15
Insect Molecular Biology (1 993) 1(3), 149-1 63 Gut-specific genes from the black fly Simulium vitfafum encoding trypsin-l i ke and carboxypeptidase-l ike proteins A. Ramos,* A. Mahowaldt and M. Jacobs-Lorena Department of Genetics, Case Western University, Cleveland, Ohio, USA Abstract In haematophagous insects digestion of the blood meal provides nutrients for survival and essential components for egg production. We have isolatedand partially characterized two gut-specific genes from the black fly Simulium viffatum. Sequence analysis re- vealed that both are highly similar to digestive proteases, one to trypsins and the other to carboxy- peptidases. RNA blot analysis indicates that the ex- pression of these two genes is regulated in a sex- specific manner; when fed the same sucrose-based diet, expression in males is substantially lower than in females. In females, expression of both genes is strongly induced by a blood meal. At 6 h after the blood meal the trypsin-like gene product was immunolocai- ired to the midgut epithelium and to the outer layers of the peritrophic matrix. Keywords: trypsin, carboxypeptidase, Simulium, gut. Introduction Black flies (simuliids) are vectors for onchocerciasis, or river blindness,which is a leading cause of blindnessin the world. In Africa the main vector for onchocerciasis are flies belonging to the Simulium damnosum species-complex, while in Central America the main vector is S. ochraceum. Neither of the above species has been cultured in the laboratory.However, the American species S. vittatum has been continually reared in the laboratory for many years (Bernard0 eta/., 1986). Fortunately, S. vittatum is a good *Present address: A.I.D.L., Colorado State University, Fort Collins, CO 80523, USA. tPresent address: University of Chicago, Department of Molecular Genetics and Cell Biology, 920 East 58th Street, Chicago, IL 60637, USA. Received 26 August 1992; accepted 17 December 1992. Correspondence: Dr Marcel0 Jacobs-Lorena,Case Western Reserve University, Department of Genetics, 10900 Euclid Avenue, Cleveland, OH 44106-4955. USA. model system for the study of onchocerciasis since it supports the development of 0. volvulus in the laboratory (E. W. Cupp, personal communication). Ingestion of a blood meal by haematophagous insects triggers the secretion by the midgut epithelium of a thick matrix which has been called peritrophic membrane or, more accurately, peritrophic matrix. Despite the almost universai occurrence of the peritrophic matrix in insects, little is known about its function (reviewed by Peters, 1976; Richards & Richards, 1977). Possible functions include protection of the gut epithelium from abrasion or from invasion by microorganisms and parasites. Because ingested food is completely separated from the absorptive epithelium by the peritrophic matrix, it is likely that this structure also functions in some capacity in the process of food digestion. The blood meal also stimulates the production and secretion of digestive enzymes by the midgut epithelium (Chapman, 1985). Digestion of the blood meal leads ulti- mately to the induction of vitellogenesis and egg pro- duction. Despite the critical role that the process of digestion plays in the physiology, fitness and survival of the fly, little is known about the relevant genes and their regulation. We have initiated the molecular characterization of genes that are specifically expressed in the gut of S. viftatum. Here we report on the isolation and initial charac- terization of two gut-specific genes that have sequence similarityto the digestive enzymes trypsin and carboxypep- tidase. Expression of these genes is sex-dependent and is up-regulated in response to a blood meal. Antibodies prepared to a polypeptide encoded by the trypsin-likegene detect the proteinexclusivelyin the midgut epithelium, both before and after the blood meal. Results /solation of Sim u li u m vittatu m midgut-specific genes In order to search for genes that are specificallyexpressed in the midgut, a S. vittatumgenomic library (Jacobs-Lorena et a/., 1988) was differentially screened with radioactive cDNAs transcribed from midgut and non-gut RNAs. Six phages that hybridized preferentially with the midgut probe 149

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Page 1: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

Insect Molecular Biology (1 993) 1(3), 149-1 63

Gut-specific genes from the black fly Simulium vitfafum encoding t rypsi n-l i ke and carboxypeptidase-l i ke proteins

A. Ramos,* A. Mahowaldt and M. Jacobs-Lorena Department of Genetics, Case Western University, Cleveland, Ohio, USA

Abstract

In haematophagous insects digestion of the blood meal provides nutrients for survival and essential components for egg production. We have isolated and partially characterized two gut-specific genes from the black fly Simulium viffatum. Sequence analysis re- vealed that both are highly similar to digestive proteases, one to trypsins and the other to carboxy- peptidases. RNA blot analysis indicates that the ex- pression of these two genes is regulated in a sex- specific manner; when fed the same sucrose-based diet, expression in males is substantially lower than in females. In females, expression of both genes is strongly induced by a blood meal. At 6 h after the blood meal the trypsin-like gene product was immunolocai- ired to the midgut epithelium and to the outer layers of the peritrophic matrix.

Keywords: trypsin, carboxypeptidase, Simulium, gut.

Introduction

Black flies (simuliids) are vectors for onchocerciasis, or river blindness, which is a leading cause of blindness in the world. In Africa the main vector for onchocerciasis are flies belonging to the Simulium damnosum species-complex, while in Central America the main vector is S. ochraceum. Neither of the above species has been cultured in the laboratory. However, the American species S. vittatum has been continually reared in the laboratory for many years (Bernard0 eta/., 1986). Fortunately, S. vittatum is a good

*Present address: A.I.D.L., Colorado State University, Fort Collins, CO 80523, USA.

tPresent address: University of Chicago, Department of Molecular Genetics and Cell Biology, 920 East 58th Street, Chicago, IL 60637, USA.

Received 26 August 1992; accepted 17 December 1992. Correspondence: Dr Marcel0 Jacobs-Lorena, Case Western Reserve University, Department of Genetics, 10900 Euclid Avenue, Cleveland, OH 44106-4955. USA.

model system for the study of onchocerciasis since it supports the development of 0. volvulus in the laboratory (E. W. Cupp, personal communication).

Ingestion of a blood meal by haematophagous insects triggers the secretion by the midgut epithelium of a thick matrix which has been called peritrophic membrane or, more accurately, peritrophic matrix. Despite the almost universai occurrence of the peritrophic matrix in insects, little is known about its function (reviewed by Peters, 1976; Richards & Richards, 1977). Possible functions include protection of the gut epithelium from abrasion or from invasion by microorganisms and parasites. Because ingested food is completely separated from the absorptive epithelium by the peritrophic matrix, it is likely that this structure also functions in some capacity in the process of food digestion.

