Gene, 75 (1989) 73-83

Elsevier

73

GEN 02858

A salivary gland-specific, maltase-like gene of the vector mosquito, Aedes aegypti

(Recombinant DNA; salivary glucosidase; tissue-specific expression; cDNA and genomic sequences; vector biology)

Anthony A. James, Karen Blackmer and Jeffrey V. Racioppi *

Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02115 (U.S.A.)

Received by S.T. Case: 27 Juiy 1988

Revised: 13 October 1988

Accepted: 16 October 1988

SUMMARY

Genomic and cDNA clones of a gene expressed specifically in the salivary glands of adult Aedes aegypti have been isolated and sequenced. This gene encodes an abundant mRNA that is transcribed throughout the male salivary gland but only in the cells of the proximal lateral lobes of the female gland. The deduced protein has many basic amino acids, several possible sites for ~-glycosylation, and displays striking similarities with the products of a yeast maltase gene and three previously unidentified genes from Drosophila melanogaster. We

propose the name ‘Maltase-like Z’ (MaN) to designate this gene. The presumed function of this gene product is to assist the mosquito in its sugar-feeding capabilities. The mosquito and fruitfly genes have similar structural features 5’ to the protein coding regions, indicating that these genes may share common control mechanisms.

INTRODUCTION

Biochemical and molecular biological techniques are being used to study the arthropod vectors of disease. A recent report of transformation in the mosquito (Miller et al., 1987) indicates that it is now possible to study the effects of a modified gene on host and parasite development. Using strong pro- moter sequences derived from endogenous genes, it

Correspondence to: Dr. A.A. James, Dept. of Tropical Public

Health, Harvard School of Public Health, 665 Huntington Ave,

Boston, MA 02115 (U.S.A.), Tel. (617)732-2028, Fax (617)738-

4914.

* Current address: Division of Science, College of Basic Studies,

Boston University, 871 Commonwealth Ave., Boston, MA

02215 (U.S.A.) Tel. (617)353-4785.

will be feasible to direct the expression of recombi- nant RNAs or proteins in target tissues in the vector hosts. As an initial effort in this direction, we have chosen to isolate genes expressed specifically in the salivary glands of mosquitoes.

The salivary glands of adult hematophagous arthropods have several features that make them attractive for biochemical and molecular analyses. Several of the salivary gland proteins are synthesized

Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA

complementary to RNA; dNTPs, deoxyribonucleoside triphos-

phates; DTT, dithiothreitol; kb, kilobase or 1000 bp; Mall,

~~~ra~e-~~e Z gene; mRNA, messenger RNA; nt, nucleotide(s);

ORF, open reading frame; poly(A)+ RNA, polyadenylated

RNA; riboprobe, radioactively-labelled, in vitro-synthesized,

single-stranded RNA.

037X-l 119~89~~~~.SO 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

in abundance during adult life (Racioppi and Spielman, 1987) and therefore the transcriptional control sequences of their genes are ideal candidates for recombinant DNA studies. Mosquito salivary glands often harbor viruses and protozoan parasites that infect vertebrates. During bloodfeeding, sali- vation provides the means for transmitting these pa- thogens to new hosts. Addition~ly, the saliva contains ~tihemostatic and ~ti-immune agents that facilitate bloodfeeding (Ribeiro, 1987) and in- crease the efficiency of pathogen transmission (Titus and Ribeiro, 1988).

A. aegypti is a tropical and subtropical mosquito with world-wide dist~bu~on. It is an efficient vector for the viruses that cause dengue and yellow fever. We have isolated the gene for an mRNA that is abundantly expressed in the adult salivary glands of this mosquito, characterized its transcriptional and tr~slational products, and noted the similarity of the deduced protein with a yeast maltase.

MATERIALS AND METHODS

(a) ~o~~ito strains

The mosquitoes used in this study were principally the Rockefeller strain of A. aegypti, although some of the work was done with a strain isolated from the Bahamas. Mosquitoes were reared using standard protocols (Ross&no1 and Spielman, 1982) and all animals used in the following procedures were usually 3-5 days old after adult emergence.

(b) ~ucIeic acid isoIation and Ia~~jiug protocois

RNA was prepared from whole adults and iso- lated tissues using a protocol modified from Chirgwin et al. (1979). Poly(A)” RNA was prepared following the protocol of Maniatis et al. (1982).

DNA was prepared from adult mosquitoes using the protocol of Bender et al., (1983). DNA was labelled by a standard nick-translation reaction (Rigby, 1977). RNA was randomly end-labelled with [ T-~*P]ATP (MaizeIs, 1976). Riboprobes were synthesized from coding sequences cloned into the plasmid, pGEM1, using 3H- and 35S-substituted ri- bonucIeotides in reaction conditions supplied by the manufacturer (Promega Biotec).

(c) Library construction and screening

Purified poly(A)+ RNA from pre~bloodfed fe- males (Bahama strain) was used to construct a cDNA library (Gubler and Hoffman, 1983) in phage Agtll (Young and Davis, 1983). The cDNA was packaged in vitro using commercial packaging ex- tracts supplied by Promega-Biotec and Vector Cloning Systems. end-labelled total salivary gland RNA, total abdominal RNA, and a clone, MElg, with mosquito sequences homologous to the riboso- ma1 RNAs (K.B. and A.A.J., unpublished) were used to differentially screen the cDNA libraries. Isolated mosqujto cDNA sequences were nick- translated and used to screen a RCharon 4 A (Blattner et al., 1977) genomic library of A. aegypti (Rockefeller strain, gift of H. Hagedorn, Cornell University).

Analyses of restriction enz~e-digested DNAs were performed according to Southern (1975).

(d) Developmental and spatial analyses of RNA ex- pression

Stage-, tissue- and sex-specific RNAs were examined by Northern analyses (Colot and Rosbash, 1982). Hybridizations in situ to tissue sections were performed as described (James and Vincent, 1986), except that the tissue was embedded in pa&in be- fore sectioning, and riboprobes were used.

(e) Nucleotide sequence determination and analysis

Subcloned genomic and cDNA fragments were sequenced by the method of Sanger et al. (1977) using a kit supplied by US Biochemicals. Oligo- deoxynucleotide primers were either obtained com- mercially or constructed with a Biosearch DNA syn- thesizer. The clones were sequenced multiple times and in both directions. Reported sequence data represent a consensus of at least three separate se- quence readings. RNA sequencing experiments were essentially identical to the DNA sequencing experi- ments except that total salivary RNA was used as the template. The primer extension reaction protocols were supplied by Dr. Brian Rymond (Brandeis University). In a typical reaction, 1 to 3 pg of total RNA was annealed with 32P-end-labelled primer for 1 h at 42’ C in a buffer containing 50 mM Tris . HCl,

pH 8.0, 40 mM KCl, 0.5 mM EDTA in a total reaction volume of 15 ~1. After cooling, one ~1 each

of 200 mM MgCl,, 2.5 mM dNTPs, 20 mM DTT,

1 mg/ml of actinomycin D and 2-6 units of reverse

transcriptase were added. The reaction was in-

cubated at 37°C for 30 min. Reactions were

phenol/chloroform extracted, ethanol precipitated,

vacuum-dried, and resuspended in 5 ~1 of 10 mM

Tris * HCl, pH 8,1 mM EDTA. Samples were run on

6% polyacrylamide sequencing gels for visuali-

zation.

Computer analyses of sequence information was

performed using the Amersham Staden software

package and the facilities of the Molecular Biology

Computer Research Resource at the Dana-Farber

Cancer Institute. The PIR (George et al., 1986) and

PseqIP (Claverie and Bricault, 1986) databases were

screened for protein homologies.

RESULTS AND DISCUSSION

(a) Isolation of a gene expressed specifically in the

adult salivary glands

We differentially screened a cDNA library to

select genes expressed in the salivary glands of

A. aegypti. Three cDNA clones were isolated that

had homology to a single gene. The longest cDNA

was named 1SGB46, and the gene was subsequently

designated B46. To determine the expression pattern

of the gene, a gel-purified B46 cDNA fragment was

nick-translated and used to probe transfers of RNA

isolated from embryos, larvae, pupae, adult salivary

glands, and adult carcasses. Carcass RNA was

made from females from which the salivary glands

were extirpated. As seen in Fig. 1, the B46 cDNA

is homologous to a salivary-gland-specific poly-

(A) + RNA of approximately 2.1 kb in length that

