EBNA-1) = Epstein-Barr Virus - Epstein-Barr Nuclear Antigen- t

EBV (oriP) = Epstein-Ban virus origin of replication RSV 3'LTR = Rouse Sarcorna Virus 3' Long Terminal Repeat SV4Op.A = SV40 virus polyadenylation signal

Figure 2: MTT Dye Cell Viability Assay Standard Curve - Live Cell Number versus MTT Dye Ahsorbance (AS7O-AÇ30)

Figure 3: Typical Screening Gel Result: .. Result of BamHl restriction digest analysis of plasmid DNA for individual colonies. Gel is 1%-agarose 1xTBVEthidium Bromide in the gel. M - Size Marker Di%

TABLE 1: Electroporation Ce11 Killing Efficiency for PCD (L0011) Ce11 Line - 46 Viability determined by MTT Cell Viability Assay

Pulse Type % viabiKy (VoltagelCapaci tance) (Expt. 1) No ~ u l s e 1 O0

(Expt. 2) (Expt. 3) 1 O0 1 O 0

*. ?

TABLE 2: Summary of Clones Obtained

1 Round # 1 N u m b e r 1 N u m b e r 1 N u m b e r 1 N u m b e r 1

* = The clones which proved difficult to sequence and are k i n g resequenced are not included in these numbers.

+ = A number of duplicates appear (approx. 70). skewing numbers

1 3 5

TOTAL

I s o l a t e d 9 6

3 00 3 04 700

Act ive 10 27 5 5 9 2

with 1nsert4 Sequenced*. 4 3

203 221 467

4 3 97 (+)

' 185 325

FUTURE DIRECTIONS

From the results obtained in Chapters 1 and 2 of this thesis a number

of future experiments and analyses could be performed. focussing around

4 specific themes. namely: a) specificity of the Hygromycin B used in

the selection medium; b) mechanism of increased sensitivity to

Hygromycin B in PCD cells versus normal cells; c) analysis of the current

clones; and d) construction of a new cDNA library in a pREP8 vector.

a) Specificity of Hygromycin B in Selective Medium - Carnitine

"RescueM Experiment

The specificity of the selective medium with Hygromycin added. for

the inhibition of growth of the PCD ce11 lines, could be explored by

carni tine "rescue" experiments performed in the presence of various

concentrations of Hyg B and increasing concentrations of carnitine

supplementation. The data presented in Chapter 1 in Figs. 6 and 7

suggests that the selective medium,' &or to the addition of Hyg B, is

selective against the growth of the PCD cell lines. As stated in the

discussion, the mechanism for the further "selection" observed. when Hyg

B is present in the medium, is unknown. The observation that there is a

slight difference in sensitivity to Hyg BT differentiating control from PCD

lines in normal medium. might suggest that this further selection is due to

a different mechanisrn. A carnitine "rescue" experiment where PCD cell

lines (L0002/L0011) and normal control ceIl lines (L0006/L0009) are

plated in selective base medium (RPMI minus asparagine. with 5 mM

galactose, 15% serum, 100 pM BSA / 100 pM palmitate) with 0, 40, and 80

pg/ml Hygromycin B and with increasing concentrations of carnitine would

help to answer this question. If the results of this experiment ~ h o w that a

complete rescue of growth is observed, then one might conclude that the

effect of the Hygromycin B is specifically related to the effect of low

intracellular carnitine concentrations. However, if the results show that

little or only moderate rescue is observed. then one might conclude that

the Hygromycin B is acting by some other mechanism.

Mechanism of Hygromycin B effect on Selection

Normal control cells are able to grow relatively well in medium

containing high concentrations of long chain fatty acids (LCFAs), whereas

PCD ce11 lines do not. Presumably. part of this effect is due to the inability

of the PCD cells to efficiently oxidize the LCFAs, but it could rlso be due to

the inherant toxicity of the LCFAs, their esters and secondary metabolites.