The blood meal also stimulates the production and secretion of digestive enzymes by the midgut epithelium (Chapman, 1985). Digestion of the blood meal leads ulti- mately to the induction of vitellogenesis and egg pro- duction. Despite the critical role that the process of digestion plays in the physiology, fitness and survival of the fly, little is known about the relevant genes and their regulation.

We have initiated the molecular characterization of genes that are specifically expressed in the gut of S. viftatum. Here we report on the isolation and initial charac- terization of two gut-specific genes that have sequence similarity to the digestive enzymes trypsin and carboxypep- tidase. Expression of these genes is sex-dependent and is up-regulated in response to a blood meal. Antibodies prepared to a polypeptide encoded by the trypsin-like gene detect the protein exclusively in the midgut epithelium, both before and after the blood meal.

Results

/solation of Si m u li u m vittatu m midgut-specific genes

In order to search for genes that are specifically expressed in the midgut, a S. vittatumgenomic library (Jacobs-Lorena et a/., 1988) was differentially screened with radioactive cDNAs transcribed from midgut and non-gut RNAs. Six phages that hybridized preferentially with the midgut probe

149

Page 2: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

150 A. Ramos, A. Mahowald and M. Jacobs-Lorena

2 0 40 6 0 I I I I I I I

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Page 3: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

Gut specific genes from the black f/y 151

were isolated. Two of these (termed T-land C-/for trypsin- like and carboxypeptidase-like; see below) were character- ized in greater detail. Southern blots of these phages probed with radioactive midgut cDNAs indicated that each phage encoded only one abundant midgut transcript (re- sults not shown). The coding-fragment from each phage was used as probes on Northern blots of size-fractionated gut RNAs. Each fragment hybridized with a unique abun- dant transcript, indicating that the two cloned DNA se- quences encode different genes (results not shown; see below).

The T-l and C-I genes encode proteins with similarity to digestive enzymes

An S. vittatum gut cDNA library was screened with the subcloned genomic fragments. The largest cDNA clones recovered were sequenced. Figure 1 presents the nucleo- tide sequence of the T-/ cDNA insert and the deduced amino acid sequence. The nucleotide sequence rep- resents about 70% of the rnRNA length as estimated from Northern blots (Fig. 5) and covers almost the entire protein coding region (Fig. 2). A sequence similarity search re- vealed that T-/ encodes a trypsin-like enzyme (Fig. 2). Sequence identity of T-/ with the other trypsins listed varies from 37% to 42%. Importantly, amino acids that are known to be essential for enzyme structure and function are all conserved in T-/. In addition, two unpublished trypsin-like mosquito sequences (GenBank accession numbers X64362 and X64363) had the highest similarity to T-l: 49.6% identity over 240 amino acids and 48.2% identity over 222 amino acids, respectively.

Figure 3 presents the nucleotide sequence of the C-/ cDNA and the deduced amino acid sequence. From the mRNA size estimated from Northern blots (Fig. 5) this nucleotide sequence covers close to 70% of the length of the transcript. Comparison to other carboxypeptidases (Fig. 4) indicates that almost the entire protein coding sequence is represented in the C-I cDNA. The predicted amino acid sequence of C-/has high similarity to a number of vertebrate carboxypeptidases (Fig. 4). Amino acid ident- ity between C-/ and the other carboxypeptidases varies between 40% and 45%. Significantly, the amino acids known to be important for enzyme function are all con- served in the C-/ protein (highlighted in Figs 3 and 4; also refer to the Discussion).

Tissue- and sex-specificity of T-I and C-l expression; regulation of gene expression in response to feeding

The regulation of T-land C-/gene expression in S. vittaturn was investigated by use of Northern blot analysis. Blots of RNA from gut and non-gut tissues were hybridized with a mixture of T-1 and C-I probes, and with a Drosophila actin probe (Fig. 5 and Methods). A 1.1 kb T-Itranscript and a 1.5 C-I transcript were detected in gut tissues (Fig. 5, lanes 1- 3) but were undetectable in non-gut tissues (lane 4). This confirmed the gut-specificity of T-/ and C-/ expression. These results also suggest two additional properties of the T-/ and C-l genes. First, there is a strong sex-specific bias of gene expression. Both genes are only weakly expressed in the guts of males (Fig. 5, lane 3) while they are strongly expressed in guts of female flies (lanes 1 and 2). Second, the T-land C-lgenes are expressed at significant levels in the guts of unfed females (Fig. 5, lane 2). This is in contrast to mosquitoes, where trypsin-like enzymes and transcripts cannot be detected in midguts of unfed females. Both genes are strongly induced by the blood meal (Fig. 5, compare lanes 1 and 2). Note that even though less RNA was analysed in lane 1 than 2 (as indicated by the weaker actin signal in lane l ) , the C-/ and T-/ bands are much stronger in lane 1, indicating a vigorous induction by the blood meal.

In summary, the T-land C-/genes are expressed exclus- ively in the gut. Expression is sex-dependent and is strongly induced in response to the blood meal.

Expression of the T-l and C-l genes in Simulium ochraceum

We investigated whether the S. vittatum T-/ and C-/ se- quences are sufficiently conserved to cross-hybridize with those of S. ochraceum, which is a major onchocerciasis vector in Central America. RNA was extracted from guts and carcasses of field-collected Guatemalan S. ochra- ceumflies and analysed on Northern blots. S. vittaturn RNA was analysed in parallel lanes to serve as a reference. As shown in Fig. 6, there was significant cross-hybridization between the S. vittatum probes and the S. ochraceum RNAs. Transcripts of approximately the same size were detected in the gut but not in the carcass of both species. Relative to actin, a weaker T-/ and C-/ hybridization signal was observed for S. ochraceum. These results may be

Figure 1. Nucleotide sequence and deduced protein sequence of a S. vittaturn trypsin-like (T-l) cDNA. The amino acids composing the 'catalytic triad' are italicized and shadowed. Other highly conserved amino acids presumed to be important for enzyme structure and function are denoted in bold underlined italics. Further details are presented in the legend to Fig. 2 and in the text.