is present in both adult males and females. We

found no evidence for this RNA being transcribed or

accumulated during the embryonic, larval and pupal

stages of development. An additional 5.1-kb RNA is

occasionally seen and may be an unspliced precursor

RNA.

Male and female salivary glands differ significantly

in size and morphology. Since only females feed on

blood, this dimorphism could reflect the different

feeding capabilities of the two sexes. Hybridization

E LE L3 L4 PE PL S SC FM

C 11,846

-5.1

Fig. 1. Stage and tissue-specific expression of the RNA homol-

ogous to the cDNA clone, ISGB46. RNA was prepared from

different stages of development and different tissues, electro-

phoresed in 0.9% agarose gels (gels were prepared with formal-

dehyde and MOPS buffer as described in Maniatis et al., 1982),

transferred to filters and probed with nick-translated 1SGB46.

Panel A: Expression of the 846 RNA in the pre-adult stages of

the mosquito; embryos (E), first- and second-instar larvae (LE),

third-instar larvae (L3), fourth-instar larvae (L4), early pupae

(PE), late pupae (PL), and female salivary gland total RNA (S).

5 pg of RNA for each stage and I pg of salivary RNA were

loaded per lane. Panel B: The filter was stripped of label and

reprobed with the clone MEZg to show the 5 S rRNA. Panel

C: Expression of the 846 RNA in adult stages. 2 fig of female

salivary gland (S) and female carcass (C) total RNA, and 1 pg

of male (M), and female (F) poly(A) + RNA were loaded per lane.

The sizes of the observed RNAs, 5. I and 2. I kb, were estimated

by comparison with EcoRI- and HindIII-digested phage I DNA

run in the same gel (not shown).

in situ to adult tissue sections with radiolabelled

nucleic acid probes was used to show those cells in

the salivary glands that were accumulating the B46

RNA. The sections were hybridized with in vitro

synthesized single-strand antisense RNA homol-

ogous to a large portion of the B46 cDNA. Control

hybridizations used sense-strand probes. The B46

RNA is sufficiently abundant that exposure times of

6-18 h with ‘H-labelled probes produced the best

results. A total of 22 experimental and 3 control

female thoraces, each containing a pair of glands,

was examined. All of the experimental thoraces

showed the same result, that the 846 RNA is ex-

pressed only in the proximal lateral lobes of the

female salivary gland. Five different preparations of

isolated female glands showed the same result

(Fig. 2). Control sense-strand RNA probes did not

hybridize. A total of 19 experimental and 2 control

male thoraces were examined and all of the slides

treated with the antisense probe showed that the B46

RNA is expressed throughout the male salivary

glands (Fig. 2). Again, control slides failed to show

any hybridization signal.

16

Fig. 2. Hybridization of RNA probes in situ to salivary glands of A. aegypti. Tissue sections of thoraces and dissected female salivary glands were analyzed for the localization ofthe B46 RNA. Panel A: A pair of female glands just after dissection. Each gland is composed of two lateral lobes and a single medial lobe (M), the lateral lobe is divided into proximal (PL) and distal (DL) regions with respect to the common salivary duct. The bar represents 0.5 mm. Panel B: Giemsa-stained section of the female salivary glands hybridized with antisense B46 riboprobe. The smail bars delimit the proximal lateral regions of the glands; scale is the same as in Panel A. Panel C: Dark-field image of(B); note that the grains indicating specific hybridization are localized over the proximal lateral regions of the glands and note that the medial lobe displays no hybridization signal. Panel D: Male salivary gland prepared as in (B). The bar represents 0.25 mm. Panel E: Giemsa-stained section of a male thorax showing portions of the male salivary gland. SD, salivary duct. The bar is the same as (D). Panel F: Dark-field image of (E); note that the medial lobe and visible portions of the lateral lobes show a strong hybridization signal.

(b) Features of the B46 cDNA and genomic sequence

The primary sequence of the ?SGB46 cDNA was determined to define the limits and properties of the coding regions. The results of the sequencing proce- dures are shown in Fig. 3 and Fig. 4. The cDNA has a 1758-nt ORE;. The 3’-end has the appropriate stop codons and consensus polyadenylation sequence. Presence of the ORF was confmed by fusing it in-frame with a 1ucZ gene (we used pUR29 1; Ruther and Milller-Hill, 1983). The recombinant gene pro- duced a hybrid protein with the expected molecular weight (J.V.R. and A.A.J., unpub~shed). Together these results suggest that the B46 RNA is an mRNA. On later comparison with genomic sequences, the

cDNA was shown to be almost full-length, missing some 44 nt from the 5’-end (Figs. 4 and 5).

We used nick-translated B46 cDNA to screen an A. aegypti genomic library and recovered clones with homology to the cDNA. Mapping the cDNA into a genomic clone, ;iAEGB46-I, confirmed that there was a large intron of approximately 3 kb near the 3’-end of the gene (K.B. and A.A.J., unpublished), and this along with the coding sequences would give a precursor RNA of approximately 5.1 kb in size. These data support the suggestion that the high- molecular-weight RNA occasionally seen in the Northern analyses is a precursor RNA.

A genomic fragment containing the 5’-end of the gene was subcloned from AAEGB46-1 and se-

A ,ksGB46 zz- c_-___I _..__d

m-_------r=_ _-f +_--_-__ = G z!E -- ----- e- - ________

I , I I I1 t I b I I I I f I I I \ 2 / 4

61 HE Xb 6 /a 10 12 / / 16 I.8

A Xb ‘RII

61 AAEGB46+/6kb

Fig. 3. Molecular maps and sequencing strategies of the cDNA clone LSGB46, map A, and a 16kb fragment of the genomic clone MEGB46-I, map B. The thick lines in A and B represent the molecular maps with various restriction endonuclease sites denoted. The thin arrows represent the regions sequenced in the direction indicated by the arrowheads (see MATERIALS AND METHODS, section e). Multiple arrows indicate the number of times each region was sequenced. The dashed arrows indicate sequences obtained using dITP instead of dGTP. The thick arrow in B indicates the start point and direction oftranscription in the genomic clone. The numbers refer to the length in kiio-

