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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
150 A. Ramos, A. Mahowald and M. Jacobs-Lorena
2 0 40 6 0 I I I I I I I
CTG ACG ACC G T T ATC AGT TAC TTT GCC CTG GTG GCA T T C GCC CTA GTC GGC G T T T C T TAC GCC ACA CCC AAA GCC L e u T h r Thr V a l I l e Ser Tyr P h e A l a L e u V a l A l a P h e A l a L e u V a l G l y V a l Ser Tyr A l a Thr Pro Lys A l a
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TCG ATC AAT GGA CGT Al l ‘ G T T GGC GGC GAA ATG ACC GAT ATC AGT CTC Al l ’ CCC TAC CAA GTG TCC GTG CAA ACC Ser I le A s n G l y A r g Val G l y G l y G l u M e t T h r A s p I le Ser L e u I le P r o Tyr G l n V a l Ser V a l G l n T h r
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GCG ATA TCC AGT TAC GGA T T C ATC CAT CAC TGT GGT GGC TCG ATC ATC AGT CCC CGA TGG GTG G T T ACG GCC GCT A l a I l e Ser Ser Tyr G l y Phe Ile His His G l y G l y Ser Ile Ile Ser P r o A r g Trp V a l V a l Thr A l a A l a
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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.
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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
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C,
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ne a
nion
ic tr
ypsi
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n (P
insk
y et
af.,
1985
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H,
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an tr
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n 2
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i eta
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se tr
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n (S
teve
nson
eta
f., 1
986)
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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
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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
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
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
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
PIY
VLK
FS
5
631. ..
. 39.7%/302aa
... CBP
B-R
HY
EV
LI
SN
VR
NA
LE
SQ
FD
SH
TR
AS
GH
SY
TK
YN
KW
ET
IE
AW
KI
G
z s 608. ..
. 37.
3%/2
95aa
...
CBPC-H
YE
IL
IH
DL
QE
EI
EK
QF
DV
KE
DI
PG
RH
SY
AK
YN
NW
EK
fV
AW
K~
G
k % K
OPT
IDENTITY
+ i
+ i!
BF
carb
Q
YH
TL
PEIY
SWL
DR
LV
QE
HPE
HV
EPW
GG
KSY
EG
RE
IRG
VK
VS
..
..
..
.
:::: :::
....
....
....
. :.
:.:.
: ..
.:
:
? z
...
....
..
..
..
: ::
:::.
.. .
:..
:: .:
::.:
. 11,
....
....
....
....
....
. 1
.:::
::
:
...
m .
.
..
.:
..
....
....
....
....
: :.
..::
:..
.:..
..
. .
.
.:..
..
....
....
....
...
:..
....
.:..
..
. ..
..
.:..
..
....
....
....
...
:..
:..
. .:
..
607 ..
.. 38.6%/295aa
... CBPC-II
YE
IL
IH
DL
QE
EI
EK
QF
DV
KD
EI
AG
RH
SY
AK
YN
DW
DK
IV
SW
KI
G
2 607.. . .
39.7
%/29
0aa
... C
BPB-B
TEHS
YEKY
NNWE
TIEA
WTEQ
VASE
NPDL
ISRS
AIGT
TFLG
NTIY
LLKV
G 3
0 m
..
.:
..
.: .
: ..
....
....
.. :
...
:..:
.:
:.
*I1
I*
I
II I
I*I
II
* I
II I
I*
I
50
60
70
80
90
100
110
120
13 0
+ +
+ i
i
i
i
+ i
BF
carb
Y-K
KG
N-P
VV
MV
ES
NI)
IT~
~L
LN
EL
LT
--S
~S
TIR
~E
~D
~IF
PV
~P
I)C
Y~
TH
TT
D~
TR
SP
NP
DS
L-C
AG
TD
P~
..
:..
...
. :::
:::.
:.::
. .
:..
....
.. :
....
: ..
....
....
....
....
....
....
... :
:.