Page 4: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

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::::

NPDLLQCVDAPVLSQADCE--AAYPGE-ITSSNICVGFLE~KDSCQODS~PWCN~L~IV~GY~~PDNP~KVCN~GWIQ~I~

YPDVLKCLKAPILSDSSCK--SAYPCQ-ITSNMFCAGYLE~KDSCQODS~PWCSGKLQGIV~GS~AQK~P~KVC~SWIKQTIASN

YPDLLQCLNAPILTNAQCN--SAYPGE-ITANMICVGYMEGCKDSCQODSOCPWCNGQLQGW~GY~~P~KVC~NAWIQNTI~

..

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-PSQLQ~IVSQSQCASSTYGYGSQIRNTMIC~--ASGKDACQODS~PLVSGG~VGW~GY~AYS~P~ADVAVLRS~ST~SI

*I

*I

II-I

II

I I*

1.

1 *1

*1*1

11**

1***

****

***I

1 I*

*

*I**

** **

* II

***

* c2

c2

sscj

T

sc3

Fig

ure

2. C

ompa

rison

of t

he d

educ

ed T

-lam

ino

acid

seq

uenc

e w

ith th

e se

quen

ce o

f sel

ecte

d try

psin

s. B

Ftry

p, T

-/seq

uenc

e (th

is w

ork)

; TR

Y2-

C,

cani

ne a

nion

ic tr

ypsi

noge

n (P

insk

y et

af.,

1985

); TR

YZ-

H,

hum

an tr

ypsy

noge

n 2

(Em

i eta

/.. 1

986)

; TR

YP

-M,

mou

se tr

ypsi

noge

n (S

teve

nson

eta

f., 1

986)

; TR

YI-

R,

rat t

ryps

inog

en I (M

acD

onal

d et

af.,

1982

); TR

YP

-B,

bovi

ne tr

ypsi

n (T

itani

et a

f., 1

975a

); TR

YP

-X,

Xen

opus

tryp

sin (

Shi

& B

row

n, 1

990)

; DR

OTR

Y, D

roso

phifa

tryps

in-li

ke e

nzym

e (D

avis

eta

/., 1

985)

. Seq

uenc

e co

mpa

rison

was

don

e by

usi

ng th

e FA

STA

pro

gram

(Pea

rson

& L

ipm

an, 1

988)

and

the

Gen

Pep

t 3 6'

3

$

data

base

, rel

ease

72.

0 (D

RO

TRY

) or S

WIS

S-P

RO

T da

taba

se, r

elea

se 22

.0 (a

ll oth

er s

eque

nces

). T-

f als

o ha

s hi

gh s

imila

rity

to th

e un

publ

ishe

d se

quen

ces o

f tw

o m

osqu

ito tr

ypsi

n-lik

e cD

NA

s (s

ee te

xt).

OP

T:

optim

ized

alig

nmen

t sco

re (h

ighe

r sco

res

repr

esen

t bet

ter m

atch

es).

IDE

NTI

TY: p

erce

ntag

e of

the

amin

o ac

ids

that

are

iden

tical

with

T-lo

ver t

he in

dica

ted

rang

e of

am

ino

acid

s. T

here

is n

o st

rict c

orre

latio

n 3 (3

3

betw

een

OP

T sc

ores

and

iden

tity,

in p

art b

ecau

se O

PT

scor

es d

epen

d on

the

leng

th o

f the

seq

uenc

e an

alys

ed a

nd b

ecau

se a

pen

alty

is im

pose

d on

the

OP

T sc

ores

whe

n a

gap

is in

trodu

ced.

[-I:

gap

intro

duce

d in

the

amin

o ac

id s

eque

nce

to o

ptim

ize

alig

nmen

t. I:]:

exa

ct m

atch

to T

-/am

ino

acid

seq

uenc

e. I.]

: 'an

ambi

guou

s m

atch

or

a m

atch

with

a c

onse

rvat

ivel

y rep

lace

d am

ino

acid

'. ['I:

exac

t mat

ch w

ith T

-l of

all

amin

o ac

ids

at th

at p

ositi

on. T

hese

am

ino

acid

s ar

e pr

inte

d in

bold

type

. [.I:

exac

t mat

ch w

ith T

-/of

six

out

of s

even

am

ino

acid

s at

that

pos

ition

. [I]: i

dent

ity o

r con

serv

ativ

e re

plac

emen

t Of a

ll am

ino

acid

s at

th

at p

ositi

on.

t , p

utat

ive

clea

vage

site

of a

ctiv

atio

n pe

ptid

e. T

, mem

bers

of t

he 'c

atal

ytic

tria

d'. S

, am

ino

acid

s in

volv

ed in

det

erm

inin

g en

zym

e su

bstra

te s

peci

ficity

. C,.

cons

erve

d cy

stei

ne re

sidu

es, w

here

n

mat

ches

pai

rwis

e th

e re

sidu

es p

redi

cted

to b

e in

volv

ed in

SS b

ond

form

atio

n. F

urth

er d

etai

ls a

re g

iven

in th

e te

xt.

3

-c

3- m D R

Page 6: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

154 A. Ramos, A. Mahowald and M. Jacobs-Lorena

20 4 0 60 I I I I I I I

CAG TAC CAC ACG CTG CCC GAA ATC TAC AGC TGG TTG GAT CGG CTC GTT CAA GAG CAT CCG GAG CAC GTG GAG CCA G l n Tyr H i s T h r L e u P r o G l u I l e Tyr Ser Trp L e u A s p A r g Leu V a l G l n G l u H i s P r o G l u H i s V a l G l u P r o

t t

10 20

80 100 1 2 0 1 4 0 I I I I I I I I

GTG GTT GGG GGC AAA AGT TAC GAA GGT CGG GAA ATT CGA GGT GTT AAG GTG TCG TAC AAA AAG GGA AAC CCG GTT V a l V a l G l y G l y L y s Ser wr Glu G l y A r g G l u I l e A r g G l y V a l L y s V a l Ser ?yr L y s L y s G l y A s n P r o Val