bases. Abbreviations: A, AvaI; C, ClaI; El, EcoRI; EV, EcoRV; HII, HincII; HIII, HindIII; Xb, XbaI; Xh, XhoI.

quenced (Fig. 3 and Fig. 5). The 1.6-kb fragment potentially contains 197 bp of transcribed sequence, 139 of which are protein-coding sequences (the ana- lysis of the 5’-end is described below). The hexanu- cleotide, TATAAA, present 30 nt upstream from the 5’ -end of the transcription start point (see RESULTS

AND DISCUSSION, section c), is the most likely RNA polymerase II recognition site. However, the region directly 5’ to this recognition site is AT-rich and another TATAAA sequence is present at nt -162.

A repeat sequence of 18 nt, 5’-ATATCGAGT- CATGGAACA-3’, was observed at nt -464 and nt -257. Additional sequences with less than perfect match were also found in the same region, defining two areas between nt -512 and -447, and -390 and -348 (Fig. 5). Within these two regions there is dyad symmetry and potential for hairpin loop formation,

(c) Determination of the 5’-end of the transcribed

region of B46

One of our goals is to isolate genomic fragments that contain the promoter and enhancer sequences of the B46 gene. To do this, we need to know where the promoter sequences are located in relationship to the

coding regions. We performed two types of experi- ments, primer extension analyses and direct dideoxy sequencing of the B46 mRNA, to determine the transc~ption~ initiation site. We compared the re- sults of our analyses with the sequence of the B46 genomic clone extending beyond the 5’-end of the coding regions. Primer extension reactions using total salivary RNA and oligodeoxynucleotides (B46-1 and B46-4, Figs. 4 and 5) complementary to regions of the RNA near its 5’-end, have identified a potential transc~ption start point (Fig. 6A). The doublet evident in the primer extension reactions suggests that although the primary start point is the C nt designated + 1, the adjacent 3’ A serves as an alternate site. Oligodeoxy~bonucleotide primers (B46-2 and B46-3, Fig. 5) overlapping the potential transcription start points failed to produce an ex- tension product confirming this interpretation.

Introns in the 5’-end of the B46 gene would affect our interpretation of the primer extension results. To check this possibility, the sequence of the 846 mRNA was determined using the oligodeox~ibonu- cleotide primers, B46-1 and B46-4. The results indi- cate that there are no introns between the primer and the 5’-end of the transcribed sequences and that the assignment of the transcription start point is most likely correct (Fig. 6B). Although we were unable to resolve the last five nt of the mRNA sequence, termi- nation of all four reactions occurred at the exact same position following the last readable sequence and this correlates well with our primer extension analyses.

(d) Putative protein product of the B46 gene

An amino acid sequence was predicted using the nucleotide sequence specified by the ASGB46 cDNA (Fig. 4). The deduced sequence is 579 aa in length, has a minimum M, of 66751 and is rich in leucine, aspartic acid, valine and serine. Lysine, glycine, aspargine and arginine are also present as significant fractions of the total protein. The amino acid se- quence has at least six sites for possible asparagine- linked glycosylation based on the aa signal se- quences, N*T or N*S (the * is a variable aa; see Wieland et al., 1987). The deduced protein has a sequence of 18-19 aa at the N-te~inus that displays the characteristic hydropathicity of a secretion signal (Von Heijne, 198 1, 1985 ; Kyte and Doolittle, 1982).

78

35

KbIfVPLLSFLtAGLTlGLDWWEHGNFYQVYPRSF

ATCGGAACAGGCAGARTSAAGAT~TlTGJl~CAClTClAAGCTTCCT~~lAGCAGGAClAA~CACCGGGllGGACTGGTGGGAACATGGAAA~llCTACCAAGTTTAC~CAA6ATCCTT~

75

KDSDGDGIGPLDGVTEKLKYLKDIGMDGV~LSPIFSSPHR

AAGGACTCCGACGGCGACGGlAlCGGGGATCTGGACGGAGl~ACCGAAAAGClGAAAlATCTGAAAGACATCGGCATGGACGGAGTTTGGTTGT~ACCGATlTTCTClTClCC6ATGGCT

115

DFGYDISNFREIQTEYGDLDAFQRLSDKCKQLGLHLILDF

GATTlTGGCTATGACAlCTCGAACTT~~GAGAGAlTCAAA~GGAATACGGGGATCTAGATGCTTTC~AGCGGTTGTCCGATAAGTGTAAGCAGCTGGG~TlGCAT~lAAlJlTAGACTT~

1%

VPNHlSDQHEYFKKSVOKDElYKDFYV~HFGVHGPNNlKV _.-- GTCCCGARTCATACTAGCGACCAACRCGAATATTrCAAAAAGl~TGTTCAAAAAGAlGAGACAlACAAGGAlTT~lACGlAlGGCAlCCGGGTGTACATGGlCCGARCAA~ACCAAGGTA

195

PPSNWISVFRGSSWEWNEERQEFYLHQFLKEQPDLNYRNP

CCGCCAlCCAACTGGATCAGlGTGTlCCGAGGAlCllC~lGGGAATGGAATGAGGAG~G~~AAGAGTllTATClGCAlCAGlllllGAAAGAACAACCAGAlCTGAACTACCGCAATCCT

235

AYV~~MKNVLRYWLDffGVSGFRIDAVPYLFESDlIDGRYR

GCCGlAGTGGAAGAGATGAAGAACGTl~llCGGTAllGGCTGGA~~GGGGAGlTT~TGGGTT~AGAAT~GAlG~AGTAC~GTAT~TJTTCGAGAGCGACATlAlCGAC6GACGATAT~Gl

275

NEPESRTTDDPENPAYLVHTQTMDQPETYDMIYQWRAVLD

AATGAACCAGAAAGlCGTACCACTCATGACCCCGAGAACCCAGCTTAllTGGTTCAlA~GCAAACCATGGATCAGCCGGAAACCTACGAlAlGAlClAlCAATGGCGlGCGGlAJT66AC

315

EYSKlDNRTRIHflTEGYlSLPKIIEFFGNATANGAQIPFN -- GAGTACAGTAAAACGGATAATAGAACTCGlATCATGATGA~lGAGGGCTACACTAGCCTTCCAAAGATCAT~GAGTT~TTTGGTAATGCtA&TGCTAACGGAGCAtAGATT~~6llCAA~

355

iEVISNVKKNSlGADFATYVKRWLDAKPANRRSNWVLGNH _ _ ._. - TTTGAAGTGATCAGTAATGTGAAGAAGAACAGCACTGGCGCTGATlTTGCCACllATGTlAAACGCTGGTTGGATGCTAAACCTGCCAACAGAAGAlCCAAJlGGGTACllGGAAA~~AC

395

DNNRLGSRLGENKIDLYNIALQTLPDIAVlYYGEEIGKtD

GACAACAATCGCTTAGGGTCCCGTTTGGGCGAGAATAAGATlGATlTGTACAACATCG~llTACAAACATTA~CGGATATTG~CGl~ACATACTACGGTGAGG&AAlCGGAAlGCT~G&T

435

Q W I P W NBV D PA AC R S D E AS Y S A Y S R D PA R T P H U W D S G K N

CAGTGGATTCCTlGGAACGAAACTGTTGATCCGGCTGClTGCAGATCAGATGAGGCTTCTTATTCCGCTTACTCTAGAGATCCTGCCCGlACTCCAATGCAGTGGGACAGCGGAAA6AAC

475

AGFSKAAKTWLPVADNYKTLNVKIQDRARKSHLKIFKKLT

GCAGGTTICTCTAAGGCGGCCAAGACTTGGCTT~CAGlGGCAGA~AATTACAAGACCCTTAA~GTAAAGAlT~AGGATCGTGC~~GCAAAAGCCATTlGAAGATATTCAAGAAGTlGAC~

515

KYRKRQILTEGDIDIRVSG ENLLVYKRKVDKVGYVVVALN

AAGTACCGCAAGCGTCAAATCTTGAC~GAAGGAGACAT~GACATTAAGGTTTCCGGGGAAAATClACTCGlGlA~AAACGTAAGGTCGAlAAGGllGGClACGTGGTCGTTGCACTGAAC

555

F GTEPVALGLSSLFDRADQRtiQVVVSSNRVSTPDNVWVDV

TTTGGTACGGAACCTGTAGCGClGGGlCTTTCCAGTTTGTTlGAlCGAGClGATCAGAGAAlGCAAGlAGTGGTATCGlCGAACAGGGlCTCCACl~CAGATAACGlATGGGTAGAlGlG

579