Lymphoblasts and fibroblasis are primarily glycolytically fueled cells.

Under conditions of oxidative substrate stress (Le. low glucose and high

LCFA) the cells must "switch" to some other method of energy generation.

In this case, with high concentrations of LCFAs in the medium, the cells

would likely switch to LCFA oxidation. Evidence from Chapter 1 - Fig. 10

show that normal cells grow relatively well in LCFA enrichedlglucose-poor

medium, whereas PCD cells do not. One question that arises is "When does

this switchover occur?". 1s it as a result of increased synthesis of key rate-

limiting enzymes in P-oxidation (CPT-1) or decreased synthesis of rate-

limiting enzymes of .glycolysis (phosphofructokinase) or is it as a result of

up- andlor down- regulation of these enzymes, respectively, by

intracellular regulatory mechanisms. With Hygromycin B present in the

medium, an additional challenge to the cells now exists. Hygromycin B

inhibits protein synthesis by binding to ribosomes and promoting

mistranslation and incomplete elongation. Experiments which ,might help

to elucidate the mechanism of the increased selection between PCD and

normal control ce11 lines could include: a) cornparison of enzyme specific

mRNA levels, enzyme activity levels and/or specific enzyme protein levels

for the major B-oxidation and glycolytic enzymes in normal control and

PCD cells in normal medium and glucose-free LCFA-rich medium; and b)

cornparison of levels of metabolic intermediates which are known to have

regulatory effects (e.g. malonyl-CoA - the specific inhibitor of CPT-1,

acetyl-CoA. etc.).

c) Further Analysis of Clones

Further analyses of the clones already obtained could be done by a

number of methods. Longer sequencing runs have been performed on

most of the "active" clones. so that addition to and subsequent analysis of

the obtained sequence would be relatively straight forward. A nurnber of

cornputer-based algorithms could be used to look at these sequences

including: a) alignment of the clones using multiple sequence alignrnent

tools; b) further BLAST-search analysis (Genbank); c) search against the

Yeast genome database; d) search against the protein motif database

(PROSITE); and e) search for transmembrane domains using

hydrophobicity / hydrophilicity plots. In addition. further restriction

digestion analysis of the inserts to determine whether similar restriction

patterns were obtained could be done to see if any of the inserts are the

same. After larger scale growth of the individual clones. grouped sets of

DNA could be re-transfected in to patient ce1 l lines followed by analysis.

Analysis could be performed by evaluation of growth characteristics in

selective medium. and then evaluation of carnitine uptake characteristics.

This would then eliminate the potential problem of isolation of, clones

which correct the growth of the PCD cells (complementation) by a

mechanism other than that related to a specific correction of the carnitine

transport defect. Together this information would provide more definitive

data as to whether these clones warrant further investigation.

d) Construction of a new cDNA Library in pREP8 vector

Construction of a new cDNA library in a pREP8 vector would remove

an important obstacle in the clone selection process. Currently the cDNA

library is constructed from normal lymphoblast mRNA in the pREP4 vector

containing the Hygromycin resistance gene. using a vector primed

synthesis strategy. The problem in using this vector is the difficulty in

predicting the degree of reduction of the "growth inhibitory" effect of the

selective medium on PCD ce11 lines that have been transfected with the

Hygromycin resistance gene. The pREP8 vector contains al1 of the same

components of the expression cassette (RSV-LTR and SV40-PA) and

episomal maintenance (EBNA-1. etc:), but has a HisD (histidinol) resistance

marker. Construction of the cDNA library using normal lymphoblast

mRNA (cell type known to express the protein of interest) into the pREP8

vector would eliminate the problem of conferred resistance to Hygromycin

B. Initially. we would choose to construct the library from lymphoblast

mRNA as construction of the library from mRNA obtained from other

tissues andfor species could cause problems due to expression,

intracellular processing, improper location of expression, etc. Finally, a

necessary control of the selection medium would be to transfect the cells

lines with the pREP8 vector containing no insert in order to assess the

effect of the vector alone.