30 40 50 t t

160 180 200 220 I I I I I I I

GTG ATG G T T GAG AGT AAC A T T CAC GCC CGC GAA TGG ATT ACG GCG GCC ACC ACG ACT TAC CTG CTG AAC GAG CTA V a l Met V a l G l u Ser A s n I l e A l a &a Trp I l e T h r A l a A l a T h r T h r T h r L e u L e u A s n G l u L e u

1 t t

60 70

2 40 2 6 0 2 80 300 I I I I I I I I

L e u T h r Ser L y s A s n Ser Thr I le Arg G l u Met A l a G l u A s n Tyr A s p T rp vr I l e P h e P r o V a l T h r A s n P r o

80 90 100

CTG ACC AGC AAA AAC TCG ACG ATT CGA GAA ATG GCC GAG AAC TAC GAC TGG TAC ATC T T C CCG GTG ACC AAT CCC

t t

3 2 0 3 4 0 360 I I I I I I I

GAC GGT TAT GTG TAC ACG CAC ACC ACC GAC CGA ATG TGG CGC AAA ACT CGC AGC CCA AAT CCG GAC AGC C T T TGT A s p G l y Tyr V a l Tyr T h r H i s T h r T h r A s p A r g Met T r p &a L y s T h r A r g Ser P r o A s n P r o A s p Ser L e u &@

t

110 1 2 0 _ _ _ _ ~ ~ ~ ~

3 8 0 400 420 440 I I I I I I I I

GCC GGC ACC GAC CCC AAC CGC AAC TGG AAC TTC CAT TGG ATG GAG CAG GGC ACG AGC TCA CGG CCT TGC ACC GAA A l a G l y T h r A s p P r o Bap &a A s n T r p A s n P h e H i s T r p Met G l u G l n G l y Thr Ser Ser A r g P r o T h r G l u

130 1 4 0 1 5 0

t

460 480 500 520 I I I I I I I

ACC TAT GGT GGT AAG AAG GCG TTT TCC GAG GTC GAG ACC AGG TCG TTC AGC GAT T T C TTG AAA ACG CTC AAA GGA T h r G l y G l y L y s L y s A l a P h e Ser G l u Val G l u T h r A r y Ser P h e Ser A s p P h e L e u L y s T h r L e u L y s G l y

1 6 0 170 180

t

Figure 3. Nucleotide s e q u e n c e of S. vfttatum carboxypeptidase-like (C-l) cDNA and deduced amino acid s e q u e n c e Conserved amino acids believed to be involved in catalytic function or enzyme structure are italicized and underlined Further details are presented in the legend of Fig 4 and in t he text

explained by one or both of the following: sequence diver- gence led to a weaker hybridization signal in s. ochraceum than in S. vittatum and/or T-l and C-/ transcripts are rarer in the gut of S. ochraceum than in S. vittatum.

Immunolocalization of fhe T-l protein to the gut

Antibodies were produced to a bacterial T-/fusion protein and used for irnmunolocalization studies. Cryosections of sugar-fed or blood-fed flies were reacted with the antibody and the distribution of the antigen was determined by indirect irnmunofluorescence. Figure 7 shows the results of

such an experiment. As expected, T-/ imrnunoreactivity was confined to the rnidgut epithelium (Figs 7C and 7D). The absence of staining with preirnmune serum (Figs 7E and 7F) served as a control for the specificity of the antibody. Interestingly, the rnidgut epithelium of flies main- tained on sugar alone reacted with the antibody (Fig. 7C), indicating that the protein had accumulated even in the absence of a blood meal. Thus, the T-I mRNA in guts of sugar-fed flies (Fig. 5 , lane 2) appears to be translated since the corresponding protein accumulates in the gut epithelium. When viewed at higher magnification, it be- comes apparent that a significant proportion of the antigen

Page 7: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

Gut specific genes from the black fly 155

5 4 0 5 6 0 5 8 0 600 I I I I I I I I

CAA ATC AAG GTG TAC TTG GCC T T C CAC TCG TAC TCC CAA CTG CTG TTG TTC CCA TAC GGT CAC ACC TGT CAG CAC G l n I l e L y s Val Tyr L e u A l a P h e jj!$g Ser E r Ser G l n L e u Leu L e u P h e P r o Tyr G l y H i s Th r Cys G l n H i s

1 9 0 2 0 0

6 2 0 6 4 0 6 60 I I I I I I I

ACC TAC AAT CAT GAC GAT TTA CAA GCA ATT GGA GAT GCG GCG GCC CGT TCG TTG GCT CAA CGA TAT GGC ACC GAT Thr Tyr A s n H i s A s p A s p L e u G l n A l a Ile G l y A s p A l a A l a A l a A r g Ser L e u A l a G l n A r g Tyr G l y T h r Asp

t

2 1 0 2 2 0 2 3 0

680 7 0 0 7 2 0 7 4 0 I I I I I I I I

TAC ACC GTC GGT M T ATC T A T GAT GCG ATC TAT CCG GCG TCG GGT GGC AGC ATG GAC TGG GCG T A T GAC ACA CTA Tyr T h r V a l G l y A s n I l e Tyr A s p A l a Ile P r o A l a Ser G l y G l y Ser && A s p T r p A l a Tyr A s p T h r L e u

2 4 0 2 5 0

7 60 7 8 0 800 8 2 0 I I I I I I L

GAT A T T CCA ATC GCG TAC ACC TAC GAA ‘ITG CGA CCG AGG GAT GGA TGG AAT GGC T T C CAG TTG CCG GCC AAT CAG A s p I l e P r o I l e A l a ‘Qr T h r Tyr ELu L e u A r g P r o A r g A s p G l y T r p A s n G l y phe G l n L e u P r o A l a A s n G l n

2 6 0 2 7 0 280

8 4 0 8 6 0 8 8 0 900 I I I I I I I I

I le I l e P r o T h r G l y G l u G l u T h r V a l Asp Ser V a l V a l T h r I l e L e u L y s G l u Ser A r g A r g L e u G l y Tyr Phe ATC A T T CCG ACT GGT GAG GAG ACC GTG GAC TCG GTG GTG ACG A T A CTC AAG GAG TCG CGT CGG CTC; GGG TAC TTC

2 9 0 3 0 0

920 9 4 0 9 6 0 I I I I I I I

AAC ACC TCT GAT TAA ATG TCA AAA CTG TAT ACC G T T TAT GTA AAT AAA AAT TGG T T C TAA AAA AAA AAA AAA AAA A s n T h r Ser A s p E n d

3 0 9

9 8 0 I I

AAA AAA AAA AAA AGG AA

Figure 3 (continued)

is located intracellularly (Fig. 8) while some immuno- reactive material is associated with the basal layers of the peritrophic matrix.