~~~~~LIGESGIVLKYLWGKNPIVS~ t

f;AlAACTACGTACTAA?CGGA6AGAGTGGCAICGTG~JGC4ATATllATGGGGAAAGAA~~~GATlGTTlCTTAATTTTGAATAGAATTTTTTGTAGTATT~TATAGAAATGGCAC~T~

AIICAATATTGCATATTTGCACAGCTTGAATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Fig. 4. Primary nucleotide sequence of the B46 cDNA and its deduced amino acid sequence. The cDNA clone, RSGB46, was sequenced

as described in MATERIALS AND METHODS, section e, and in Fig. 3. The deduced start codon (ATG) and polyadenylation signal

sequence (AATAAA) are in bold letters. The asterisks mark in-frame stop codons in the 3’-end regions of the cDNA. The deduced

amino acid sequence is listed just above the coding region in each line (numbering refers to the amino acid sequence). Six consensus

sites for possible N-glycosylation of the deduced protein are underlined.

A search of the PIR and PseqIP protein databases ing three, the H, D and L gene products, were from

ident~ed four proteins with sequence features D. me~u~ogaster (Snyder and Davidson, 1983). The

notably similar to the B46 gene product. One of the function of the Drosophila genes was not inferred at

four proteins is a maltase from Saccharomyces carls- the time of their discovery, but recently Henikoff and

bergensis (Hong and Marmur, 1986) and the remain- Wallace (1988) have noted their similarity to the

+1 $ t : t98

TCCRTTGdTTRGCGAACATGGTCATCATTCbdCTGACATCGGTTGCTCT~GGC~T~G~GTGC~CTG~TCGG~~C~GGC~G~nl~~~G~TCTTTGTlCC~CTTCT~~GCTTCCTCCT~GC~ GlRtTRRGllGACTGTAGCCRRCGAtRTCC 846-2 GARGAlICGARGGAGGATCG7-

GlRACTRdTCGCllGlRCCAGl~GI~~GlT 846-3 ClTCTRGAARCAAGGTGRlG 846-d

+I98 GGRCTBACCRCCGGGTTGG~GGTGGGdACATGG~~~CTTCT~CC~~GTTT~CCC~~G~TCCTTC~~GG~CTCCG~CGGCG~CGGl~~TCGGGG~TCTG __-__ CCTGATTGGTGGCCCAACCTGGCCACCCCrlG 846-l