TISSUE K m N a E n e r g y C h a r a c t e r i s t i c s l I n h i b i t o r s (Species - prep) (PM) d e p e n d . d e p e n d .

SKELETAL MUSCLE *

-Human - primary cultured (21)

-Rai - isolated (22)

-Rat - isolated (23)

K I D N E Y -Rat - cortex sfices (24)

-Rat - cortex slices (25)

-Rat - brush border membrane vesicles (26)

-Rat - brush border membrane vesicles (27)

Characteristics - showed iwo transport systems exist in muscle and fibrobtast (hi@ and low affinity)

6 0 Yes Yes Inhibitors - 2,4-DNP, azide, anoxia, ouabain, carnitine analogues, Nat deplet ion

Characteristics - suggests stereochemistry of 9- hydroxy group important for uptake process

Yes Inhibitor - y-butyrobetaine Characterist ics - temperature depend. (active transport)

Yes Inhibiiors - anoxia, CCCP, low temp. Characteristics - dibutyryl CAMP increased uptake

(suggests possible hormonal control?)

9 0 Yes Yes lnhibitors - anoxia, CCCP, 2.4-DNP, low temp,, carnitine analogues, N-ethylmaleimide, ouabain, KCN,

Characteristics - high affinity (90pM) and fow affinity (333pM) transport system present

110 Yes ( t ot a i )

5 5 (Na. dep)

17.4 Yes 15000

Inhibitors - 7-butyrobetaine, D-carnhine, carnitine analogues

Characteristics - Km for total (11OpM) and Na-graâient dependent (55pM) transport

-tram-stimulation of uptake -recognition sites for carboxy, trimethylamino groups

Inhibitors - carnitine structural analogues Characteristics - demonstrated t wo Na-dependen t

transport systems exist (high and low affinity)

TISSUE K IN N a E n e r g y C h a r a c t t r I s t i c s / I n h i b i i o r s (Species - prep.) (PM) d e p e n d . d a p e n d .

Kidney (cont'd) -Rat - cortex mRNA (expressed in 149 Y e s

Xenopus laevis oocytes) (28)

LIVER -Rat - perfused whole (29) 2 7 0

(camitine ou tward transport) -Rai isolated cells (30) 5 6 0 0

-Rat - perfused whole (31) (carnitine uptake)

Inhibitors - trimethyl-lysine, D-carniiine, carnitine analogues

Characteristics - mRNA fraction giving maximal upialte approx. 2kb

No Inhi bitors - mersalyl (inhibited efflux) Characteristics - decreased efflux afier starvation

Yes Inhi bit ors - carnitine analogues, 2,4-DNP Characteristics - suggests carnitine and buiyrobetaine

transportcd by sarne transporter

2590 Yes Yes Inhibitors - 2,4-DNP, KCN (inhibited uptake) (fasted) - mersalyl (inhibited efflux from liver)

4220 Characterist ics - fasting causes decreased carni t h e (fed) uptake Km

D R A I N -Rat - cerebral cortex slices (32) 2 8 5 0 Yes Yes liihibiiors - anoxia, glucose deprivation, 2,4-DNP, CCCP,

KCN, N-ethylmaieimide, ouabaiti

-Mouse - synaptosomes (33)

INTESTINE -Human - proximal small intestine (34)

-Rat - in-vivo perfusion small intestine (35)

-Rat - jejunal brush border microvillous membrane vesicles (36)

-Rat - everted riagshacs of small intestine (37)

Yes Yes Inhibitors - ouabain, NaCN - GABA (cornpetitive)

9 7 4 Yes Characteristics - saturable system and a significant diffusional componen t

1035- Characteristics -pariially saturable (?) 1267

Characteristics -no carrier-mediated transport system ohcwcd - only passive diffusion

206- Yes Yes Inhibitors - 2.4-DNP, anoxia, KCN 3 1 6 Characteristics - duodenum and jejunum dernonsttated

active transport and a linear diffusion component

P P-

TISSUE K m N a Energy C . h a r a c t e r i s t i c s / I n h i b i t o r s (Specles - prep.) (PM) d e p e n d . depend .