Discussion

Trypsins are members of a large family of serine endopep- tidases. They differ from other serine proteases by their specificity for arginine and lysine residues and by their ability to activate other zymogens. Amino acid sequence comparison strongly argues that T-/ encodes a trypsin enzyme. Many of the key residues for enzyme structure and function (Kraut, 1977; Huber & Bode, 1978) are con- served in the s. vittatumgene. These include the members of the His-Asp-Ser ‘catalytic triad’, where His-76 functions

to transfer a proton from Asp-120 to Ser-216. Asp-205 forms a salt bridge with amino terminal lle-31 to stabilize the catalytic site. Asp-210, Ser-211, Gly-235 line the sub- strate binding pocket and determine substrate specificity by establishing ionic and hydrogen bonds with lysyl and arginyl side chains. In particular, the negatively charged Asp-21 0 ionically interacts with the positively charged Lys or Arg residue of the substrate. All trypsins have an Asp (or Glu) at this position, while in other serine proteases the corresponding amino acid is uncharged (in chymotrypsins it is usually Ser or Gly). For this reason we believe that T-/ actually encodes an enzyme with specificity closely resem- bling that of trypsins and not of other serine proteases. Of the twelve cysteine residues that are known to form disul- phide bonds in vertebrate trypsins (Kauffman, 1965), six

Page 8: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

10

20

30

40

+ P

-8 720 ..

.. 44.7%/300aa

... CBP

2-R

ED

VQ

VL

LD

QE

RE

EM

LF

NQ

QR

ER

GG

NF

NF

NF

EA

YH

TL

EE

IYQ

~~

VA

EN

~L

VS

K~

SS

F~

P~

KF

S

719 ..

.. 44

.7%/291aa

... C

BPB-A

MDWTSYHDYDEINAWLDSLATDYPELASVEDVGLSYEGRTMKLLKLG

....

...

....

...

..

0” B 68

8 ..

.. 45.3%/296aa

... C

BPA-B

DV

QSL

LDEE

QEQ

MFA

SQSR

AR

STN

TFN

YA

TYH

TLD

EIY

DFM

DLL

VA

EHPQ

LVSK

LQIG

RSY

EGR

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VLK

FS

5

631. ..

. 39.7%/302aa

... CBP

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LI

SN

VR

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LE

SQ

FD

SH

TR

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GH

SY

TK

YN

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3%/2

95aa

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RH

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AK

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NW

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K~

G

k % K

OPT

IDENTITY

+ i

+ i!

BF

carb

Q

YH

TL

PEIY

SWL

DR

LV

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HPE

HV

EPW

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..

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60

70

80

90

100

110

120

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carb

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NI)

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PV

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DCGI

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WSPA

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FVYQ

ATKT

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Page 9: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

140

150

160

17 0

180

190

200

210

220

+ +

+ +

+ +

+ +

+ BFcarb WNF

HWME

QGTS

SRPC

TEmG

OKKA

FSEV

ETRS

FSDF

LKTL

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::

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CBP2-R WD

ANFG

GPGA

SSSP

CSDS

YHGP

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SYSQ

LLLY

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. :.

..:.

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.::.::

CBPB-R FN

AGWC

EVGA

SRSP

CSET

YCGP

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. ...

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::.:

1.

CBPC-H FN

ASWN

SIPN

TNDP

CADN

YROS

APES

BKET

~~FI

RSHL

NEI~

ITFH

SYSQ

MLLF

PYGY

TSKL

PPNH

EDLA

KVAK

IGTD

VltS

TRYE

TRYI

..

: ..

... ::

..

I :

. :: ::.

. ..

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. ...

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. CBPC-R4

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zs

230

240

250

260

27 0

280

290

300

3 10

+

+ +

+ +

+ +

+ +

BFcarb VON

IYDA

IYPA

SOGS

MWQA

YDTL

DIPIAYTYEtRPRM=WNDPQLPANQIIPTGEETVDSWTILKESRRLGYFNTSDYMSKLYTVYV"WF

..

:: :...::.:::::.:::::

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: CBP2-R VO

PICS

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SIWQ

AYD-

LGIKYSFAFELR-DTAFYGFLLPAKQILPTAEETWLGLKTIM

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CBPB-A IOSSTNTIYAAAOGSDWQAKGEGGVKYAYTIEIJUYIY=NY-QPLLPENQIIPTGEETFEGVKWANFVKDTYS

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S

S

S

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0

C B

n P

Flgu

re 4.

CO

mPa

riSO

n Of th

e de

duce

d C

-1 a

min

o ac

id s

eque

nce

with

the

sequ

ence

of s

elec

ted

carb

oxyp

eptid

ases

. BFc

arb,

C-/

sequ

ence

(thi

s w

ork)

; CB

P2-

R,

rat p

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eatic

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boxy

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idas

e A

2 (G

arde

ll et

a/.

,

CB

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an m

ast c

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989)

; CB

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mou

se m

ast c

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nold

s et

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1989

); C

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vine

car

boxy

pept

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e B

(Tita

ni e

ta/.,

197

5b).

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enC

e C

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rison

was

don

e by

usi

ng th

e FA

STA

pro

gram

(Pea

rson

& L

ipm

an, 1

988)

and

the

SW

ISS

-PR

OT

data

base

, rel

ease

21 .O. 2,

S an

d C

repr

esen

t am

ino

acid

s in

volv

ed in

zin

c-bi

ndin

g, s

ubst

rate

posi

tions

bec

ause

of g

aps

intro

duce

d in

the

amin

o ac

id s

eque

nces

. For

the

mea

ning

of o

ther

sym

bols

and

ent

ries

refe

r to

the

lege

nd o

f Fig

. 2.