:.::
:::
CBP2-R
T-C
GD
K-P
AIW
LD
AG
IHQ
AT
AL
~~
KIA
SD
YG
TD
PA
ITS
LL
NT
LD
1F
LL
P~
PI)
C~
FS
Q~
N~
TR
SK
RS
GS
G-C
V~
DP
~
.. .
......
......
......
......
...
:...
: ..
.:
....
....
....
....
....
....
....
...
:.::
:::
CBPB-A
KC
GA
DK
-PII
FID
OG
IHA
RE
WIA
PS
TV
mIV
NE
FV
---S
NS
A~
DD
ILS
NV
NF
YV
MP
TIN
PI)
CY
AY
TF
TD
D~
~T
RS
E~
S~
~K
GA
DP
~
....
...
.::.
::::
:.::
....
..
....
....
....
: ..
....
....
....
.:.
::::
::
....
. :.
:.:.
:::
CBPA-B
T-G
GS
NR
PA
IWID
LG
IHS
RIT
QA
n=
VW
FA
KK
FT
ED
YG
QD
PS
FT
AIL
DS
~IF
LE
I~P
I)C
FA
FT
HS
Q~
~T
~~
SS
SL
-CV
~~
..
. :.
....
...:
::::
:..:
..
....
..
....
...
:.:.
.::.
: I::::: :.:
::::
::::
. ..
: : 1
. :::I
CBPB-R
KT
RP
NK
-PA
IFIM
JG~I
SP
AF
CQ
WF
VR
EA
VR
TY
NQ
EIH
MK
QL
LD
EL
DF
YV
LP
VV
NID
DY
VY
TW
TK
D~T
RS
TS
S-C
LO
VR
PN
RN
:.
..
....
... :I::::...:
..
..
..
. :...
...
....
....
. ..
... :
::..
. :.
.:::
::.:
: :.
.: : :::
t:
:
CBPC-IX
E-KN
ERRK
AIFM
DCGI
HARW
WSPA
FCQW
FVYQ
ATKT
YGRN
KIMT
KLLD
RMNF
YILP
VFNV
WYIW
SWTK
NRMW
RKNR
SKNQ
NSK-
CIGT
DL”
....
....
... ::
::::
:..:
....
....
....
....
....
...
CBX-M K-KDGERKAIFMDCGIHARWQISPAFCQWFVYQATKSYGKNKIMTKLLDRMNFnnPVFNVWYIWSWTQD~RNQNST-CIGTDL“
....
....
....
i:::::..:
....
..
....
:. .
....
....
.. ::
:.:: ::
.:::
::::
:...
.:
1.:
::
I::
C8PB-B
KP
GS
NK
-P
AV
FM
DC
GF
IS
PA
FC
QW
FV
RE
AV
Rm
GR
....
....
. ..
....
....
..
...
....
....
....
....
..
..
..
..
....
..
..
..
.
..
..
:::..
. : ::
::::
.::.
:..:
: ::: :::
..
.
..
..
.
.
II
II I
I Il
****
*lll
* I
I I1
11
11
I 11
*
**
Ill
1*1***
1**
I *I
**
* z sz
S
C S
140
150
160
17 0
180
190
200
210
220
+ +
+ +
+ +
+ +
+ BFcarb WNF
HWME
QGTS
SRPC
TEmG
OKKA
FSEV
ETRS
FSDF
LKTL
KGQI
~LAF
IISY
SQLL
LFPY
GH~Q
H~~D
DLQA
IGDA
AARS
LAQR
YGTD
YT
:. .
....
....
::...:
t :.
x:::
..:.
::
.:.
....
....
....
....
....
....
....
..
t.
..
..
::
..:
. .:
:.:.
CBP2-R WD
ANFG
GPGA
SSSP
CSDS
YHGP
KPNSEVKVKSIVDFIKS-HGK~FITLHSYSQLLMFPYGYKCTKPDDFNELDEVAQKAAQALKRLHGTSYK
:.::
: :
:.:..:
:...:.
: ..