Intes t ine (cont9d) -Guinea pig - enterocytes (38) 6 Yes

FIBROBLAST -Mouse - heart fibroblasts(39) 15.6

-Human - Skin (40)

-Human - Skin (41)

-Human - Skin (42)

-Human - fetal lung (43)

LYMPHOILAST -Human - Transformed B ce11 (44)

Yes

Characteristics - suggested to be facilitated diffusion rather han active transport

Characteristics - juvenile visceral steatosis (JVS) mouse proposed as a modcl for prirnary systemic camitine deficiency because no saturable

uptake observed in JVS fibroblasts Characterisiics - showed patients had <2% control uptake

velociiy at Km concentration of cmitine and parents had intermediate Vrnax values

(13.44% control) with normal Km values - evidence for autosomal recessive inberilance

Characteristics - direct clinical evidence for high dose carnitine correcting defect in ketogenesis

Possible Inhibit ors - 2,4-DNFB, N-ethylrnaleimide. mersalyl, rotenone, antimycin A, KCN, nigericin,

Characterisiics - replacement of extraceflular Na+ with Li+, K+ or Rb+ ions tesuited in a

dramatic reduction of uptakt Inhibitors - N-ethylmalcimide, D-carnitine, octanoyl-D-

carnitine Characteristics - late passage cells took up carnitine

more rapidly than early passage cells

Characteristics - showed Uiat PCD patients had <2% of normal control uptake velocity at Km concentration of carnitine

KEY: CCCP = carbonyl cyanide rn-chlorophenylhydrazone, 2.4-DNP = 2.4-dinitrophenol, KCN = potassium cyanide. 2,4-DNFB = 2,4-dinitrofluorobenzene, PCD = plasmalemmal carnitine transport defect, GABA = y-aminobutyric acid

Clone Summary - Round 1

( Clone # 1 Size 1 Status 1 Origin 1 Nuc.lProt. BLAST Results 1 E.S.T. or Yeast BLAST Results 1 Corn ments 1

N.M. b Protein

HepG2iD-2 microglobulin

match not close - Fragile X pDR2 vector - aph pene phosphatase (tyrosine) cDNA (no funciion) cbNA (no function) pDR2 vector - aph gene glycine decarboxylase

I

Protein Protein

1

Coding of BLAST Results / Commenis: F - Finished (eliminated after BLAST search anaiysis) ' a.

A - Active (remaining after BLAST search analysis) . t . .

N.M. - (no match) - BLAST-search of nucleotide database did not provide a match M.N.C. - (match not close) - BLAST-search of nucleotide database produced match, but degree of

similarity was low cDNA (no function) - matched cDNA clone in database, but no funciional characteristic are attached Seguence 1 Protein - clone is to be sequenced, or cornputer translated, respectively SMA1S.M.A. = Spinal Muscular Atrophy

t

cDNA (no function) mitochondrial eno orne DNA cDNA clone (NF) Alu repeti~ive sequence

Protein

CODING KEY for APPENDIX TABLES 3 and 4

STATUS: NS = Not sequenced FN = Finished/eliminated after nucleotide BLAST search FP = Finished/eliminated after protein BLAST search A = Active Dup = Duplicate DNSW = Did not Sequence well on first attempt

i L

ORIGIN CODE: -First number indicates PCD line number into which library was

transfec ted -Second number indicates concentration of Hygromycin used in the

formulation of the selective media -Ab indicates presence of antibiotics (standard -pnnicillin /

streptomycin concen nations)