* 19

88);

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PB

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cray

fish

(Ast

acus

f/uv

iati/

is) c

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ptid

ase

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itani

eta

/., 1

984)

; CB

PA

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bovi

ne c

arbo

xype

ptid

ase

A (H

uero

u et

al., 1

991)

; CB

PB

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rat c

arbo

xype

ptid

ase

B (C

laus

er e

ta/.

, 198

8);

bind

ing

and

disu

lphi

de b

ridge

form

atio

n, re

spec

tivel

y. C

-/am

ino

acid

1 c

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spon

ds lo

the

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ne c

arbo

xype

ptid

ase

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min

o ac

id 1

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a/.,

1991

). H

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his

corr

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nden

ce is

not

mai

ntai

ned

at a

ll

B

-L Ln

4

Page 10: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

158 A. ffamos, A. Mahowald and M. Jacobs-Lorena

Figure 5. Northern blot analysis of trypsin-like (T-l) and carboxypeptidase-like (C-/) gene expression. RNA from dissected mid- guts or carcasses (non-gut tissues) was fractionated by electrophoresis on a denaturing gel, blotted onto a nylon membrane and hybridized at high stringency with a mixture of 3zPP-labelled T-land C-lcDNA probes. After washing, the filters were rehybridized at low stringency with a Dros- ophila actin (Ac) probe to control for the amount of RNA loaded and RNA integrity. Lane 1 : RNA from blood-fed female midguts; Lane 2: RNA from sugar-fed female midguts; Lane 3: RNA from sugar-fed male midguts; Lane 4: carcass RNA.

are conserved in S. vittaturn and probably form disulphide bonds as indicated in Fig. 2. Although methods are avail- able to predict the probable site of signal peptide cleavage (von Hijne, 1986), this site cannot be identified with confi- dence in T-l. By homology to the vertebrate counterparts, activation of the enzyme is likely to occur by cleavage after Arg-30 to generate the conserved N-terminal isoleucine of the mature enzyme. However, no information is available in invertebrates on how or where this cleavage takes place.

Carboxypeptidases belong to a family of zinc-containing exopeptidases that catalyse the hydrolysis of carboxyter- minal amino acids. Carboxypeptidases A exhibit a strong preference for cleaving uncharged amino acids with an aromatic or aliphatic side chain. The closely related car- boxypeptidases B specifically cleave C-terminal lysines and arginines. By analogy with other carboxypeptidases, the following C-l amino acids are predicted to be important

Figure 6. Expression of T-/ and C-/ in Simulium ochraceum. S. ochra- ceum and S. viffatum RNAs were analysed as in Fig. 5. Lane 1: RNA from sugar-fed S. ochraceum midguts; Lane 2: RNA from S. ochraceum carcasses; Lane 3: RNA from S. vittafum carcasses; Lane 4: RNA from S. viftatum midguts.

for enzyme function (numbers in parentheses indicate the corresponding positions in mammalian carboxypepti- dases; Clauser et a/., 1988). His-58(69), Glu-61(72) and His-1 84(196) participate in zinc-binding; Arg-60(71), Arg- 114(127), Arg-132(145), Tyr-186(198), Tyr-236(248), Glu- 259(270) and Phe-269(279) are involved in substrate bind- ing or catalysis as follows. Arg-132 establishes an electro- static bond with the target C-terminal amino acid. Tyr-236 acts as a proton donor for cleavage of the C-terminal amino acid and Glu-259 acts as a nucleophile in the initial attack of the carbonyl group of the substrate at which cleavage will take place. Finally, the tertiary structure is maintained in part by a disulphide bond between Cys-125 and Cys-148. Carboxypeptidases B have an aspartic acid residue at position 243(255) that presumably interacts electrostati- cally with the C-terminal Lys or Arg residue to be cleaved. By contrast, carboxypeptidases A have an uncharged amino acid at that position (usually isoleucine or leucine). The presence of methionine at position 243 suggests that C-l may encode a type A carboxypeptidase.

~

Figure 7. lmmunolocalization of the T-/protein in fly cryosections. Cryosections were prepared from sugar-fed (left panels) or blood-fed (right panels) flies approximately 6 h after the blood meal. Panels A and B show bright-field images from sections of a sugar-fed and a blood-fed fly, respectively. Panels C and D show views under fluorescence optics of the same sections as A and B, respectively. Both fly sections were incubated with chicken polyclonal antibodies to a [TrpEif-l] bacterial fusion protein. The secondary antibody was a fluorescein-conjugated anti-chicken antibody. The gut epithelia of both sugar-fed and blood-fed flies, react with the anti-T-/antibody. Panels E and F are adjacent sections stained and viewed as for C and D, except that preimmune rather than immune serum was used. No fluorescence above background was detected. Arrowheads point to the gut epithelium. 'a' = abdomen; 'b' = blood meal; '1' = thorax. The bar in panel A represents 1 mm.

Figure 8. Distribution of the T-/gene product between the epithelium and the peritrophic matrix. This is a high-power view of the section shown in Fig. 7D. The midgut epithelium (e) has artifactually separated from the peritrophic matrix (Pm). The same field is viewed with bright field optics in panel A and with fluorescence optics in panel B. The T-/protein is detected within the epithelial cells and in the basal part of the peritrophic matrix (double arrows in panel B). Magnification is x 1000.

Page 11: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

Gut specific genes from the black fly 159

Page 12: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

160 A. Ramos, A. Mahowald and M. Jacobs-Lorena

Protease gene expression in insects and other inverte- brates is not as well characterized as for their mammalian counterparts. Most of the available information comes from measurements of enzyme activity in crude insect midgut extracts (reviewed by Chapman, 1985). As a rule, proteo- ly-tic activity is low or undetectable in midguts of adult haematophagous flies before a blood meal and this activity raises to high levels after the blood meal. That enzymes are stored in the form of an inactive precursor proenzyme is still a possibility at this point.