Fig. 5. Primary sequence of a 5’-end, genomic subclone of the B46 gene. A 1.6-kb DNA fragment of the genomic clone, MEGB46-1

was sequenced as described in MATERIALS AND METHODS, section e and in Fig. 3. The genomic sequences that overlap with the

cDNA sequences in Fig. 4 are underlined. A consensus eukaryotic TATAAAA sequence and the start (ATG) are in bold letters. The

18.bp direct repeats (ATATCCAGTCATGGAACA) in the region 5’ to the protein-coding sequences are also in bold letters. The

underlined sequences are direct and inverted repeats with 70% sequence identity to the 18-bp repeat motif. The sequences in italics

are regions with 60% identity with a consensus repeat found at the 5’-ends of the D. melunogasrer HDL gene family. Additionally, the

sequences of four oligodeoxynucleotide primers used to define the 5’-end of the RNA are listed and aligned with their complementary

sequences. + 1 marks the C nt that is the limit of the primer extension reactions using both primers, B46-1 and B46-4. Primers B46-2

and B46-3 failed to generate any extension products. The T at nt + 6 is the limit of readable sequence from direct dideoxy sequencing

of mRNA using the B46-1 primer. The asterisks mark in-frame stop codons in the 5’untranslated region of the genomic sequence.

yeast gene. All five proteins show considerable

similarity in their N-terminal regions (Fig. 7, aa

26-232). In the region between aa 33-137, there is

74x, 87%, SO%, 87%, and 64% sequence identity

between the mosquito, fi-uitfly H, L, and D, and yeast

proteins, respectively, and the consensus sequence.

Because there are three fruitfly proteins, we expect

the consensus sequence to be skewed in their favor.

However, even with this apparent bias, the similarity

between the mosquito and yeast proteins is remark-

able. Following this region of high similarity, the

yeast protein no longer resembles the Dipteran pro-

teins, with the notable exception of the region

between aa 385 and 408. However, the B46 protein

is very similar to the Drosophila H and L proteins

over most of their length.

(e) Conclusions and discussion

The B46 cDNA and homologous gene were

isolated and cloned using standard differential

screening techniques with tissue-specific RNA

probes. The isolated gene encodes an mRNA that is

abundantly expressed in adult salivary glands. This

Fig. 6. Primer extension analysis and direct sequencing of the 5’-end of the B46 mRNA. Reactions were carried out as described in MATERIALS AND METHODS, section e. Panel A: Primer extension analysis of the limit of the 5’-end of the B46 RNA. Lane 1 shows the extended product of the end-labelled B46-4 oligodeoxynucleotide primer (see Fig. 5). Lanes 2-5 (T, C, G, and A sequencing reactions, respectively) are the sequence of the B46 genomic clone in the region of transcription initiation. Panel B: Direct dideoxy sequencing of the 846 mRNA using the oligodeoxynucleotide primer, B46-1. Lanes l-4 are the T, C, G, and A reaction lanes, respectively.

gene is expressed in all of the secretory cells of the male salivary gland, and only in the proximal regions of the lateral lobes of the female gland.

The manner in which the B46 cDNA was isolated, the short time it took to expose the in situ hybridi- zations, and the hybridization signal on Northern analyses indicate that the B46 mRNA is abundantly expressed. Because we plan to use the sequences controlling transcription to direct the expression of a hybrid gene, it was necessary to define the tran- scription start point with certainty. The primer ex- tension analyses and dideoxy sequencing of the B46 mRNA indicated the same transcription start points. The C and A nt at + 1 and + 2, respectively, are the only transcriptional initiation sites elucidated by the primer extension. These particular nucleotides are a characteristic feature of many prokaryotic (Rosen- berg and Court, 1979) and eukaryotic (Cordon et al., 1980) transcription initiation sites.

The deduced amino acid sequence of the B46 gene product predicts a basic protein cont~ning a se- cretion signal peptide and several potential glycosy- lation sites. These predictions are supported by ob-

servations that the majority of salivary gland pro- teins can be labelled with [ ‘Hlmarmose (I.V.R., un- published). The putative signal sequence indicates that this is probably a secretory protein.

The sequence similarities between the mosquito, yeast and fiuitfly proteins are compelling evidence that suggests that the mosquito and fruitfly proteins are probably glucosidases. These proteins appear to be composed of two domains, an N-terminal region, which could provide the glucosidase functions and is conserved between yeast and the Diptera, and a C-terminal region that is similar between the mosquito and two of the Drosophila proteins. Notably, the putative mosquito and fruitfly proteins conserve some of the structural and functional amino

acids and regions identified in related amylases, cl-glucosidases and transglucanosylases. Svensson (1988) identified four sites in yeast maltase that con- served substrate-binding, inhibitor-binding, and catalytic aa found in other carbohydrate metaboliz- ing enzymes. Three of the four sites are towards the N-te~inus of the yeast maltase and appear con- served in the Dipteran proteins as well (Fig. 7). The fourth site is the most distal from the N-terminus and is the least conserved between the Dipteran and yeast proteins. Within that region, however, all live proteins share the amino acid sequence, asparagine- histidine-aspartic acid (NHD), that has been identi- tied as a substrate-binding and catalytic site in 15 other related proteins (Svensson, 1988). Because of the similarities of the mosquito protein with the yeast protein, we propose the name, ~~altase-like I’ (Malf), to designate the gene. The dissimilarities in the C-termini of the five different proteins examined could be an indication that these portions of the proteins are not involved directly in metabolizing

sugar. The similarities between the mosquito and fruitfly

deduced proteins indicate that they are most likely the products of homologous genes. Further evidence for this conclusion is provided by the sequence of the noncoding portions of the genes. In the analysis of the L). ~eZa~og~ter H, D and L genes, Snyder and Davidson (1983) observed a conserved sequence in the regions 5’ to the coding portions of the three genes. Similar DNA sequences exist in the two re- peat regions described in the results and Fig. 5. Dyad symmetry is a characteristic of many DNA se- quences that bind truns-acting regulatory proteins