BLAST Search RESULTS: N = ResuIts/match after nucleotide BLAST search P = Results/match after nucleNde BLAST search E = Results/match *after EST ~ L A S T search cDNA1N.F. = matched to cloned cDNA with no functional

characteris tics N.M. = No match p

Appendix Table 3: Clone Summary - Round 3

1 Clone # 1 Size 1 Status 1 Origin' BLAST Search Results

Nucleo tide(N-) 1 Expressed Seqpence Tag Comments andfor Protein(P-) andlor Yeast

E-yo64d01 .r 1 clone 1 182689 a

N-ribosomal L3 7a protein 1 I

N-?? 1 E-no match 1 1

I - E-EST01618 similat to

t , . Alu elements I 1 m

NI?? 1 E-no match ' ' . * 1 1

N-ubiquitin conj. enzyme E-yj07c03.r 1 clone P-?? 148036

1

N-CLP mRNA N

N-33

N-MHCII-gamma c h a h

No?? P-MCAD precursor(7)

Commenis

.. 37

L

38 39 40 42 49 50 5 1 52 53

1

54 55 56 57

r

58 59 60

l

6 1 62

r

63 65 67 68 69 ,

7 1 72 76 78 79

I

80 8 1 w

82

E.S.T./Yeast BLAST Result Clone # 36

1.5 0.7 0.8 0.4 0.6 1.0 1.1

0.65 1.1 1 .O 1.1 1.1 0.7 1.1 1.1 '

1 .O 1 a 1 1 .O 1 .O 0.9 1 .O 1 .O 1.1 1.1 1.1 1.1

1 . 0 t .O 1 .O 1 .O 0.9 1 ,O

Origin 11/80

Nuc./Prot. BLAST Hesults N-N.M.

She 1 ,0/Ob5

FN NS NS NS NS

NS

NS

to read

DUO DUD

' Dup

Status A

f 1/80 11/80 ., 11/80 11/80 1 1/80 11/80 11/80 11180

A

P-37 N-nucleolar phos.-prot.-823

a

t.

-same as 72 DUD -

11/80 11/80 1 1/80 11/80 11/80 11/80 11/80 11/80 11/80 11/80 11180 11180 11/80 11/80 11180 11180 11/80 11/80 11/80 11/80 1 1180 11/80 11/80 11/80

.

-

1

. . -same as 72

- - - - -

-same as 72 -same as 72

1

t

,

m

1

1

d

d

I

I

Clone Surnmary - Round 3

I

1

,

,

1

83 84 9F

85 86 87 88 89 90 91 92 93

v

94 95 96 97 98 99 100

, 101 102 103 104 105

, 106 107 108 1 09 110 11 1

I

112

113 114

1 .O 1.1

1 ,O 1 .O 1 #O 1 .O 1 *O 1 ,O 1 .O 1 ,O 1 .O 1 .O 1 .O 1 .O 0.9

0.810.9 0.8J0.9

0.8 0.8 0.8

0,8/0,9 0.8f0.9 0.8/0.9 0.810.9 0.810.9 0.8/0.9

0.8 0.85 0.9 3,O

0.9 0.9

DUD A

Dup NS NS NS

DUP NS NS NS NS

DNSW DNSW DNSW NS NS DUP FN

. DUP NS NS

Dup Dup NS NS NS NS

1 1/80 11/80

11/80 11/80 11/80 11/80 11/80 11/80 11/80 11/80 11/80 11/80

. 11/80 11/80 1 1/40 1 1140 1 1/40 11/40 1 1/40 1 1/40 11 /40 11/40 1 1/40 1 1/40 1 1/40 1 1/40 1 1/40

-same as 100 N-crlpastatin P-ca lpastath

Nuc./Prot. BLAST Resul ts E.S.T.Neast BLAST Result -same as 72 N-N.M. Po??

'.-same as 72

NS Dup

, FN

NS NS

-same as 100 ' 1

-same as 100 N-profilin -same as 100

-same as 100 -same as 100

1 1 140 1 1/40 11/40

1 1/40 1 1 140

a

J .