Among invertebrates, the best characterized enzymes are the mosquito trypsins. Trypsin activity is insignificant in the guts of unfed flies (Spiro-Kern, 1974; Briegel & Lea, 1975; Berner et a/., 1983; Borovsky, 1986) and is greatly stimulated by a blood meal (Graf & Briegel, 1989; Barillas- Mury eta/., 1991). Two forms of trypsin were recognized in the latter studies: a group of ‘early forms’ of 32-36 kD that are synthesized immediately after the blood meal, and a ‘late form’ of around 30 kD that is only detected 10 h later. Using an anti-trypsin antibody, Graf et al. (1986) localized the enzyme in mosquito midguts as a function of time after the blood meal. Strong immunoreactivity was initially found in the epithelial cells and in the peritrophic matrix. At later times the immunoreactivity extended progressively further into the food bolus. Eventually the enzyme was excreted. The ‘late’ mosquito gene has recently been cloned. By use of nucleic acid probes and trypsin antibodies Barillas-Mury et a/. (1991) have demonstrated that the late mosquito trypsin is induced from undetectable levels to peak at 24 h (mRNA) or 36 h (protein) after the blood meal. The simi- larity of T-/ with this late trypsin gene was relatively low (31 5% identity over 248 amino acids) while a much better match was found to two unpublished mosquito trypsin cDNA sequences (see Results). It is possible that the latter mosquito cDNA sequences correspond to the ‘early’ tryp- sin genes as opposed to the late gene isolated by Barillas- Mury efal. (1991). It is not known whether S. vittatumalso has a similar set of ’early’ and ‘late’ genes.

Hardly any information is available on the regulation of trypsin or other protease gene expression in black flies. Yang & Davies (1968) localized trypsin activity to the midgut of black flies. In contrast to mosquitoes, these authors detected significant trypsin activity in the midgut of sugar-fed females and males. These results are in agree- ment with our finding that T-/ transcripts are present in the midguts of female and male flies before the blood meal (Fig. 5, lanes 2 and 3) and that the midgut epithelium of sugar-fed females contains immunoreactive material when probed with anti-T-/antibodies (Fig. 7C). Similarly, Lehane (1976) detected protease activity in guts of the stable fly Stomoxys calcitrans prior to the blood meal. Yang & Davies (1968) found that the trypsin activity in midguts of black fly males and sugar-fed females is of comparable magnitude. In contrast, we found that T-/ transcripts were significantly

more abundant in midguts of sugar-fed female than male flies. This discrepancy between enzyme activity and tran- script levels could be explained in several ways. It is possible that it is due to post-transcriptional regulatory events (e.g. translation, protein stability, activation of an inactive proenzyme, etc.), to the expression in males of other trypsin genes not detected by our probe, or to species differences (trypsin activity was not measured by those authors in the midguts S. vittatum males). In any event, the difference in transcript levels in the guts of females and males appears not simply to be a result of the different dietary intake because females express T-/at much higher levels than males, even though both were kept on the same sucrose diet (Fig. 5, compare lanes 2 and 3). Most likely, this difference reflects a sex-specific bias of gene ex- pression. This could be due either to higher transcription rates in females than in males, to differential efficiency of transcript processing, or to differential mRNA stability. lmmunostaining of frozen tissues revealed that a large proportion of the immunoreactive material is located within the midgut epithelial cells at about 6 h after the blood meal. It is possible that as time after the blood meal progresses, a larger proportion of the protein will be found extracellularly associated with the peritrophic matrix and with the blood meal, as is the case for mosquitoes (Graf et a/., 1986). Larvae of the silkworm (Eguchi & Iwamoto, 1976; Eguchi et a/., 1982) and of the cassava hornworm Erinnyis ello (Santos & Terra, 1984) also appear to have sub- stantial trypsin activity associated with midgut epithelial cells.

Little is known about insect carboxypeptidases. Gooding (1 969) was unable to detect carboxypeptidase activity in the guts of blood-fed Aedes, Culex or Melophagus but detected carboxypeptidase activity in the gut of Rhodnius. Carboxypeptidase activity in Rhodnius was also observed by Houseman & Downe (1981) who tentatively proposed that the enzyme is secreted into the gut lumen. Spiro-Kern (1 974) demonstrated carboxypeptidase A activity in the guts of Culexlarvae, but this activity was hardly detectable in the guts of sugar-fed adult flies. Induction of the enzyme by a blood meal was not reported in that work. Houseman etal. (1 987) identified carboxypeptidase A and B activity in the midgut of the stable fly Stomoxys calcifrans. Based on gel exclusion chromatography they assign a 26,000 and 28,500 molecular weight respectively, to these enzymes. These authors suggest that the enzymes are secreted into the gut lumen. However, Terra etal. (1979) suggest that in Rhynchosciara larvae both carboxypeptidases are re- stricted to intracellular locations. So far, the only insect carboxypeptidase-like gene to be sequenced is a mosquito yolk-associated serine carboxypeptidase (Cho et a/., 1991). The enzyme is stored in oocytes and is apparently activated during embryonic development when it is pre- sumed to function in yolk degradation. This mosquito

Page 13: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

Gut specific genes from the black fly 161

carboxypeptidase gene is not expressed in the gut. Thus, C-l is the first insect gut carboxypeptidase to be cloned.

In summary, we have isolated two gut-specific genes encoding proteins with similarity to digestive enzymes. The genes appear to be coordinately expressed and regulated according to sex and food intake. The materials generated by this study provide useful tools to explore the molecular basis of gene regulation in the gut of insects.

Experimental procedures

Live materials

Rearing of S. viffafum and blood-feeding were done as described (Bernard0 eta/., 1986). S. ochraciumflies were a kind gift from Dr E. Cupp.

Isolation of midgut specific genes

Four genomic equivalents of a S. viffafum genomic library (Jacobs-Lorena eta/., 1988) were differentially screened with 32P- labelled cDNA probes made from polyadenylated RNA extracted (see below) from midgut and non-midgut tissues. Six clones were recovered that hybridized preferentially with the midgut probe. The coding region(s) within the genomic DNA inserts were mapped as follows. DNA from each clone was digested singly or doubly with Sal I and Barn HI, and the fragments from each clone were fractionated in duplicate by electrophoresis on 1 % agarose gels. One gel was transferred to a nitrocellulose filter and the other one was kept at 4°C. The blotted DNA was hybridized and washed under high stringency conditions (see below) with midgut 32P- labelled cDNA to identify the mRNA-encoding fragments. One strongly hybridizing fragment from each phage was recovered from the gel that was stored at 4°C and subcloned in the pGEM4 vector (Promega). The midgut-specificity of each of the six clones was verified by hybridization of random-primed 32P-labelled probes (Feinberg & Vogelstein, 1983) to Northern blots of midgut and non-midgut RNAs. Two of the positive subclones were used to screen a cDNA library. The library was constructed in the Lambda ZAP I vector (Stratagene) using S. vittafum polyadenylated RNA from midguts of blood- and sugar-fed flies (Ramos & Jacobs- Lorena, unpublished). The cDNAs with the largest inserts were further characterized.