::::
: :.
.:
..
..:::
::.:::
::::
....
....
.. .:
:...
...:
. .:
.. .
:: :
. CBPB-A WSFHWDEVGASDSPCSDIYAGPEPFS~~IL~~I~LT~SYSQL~PWG~SDLPDDWQDL~~A~AtTA~GTRYE
:.
. ..:.::.::.:::
:: :
::::..
:. ::
.:.
......
......
......
......
: ..
... :
....
..:.
.:
:::.
:.
CBPA-B WD
AGFGKAGASSSPCSETYHOKYA
NSEV
EVKS
IVDF
VKD-
HGNF
)tAF
LSIH
SYSQ
LLLY
PYGYTTQSIPDKTE~QVAKSAVEAtKSLYGTS~
.: : : :.:
..::.:
:: : .
1:
::..
....
...
....
....
:::::
..:.::
.....
. :.
..:.
:. .
.::.
.::
.::.::
CBPB-R FN
AGWC
EVGA
SRSP
CSET
YCGP
APESEKETKALADFIRNmLSTIlCAYLTIHSYSQ~YPYSYDYKLPENYEElClNALVKGAAKELATLHGTK~
....
...
t:
..
.:
:.
. :: ::
.. .
. :..
. ...
......
......
......
....
. ::.::. .
....
1.
::.:
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 :
. :: ::.
. ..
:...
. ...
......
......
......
.
. ::
.::
..
:. .:
. ::
.: :
. CBPC-R4
FDVSWDSSPNTNKPCLPAPESEKETKA~FIRSHLNSI~YITFHSYSQMLLIPYGYTFKLPPNHQDLLKV~IATDAtS~YETRYI
.. : ..
....
::.:::
1.
: ::
x...
..
....
. ::
.::.
.:z:
::..
:.::
....
. .
:. .:
....
..:.
..::
.::.:.
CBPB-B FDAGWCSIGASNNPCSETYCOSAAESEKES~V~FI~LSSI~YLTIHSYSQ~YPYSYDYKLP~E~L~GAVKK~LHG~S
...
..
..
....
....
..
...
..
....
.
. .
.
..
II
I I
111
** 1
1*
I **
* I
I 1.
1 I
I l*
llll
I***
**I
II*
*I
I I
I I*
1
*I
* Il
* *
I c
s
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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ork)
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,
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an m
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ell c
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nold
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se m
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ell c
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xype
ptid
ase
A (
Rey
nold
s et
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1989
); C
BP
B-B
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vine
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boxy
pept
idas
e B
(Tita
ni e
ta/.,
197
5b).
SeqU
enC
e C
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rison
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don
e by
usi
ng th
e FA
STA
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gram
(Pea
rson
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ipm
an, 1
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and
the
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ISS
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OT
data
base
, rel
ease
21 .O. 2,
S an
d C
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esen
t am
ino
acid
s in
volv
ed in
zin
c-bi
ndin
g, s
ubst
rate
posi
tions
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ause
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aps
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duce
d in
the
amin
o ac
id s
eque
nces
. For
the
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ning
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ther
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bols
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ent
ries
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r to
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lege
nd o
f Fig
. 2.
* 19
88);
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fish
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acus
f/uv
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is) c
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xype
ptid
ase
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itani
eta
/., 1
984)
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ne c
arbo
xype
ptid
ase
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uero
u et
al., 1
991)
; CB
PB
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rat c
arbo
xype
ptid
ase
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laus
er e
ta/.
, 198
8);
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ing
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lphi
de b
ridge
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atio
n, re
spec
tivel
y. C
-/am
ino
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the
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ne c
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xype
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min
o ac
id 1
1 (H
uero
u ef
a/.,
1991
). H
owev
er, t
his
corr
espo
nden
ce is
not
mai
ntai
ned
at a
ll
B
-L Ln
4
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.
Gut specific genes from the black fly 159
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
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.
162 A. Ramos, A. Mahowald and M. Jacobs-Lorena
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