Cluiic Suiiiiiiiiry - Ihwiirl 3

Comments 1

, 286 29 1

I

, 293 , 294

297 298

E.S.T.IYeast BLAST Result E-EST35635

Clone # 285

1.6 1 .4

1.3 1.2 0.7 1 .O

Site 1.4

FN A

FN FN NS FN

Status A

1 1/80 S 1/80 '*

1 1/80 1 f 180 11 180 1 1/80

Origin 11/80

Nuc.lProt. BLAST Results N-N.M. Po?? N-VH5 i rnmuno~ lobu l in N-?? P-21 N-initiation factor 4AI N-cap- blnding protetn

N-cap binding' proiein

E-yx42b07.r 1 clone 264373

a

b

Appendix Table 4: Clone Summary - Round 5

Size 1 Stitus Origin BLAST Search Results

Nucleotide andlor Protein 1 Expressed Sequence Tag 1 Comments

N-40s ribosomal prot. S18 1 1 œ

N-60s ribosomal protein N-cDNA/N.F. ' P-channel homology

1 1

N-sn ribo.nuc.prot.-E N- l9K CAMP-reg. phosprot P-?? * :

P-memb. prot. homoIoaies N-cDNA/N.F.

1

15 1

16 17

18

0.8

1.4 O S

20

21

w 22

3.5

A

FN FP

0.5

0.85

1 .Y

A

2 180

2/80 2/80

FW

FP

P-ORF/Ca2+ bindina prot. N-chloroplast DNA(??) P-memb. prot. homologv N-HLA-DRa (p34) N-??

2/80 P4OS ribosornal mot. S21 N-cDNA/N.F.

2/40

2/40

2/40

PI?? N-mitochondrial DNA (cyt.ox.) N-ribosomal po t . YLlO P-ribo prot. 60s LIS

Clone Sunirnary Rouiid 5

P-?? 2 5 0.7 FN 2/80 N-40s ribosomal S17 protein 2 6. 1.3 FN 2/80 N - A h sequence 28 3 .5 2/80

i

29 3 as A 2/80 N-NOMb

Corn ment s E.S.T./Yeast BLAST Result

3 1

32 33

L

35 36 37

b

38

Nuc./Prot. BLAST Results N-N.M.

1

39

Clonc # 23

1.3

3 .O 2.5

0.9 1 *3 0.8 1 .3

41 42

S tatus A

Sizc 0.7

1.3 FN

43 ,

45 46 49

50

53

54 L

55

O.rig in 2/40

A

FN FN

Dup. FN FN

1.2 1.4/0.5

2/80

0.4 0.7 0.7 2*0

0.8

1.3

O .9 1.3

2/80

2/80 2/80

+ 2/80 2/80 2/80 2/ 80

Dup. . A

isomerase N-Triose-phosphate .

FN NS FP A

A

,

FW FN

P-60s L 13 6

N-CAMP-reg. phos.-prot. P-73 N-CD20 antipen N-EBV G-coup. recepi. (EB12)

N-CAMP-reg. protein(r5-3 1) N-60s ribosomal L12 protein N-Triose-phosphate

2 /80 2/80

l<< .

isomer ase N-CAMP-reg. protein(r5-3 1) N-cDNA/N.F.

2180 2/80 2180 2/80

2 / 80

2 /80

2/80 2 / 8 0

a

. -

1

I

I

I

PI?? N-proteasorne subunit-DDS

same as r5-30 N-N.M, P-31 ~-ch&$aS ~DNA?~: P-vro tease?? N-737 P-collagen/ri bo. 60s L 1 3 cDNA/N.F. (same? as r5-54) N-60s ribosomal. L3 protein

1

- .

A

Clone Surnmary - Rouiid 5

1 Clone # 1 Size Status 1 Origin 1 Nuc./Prot. BLAST Results 1 E.S.T./Yeast BLAST Resultl Comments 1 FN 2/00 N-60s ribosomal L3 protein . A 2/80 N-cDNA1N.F.