Nucleic acid preparation and analysis

RNA was extracted by the method of Hough-Evans eta/. (1980). For Northern blot analysis RNA was fractionated in 1 5% agarose- formaldehyde gels and transferred to Genescreen nylon mem- branes (DuPont-New England Nuclear). The blots were first hy- bridized with a mixture of T-/ and C-/probes under high-stringency conditions as follows. Hybridization in 1% BSA/1 mM EDTA/O.5 M

NaHP04/7% SDS, pH 7.2 (Church & Gilbert, 1984) at 65°C and final wash in 0.1 x SSC/O.l% SDS at 65°C (1 x SSC is 150 mM NaClll5 mM sodium citrate). The blots were then hybridized and washed under low stringency conditions with a Drosophila actin probe (Kay & Jacobs-Lorena, 1985) using the same buffers but lowering the temperature to 42°C. DNA sequencing was per- formed with the Sequenase kit of United States Biochemical Co. following the manufacturer's protocol. DNA sequences were ana- lysed with the MacVector program (International Biotechnologies Inc.).

Production of a T-l fusion protein and antibodies

A 0.71, kb Eco RI T-/ cDNA fragment was subcloned into the expression vectors PATH 1, 3 and 11 (Hardy & Strauss, 1988). Each of these PATH vectors links the inserted DNA to the E. coli frp€ gene in a different reading frame. The constructs were transformed into DH5alpha E. coli cells and several independent colonies from each construct were tested by inducing the pro- duction of fusion protein with P-indoleacrylic acid (Hardy & Strauss, 1988). The proteins were extracted, fractionated in 10% SDS acrylamide gel and stained with Coomassie blue. Several clones carrying the PATH 1 1/T-/construct (but none of the PATH 1 or 3 constructs) produced a fusion polypeptide with a mobility corresponding to a 65 kD protein (the frpE portion of the fusion protein is about 37 kD). The fusion protein from one of the PATH l l /T - l clones was purified by fractionation on 10% SDS- acrylamide gels. After electrophoresis the gels were immersed in cold 0.1 M KCI to visualize the proteins (Nelles & Bamburg, 1976) and the 65 kD fusion protein band was excised from the gel and used for immunization of chickens (done by Pocono Laboratories, Canadensis, Pa.). Recovery of antibodies from the egg yolks was essentially as described by Polson eta/. (1 980).

lmmunolocalization of the product of the T-l gene

lmmunolocalization studies were performed as follows. Flies were dipped in 0.5% Kodak photoflow solution, transferred to a solution containing 4% paraformaldehyde in PBS and the head and anal plate were removed. After 1 h in the fixative flies were transferred to a solution containing 20% sucrose in PBS and left overnight at 4°C. The flies were then immersed in OCTand flash frozen in liquid nitrogen. Sections, 15 pn thick, were cut with a cryotome. The dried sections were stored at -20°C until needed. Sections were warmed up to room temperature and incubated with a biocking solution containing 3% goat serum (Cappel) and 0.1% Tween-20 in PBS for 30 min at room temperature. The immune and pre- immune chicken sera were incubated with purified frpE protein (not the fusion protein) for several hours at 4°C and then diluted 1 :lo00 with blocking solution. The diluted antibodies were incu- bated with the tissue sections for 4 h at room temperature. After several washes with blocking solution, a flourescein-conjugated anti-chicken secondary antibody (Cappel) diluted 1 :2500 in blocking solution was added and incubated at room temperature for 2 h. Finally the sections were washed with PBS containing 0.1 % Tween-20 and mounted with glycerol containing 2% n-propyl gallate (Giloh & Sedat, 1982). The sections were viewed and photographed (same exposure for all sections) in a microscope equipped with epifluorescent optics.

Acknowledgements

We are indebted to Dr E. Cupp for providing the black flies used in this research. Expert technical assistance by M. Doman is grate- fully acknowledged. We are also grateful to Ms Ann Riedl for comments on the manuscript. This work was supported by funds from the John D. and Catherine T. MacArthur Foundation and by a grant from the National Institutes of Health.

GenBank accession numbers

L08428, trypsin; L08481, carboxypeptidase.

Page 14: Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

162 A. Ramos, A. Mahowald and M. Jacobs-Lorena

References

Barillas-Mury, C., Graf, R., Hagedorn, H.H. and Wells, M.A. (1 991) cDNA and deduced amino acid sequence of a blood meal- induced trypsin from the mosquito, Aedes aegypti. Insect Bio- chem 21 : 82-31,

Bernardo, M.J., Cupp, E.W. and Kiszewski, A.E. (1986) Rearing black flies (Diptera: Simuliidae) in the laboratory: colonization and life table statistics for Simulium vittatum. Ann Entomol Soc Am 79: 61 M 2 1 .

Berner, R., Rudin, W. and Hecker, H. (1983) Peritrophic mem- branes and protease activity in the midgut of the malaria mos- quito, Anopheles Stephens; (Liston) (Insecta: Diptera) under normal and experimental conditions. J Ultrastr Res 83: 195- 204.

Borovsky, D. (1986) Proteolytic enzymes and blood digestion in the mosquito, Culex nigripalpus. Arch Insect Biochem Physiol3:

Briegel, H. and Lea, A.O. (1975) Relationship between protein and proteolytic activity in the midgut of mosquitoes. J Insect Physiol

Chapman, R.F. (1 985) Coordination of digestion. Comparative Insect Physiology, Biochemistry and Pharmacology, Vol. 4 (Kerkut, G.A. and Gilbert, L.I., eds), pp. 213-241. Pergamon Press, New York.

Cho, W.-L., Deitsch, K.W. and Raikhel, A S . (1991) An extraovar- ian protein accumulated in mosquito oocytes is a carboxypepti- dase activated in embryos. Proc Natl Acad Sci USA 88: 10821- 10824.

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