81

CONSENSUS

d.a. 846

D.ra. ii D.s. L D.m. D 5.c. ma1

CONSENSUS

A.a. 640 D.m. H 2.m. L D.in. C 5.c. ma1

CONSENSUS

A.a. 846

0.1. H

3.8. t

D.I.Q. Ii

S.C. ma:

CONSENSUS

A.a. 046

D.fl. H

D.m. L D.m. D 5.c. ma1

CONSENSUS

A.a. 846

D.8. H 3.m. L D.N. D S.C. ma1

1 25 26 50 51 75 76 100

___.._Swa7 Swence_ __________ _________ __-.________ ._ DWWEtGNt-YPIYPRSFKDSDGDGIG DLKGIT~KLQYLKDIGITRTWLSPI FTSPHADFGYDISNFY*IDPtYGTt

MKIFVPLLSFLLAGLTlkl H V DVE K IIDGV S RE QTE DL

MRPOSAACLLLAIVGFVGAT E S Y R NVE FG K V DQHE Ii

I'I~KLLVLSCLLALALPSL~V G KT Q V I QQP E VRDLKG IF H

~PKUAHfGLAAlllISllQEGTADI NRSL Q SRG E S D IF L

\TISDHPETEP K KEATI A NN W S I L VD I VC F YD QQ fl YEKVW T N (____2_

101 125 126 150 151 175 176 200

E_DERLI'KRKELGlK~ILDFVPII~ $DEtEWFnljSV*S*E*YK-DFYVW H*GKVNNETG*R*PPSNWVSVFRGS tWTWNEERQtYYLHQFR*KQPDLNY -__-- _-. ._-- -__--__- DA Q SD C Q LHL 1 QHY QKD 1 - P - HGPNNTKV I SE EF LKE

HA v S 1 N 1 D DPV - I D I E E NEYAE VQ IQ A

(I LRR D 1 CD IR AAGE E - PT V -- I Q 1 n A HP,

DD VE S V S NV E NREDG D- DD LE AD SPt4 KQF QV F

CFE D THK 1 F 1 L I C 1 H E RS KTNP R WFF APP GYDAE KPI N K F G A FD TTNEF RL SR V W

-) (_______3____ ___)

201 225 226 250 251 275 276 300

RtJf#YVE*tlK*VL-RFWLDtGY*GF RID#/P*L*Et**DADG*YPDEPS* ***S*PNDYD---Y*QHIYT*DQPE TtDUYQWREVLDEYtAENGGDrRY -___ EN-Y RS Y F SDIIDGRYRNEPE R TTDDPE PAYLVH-T --- II Yl41 R SKTDNRT- I NE NI- GK S Y F VDL RYNQ LT NDSVNCP P DHC T Q II I tl LV FHV K t

K AD- AK AY HVY IPA NW RN EAV D E T--- L T LEL AF D IE ID L D

1 M R HLD -K R D HIY -HRN S VS GWG D A --- HD K AVLHE FNRQ S

E EDCRRAIFESAVG H D TAGLYSKRPGLP SPIF KT K LQHPNWGSHNGPRIHEYHQELHRFM KNRVKDGREIHRVGFV HGSDNRLY (__-___._ 4______,

301 325 326 350 351 375 376 400

L*TERYtSLEV***Y*GNGiHNGSQ IPFNFErLtrItrSStAtrtttIIK *WLDA*PEGQ*ANWVLGMiDNNRV* SRLGRD*IDLLNI*LQTLPG**VTY HM- k T PKiIEFF AA R VISNVKKN TGdDFATYV R K ANRRS LG ENK Y A DIG I T F NIMT Y VR H DF TS NNA K GEYVKH K M S VY K A F VQRT I L HR

t SP LMQ Y L L AQ SY D YHYSEL H N NH V F QS IG R AC HIILG VS

LR S V TLSA F S 0 I t M QLtlYLSGY TAKDVVGS DY WtlNTtlWK H T V T A D ti HKV VIVNA AS

TSAAR EVC FSFTHVEVGTSPFF RYNIVPFT--LKQWKEA-----IAS NF FINGTDSW TTYIE QA SI T FAD SPKYRK SGKL TLtECSL (________6_______ _)