P-polyketide synthase? I

A 2180 ., N-cDNA1N.F.

DUP 2/80 N-cDNA1N.F. (same as r5-30) P-??

A 2/80 N-sim. to mito memb, prot, P-13 b

DUP 2/80 N-CAMP-reg - phosphoprotei n

FP 2/80 N-sim. to Huntington's locus 4

P- Alu sequence A 2/80 N-N.M. (phospholiposc-C)

Y - ? ?

FN 2 / 8 0 N-ribo. prot. 40s SI2 Dup 2/80 sameas36,41,65

A 2/80Ab N-N.M, P-??

FN 2/80Ab N-B-2 micro~lob . precursor I

FN 2/80Ab N-same as 75 I

A 2180A b N-cDNA1N.F. P-??

FN 2/80Ab N-xs25/ pyruvate kinase 2/80Ab

FN 2/80Ab N-8-2 microglob. precursor A 2/80Ab N-cDNA/N,F.

' P-?? NS 2/80Ab

' A 2/80Ab N-N.M. P-??

Clone Summary - Round 5

,

E.S.TJYeast BLAST Result

a

. a,

E-no match

E-yd93f06.r 1 clone 115811

Clone # 85

86 89 90 ' 91 92

94

96 97

98 r

99 1 O0 102 103

1 04 , 109

110 1 1 1 112 113

1 l 4 116 117

118

122 123 124

Comments

I

1

i1

I

1 Size 1.3

1 .5 1 .O 0.7 0.6 0.7

? ?

2 ,O 2.3/1.3/

0.6 1 .O 0.7 0.8 0.9 0.9

0.6 1 .O 0.6 1 .O 1 ,O 1 .O

0.6 0.8 2 .O

1.5

0.6 1 .S 0.6

Status A

FN

NS NS FP

FP

NS NS FN A

NS

NS FN

A

NS NS FP

A

NS FN NS

O t i ~ i n 2180Ab

2/80Ab 2/40 2 /40 2 /40 2/40

2/40

2/40 2/40

2/40 2/40 2/40 2/80 2 /80

2 / 80 2/80 2/80 2/80 2/80 2/40

2140 2/40 2 /40

2 /40

2/40 2/40 2/40 -

NucJProt. BLAST Results N-DNA binding protein P-growthldi f f 'n teceptor same as 85

No?? P-proteasorne subunit p40 N-cDNA1N.F. ' , P-??

No?? P-kinesin

N-cathepsin C N0N.M. Po??

contains Alu WAIu sequence

N-31 P.??

N-73 P-AIu sequence N-?? P-37

N-Histone H2A-X

JClone # I Size 1 Status 1 O r i g i n I

El-laminin bindinp proiein

Nucb/Prot. BLAST Results 1 E.S.T.Neast BLAST Result I

N - E-yd3Sa09.r l c.lone P-ROSH5.5 1 calsequesirin 110200 N-acidic ribo.phospho.prot,

Comment s

N-KIF2 protein (f n??) N-?? E-no match Po77

N-heat shock protein N-33 E-EST04809 (L 1 repeat)

N-N,M. E-EST54804 P-?? (K channel) No?? . E-ys13e08.r l clone P-??(SH3 btnding) 2 14694

N-Int-6 pseudogene P-??/ribo 60s L24 N-nucleolar phos.prot.-B23 . .

N-thymidine kinase N-p3 8-2G4 E-EST63792 P-77 N-CD6 an tigen 1 N-HRCl (DNA binding prot.)

N-?? 1 E-ye94c0 1 .r 1 clone 1

Clone Suniniuy - Itouiid 5

N-ATP synthase y

Comment s l Nuc./Prot. BLAST Results

N-HRC 1 (DNA binding) N-?? P-??

E.S.T.1Yeast BLAST Result

P-?? 1 N-HRC 1 (DN A bindinp) N-TRK-T3 oncogene

1

N-apolipoprotein

E-yx8l hOl .r l sim. to. 19K CAMP phosphoprot,

E-no match a

P-Dvl protein N-3 ? I

,

P-T3 oncoprotein N-N.M. P-32 N-DvI-2 mRNA

P-Alu sequence

N-Int6 protein P-?? N-cosmidslplasrnids P-reverse transcriptase

N-Dhm i protein P-Dhm 1 protein N-proteoal ycan .

N-MBl gene (fn??) N-Dhm 1 protein (f n??)

l

Clorie Suniiiiary - liouiid 5

Status /( Nuc./Prot. BLAST Results 1 E.S.T./Yeast BLAST G u l t ( Comments 1 - -- - -- - -

N-?? 1 E - ~ ~ ~ 0 5 6 7 4 clone 1 1

N-DNA binding protein N-importin subunit

P-77 N-MAR/SAR DNA blnding ( ? )

P-?? - N-ATP synihase . y subunit

HFBEOIS E-no match

N-elongaiion factor la

rn

N-ser. hydroxyrneih. transf. N-eloneation factor 1 y

N-77 1 E-zb24d09.r 1 clone 1 1

4

N-N.M. 1 E-yb8ShlZ.rl clone 1 1

Po?? N-?? Po?? N-MHCII-Y chain 4

302993 E-yxB1hOI.rl sim. to 19K CAMP phosphoprotein

P - 3 3 N-acldic ribo.phospho. p r o t e i n

N-mito. aenome DNA N-alutathione-S-transf. N - ? ? / d y s t r o p h i n N-13 P-?? N-Int6 P m ? ?

N-ribosomal proiein L5

78023 a

E-JO495 sim. to mito. seq.

E-EST64858

I

. ,

Cloiie Suiiiniury - R o u d 5

1 Clone # Size tat tus 0 r i g i n 272 1 . 1 A 2/80

280 , 2 8 1

282

~ u c . / ~ r o i . DLAST Results N-?? P-71 0 N-31 P-?? N-prolif. cell nuc. antig.

N-?? a

, 274 275

276 , 278

2 7 9

283

2 84

1 .O 1.3 1 ,O

285 286

E.S.T./Yeasi BLAST Resuli E-yv52f07.r 1 clone 246373

E-GEN-093D03

E-no match

1.3 2,0/1.3

1.1 1.3 1.1

4.0

1 .O

, 287 , 288

289 I

, 290 , 291

292 , 294 , 295

297 , 298

299

301

Commcnis 1

a

FN FN A

0.9 4 .O

FN A

FN

A

FP

FP

2.0 0.7 1 .O

1.1 2.1 0.8 0.7 2.1 1 3 3.5 1 . f 0,9

2/80 2/80 w

2/80 2 /80 2/80

2/80 2/80 2/80

NS A

2/80

2/80

FN NS A

FN

NS NS

FN FN A

FN

P-?? b

N-HLA-DI1 aniiièli N - t u b u l i n N-vimentin (?)

2/80 2/80

P-?? N-?? P-putative G6P isomerase N-??

2/80 .

2/80 2/80

2180 2/80 2/80 2/80 2/80 2/80 2/80 2180

2 18 0

E-yf49a04.r 1 clone 25282 1

P-serine/threoirine kinase

N-37

,

E-no match I . P-31 N-8-act in

N-31 P-13 (some transp. homol.) N-GAPDH

N-elongation factor I -a N-CD20 antipen N-?? P-?? N-elongation factor 1-6

E-ym24a09.r l clone 48782

E-yx8lhOl.rl sim to. 19K CAMP phosphoprot.

,

I

I

Clone Sumrnary - Round 5

Comments

204 I

E,S.T./Yeast BLAST Result E-za68e04,rl clone

Clone # 3 02

1.0

Size 0.9

M

Status A

. 2/80 -, &

Origin 2180

NucJProt. BLAST Results N-?? P m ? ? N-23kD highly basic proi.

297726 ,

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