401 425 426 450 45i 475 476 500

*_trit*~~GEEIGMTDVW!SWEtTV DP*RC*S*E**Y*A*SRDPARxPte WD*GtNAGFS*AS*TWLPVAD*YKi LNVKKQ*RAPKSHLNIFKKL***RK _ _ _ _ _ _ _ Y LO PNE A RDASSY TM SK K RK N IDR K TKY _______N ,_ D N N DPDN Y R S Y ASSK TS DH D N ALQ L R Q XRV __---__Q n D Q Q N QEFERLT V T F SDEV N V SN LV ERGIRL VY Q RAL D _______ Y SN DVECTGDS CEDRDGERTPMQWTRGKNADFSDGES TWLPLSPEYQRYNVQTERGVSRSS IF GLQEL SSAFLAFKEDGGF

TGTLYVYP Q QINFKEWPIEKY EDVDVKNNYEIIKKSFGKNSKEHKDF FK IRLLSRDH R PH WTKDKPN AGFTGPDVK WFF ESFEQGINVE

94

94

95

96

100

86

193

192

194

193

199

186

289

206

293

209

294

296

389

386

393

389

3’94

379

482

479

486

402

487

479

Fig. 7. Amino acid sequence similarities between the deduced proteins of the Aedes aegypri B46 gene (A.a. B46), the Saccharomyces carlsbergensis maltase gene (S.C. mal), and three clustered genes of D. melunoguszer (D.m. H, L, and D). The five amino acid sequences

were aligned for maximum similarity and a consensus sequence derived. Asterisks denote positions where no consensus was derived

and dashes represent shifts in the sequences to compensate for extra amino acids and to achieve maximum alignment. The N-terminal

sequences listed in italics have properties consistent with transport signal peptides. The four regions (2, 3,4,6) delimited by brackets

are the conserved regions identified by Svensson (1988). The amino acids within these regions that are conserved in carbohydrate

metabolizing enzymes are listed in the consensus sequence in bold.

(Wingender, 1988) and the presence of these se- those species that overwinter (Van Handel, 1984).

quences in the mosquito and fruitfly suggests that The observation that the putative mosquito glu-

they have a role in regulating their respective genes. cosidase is expressed in the salivary gland is interest-

Sugar-metabolizing enzymes have been described ing since most glycosylases and glucosidases de-

in many hematophagous insects (Gooding, 1975). scribed in insects are produced in the gut. Indeed, we

Sugars provide the mosquito with the necessary en- found only a single report of a sugar metabolizing

ergy for the flight muscles, and provide nutrients for activity being localized in the salivary glands of a

82

mosquito, Culex tarsalis (Schaefer and Miura, 1972). Salivary gland extracts of this mosquito were able to cleave sucrose, maltose and melezitose, all al-4- linked di- and trisacchatides. Female A. aegy& swallow saliva during feeding (Ribeiro et al., 1984, Rossignol and Leuders, 1986) and the secretion of a glucosidase from the salivary gland would enable sugar digestion to commence in the crop.

The adult-specific expression of the MaN gene further correlates with the putative sugar meta-.. bolizing role of this gene and with the feeding habits of adult A. aegypti. Larval and adult feeding habits are significantly different in that larvae feed on bacte- ria that thrive on the organic material present in the aqueous environment, while adults (males and non- bloodfeeding females) feed on honey, nectar, or sugar solutions (Christophers, 1960). This function-related expression of the putative glucosidase is further emphasized by the pattern of expression of the similar genes in D. melanogaster. The H, D and L genes are expressed in all the larval and adult stages, but not in the embryonic or pupal stages (Snyder and Davidson, 1983). Larval and adult D. mezanogaster can feed on the same food source and potentially utilize similar digestive enzymes. The control of the expression of the mosquito gene is unknown. As in other Diptera, the salivary glands of the larvae and adult have different developmental origins. Larval glands are histolyzed during met~orphosis, and replaced by the adult glands (Trager, 1937). Control of expression could be achieved by limiting the expression of the Ma@ gene to adult tissues.

The spatial expression of the Mall gene in adult male and female salivary glands most likely reflects differences in the feeding capabilities of the two sexes and in the development of the glands. The male salivary gland has three lobes composed of secretory cells all of approximately the same size. The female salivary gland is much larger than the male gland, and the medial and distal lateral lobes appear to be sex-specific. The secretory cells in the proximal distal lobe are similar in size to the cells in the male gland whereas the cells in the other parts of the gland are significantly larger. The expression and accumula- tion of the Mall mRNA in the male and female glands correlates well with these morphological observations. Additionally, the expression pattern of the MuIf mRNA is consistent with histochemical studies that showed that the male salivary lobes and

proximal lateral lobes of the female salivary glands contain similar staining material (Orr et al., 1961).

In conclusion, the cloning of the A4ulI gene pro- vides us with a tool to direct the expression of exo- genous genes in vector mosquitoes. Although it is expressed only in a portion of the female salivary gland, it is abundantly expressed and secreted and should be a versatile substrate for genetic modifica- tion.

ACKNOWLEDGEMENTS

The authors thank their colleagues in the Depart- ment of Tropical Public Health, Drs. Randy Smith, Osvaldo Marinotti and Ethan A. Lemer, and Ms. Yvonne Kwong for help in preparing the manuscript. This work was supported by a grant from the John D. and Catherine T. MacArthur Foundation, the Milton Fund of the Harvard Medical School, and a National Institutes of Health BRSG grant awarded to the Harvard School of Public Health.

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