Gene. 112 (1992) 205-211 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

205

GENE 06337

Human cleavage signal-t protein; cDNA cloning, transcription and immunological analysis (Immunoblot; immuno-infertility; sperm antigens; fertilization; embryogenesis; contraceptive vaccine; in vitro translation)

Ali A. Javedb and Rajesh K. Naz" "Deparnnent of Obstetrics and Gynecology, Division of Reproductive Endocr#wlogy mid InJbrtility, and b Division of Reproductive Genetics,

Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) Received by J.A. Engler: 22 August 1991 Accepted: 21 October 1991 Received at publishers: 7 December 1991

SUMMARY

The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-I eDNA and its expression in vitro to c,btain the recombinant protein (reCS-1) molecule. The CS-I cDNA clone was isolated by immunological screening of a human testis ).gtI 1 cDNA library with monospecific polyclonal antibody against CS-I. The eDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845-847). The eDNA isolated has a 97-bp 5' and a 984-bp 3' untranslated region. The potential polyadenylation signal (5'-AATAAA) is at bp 1803-1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-I is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-I IZf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-I RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-I protein, as assessed by migration in a SDSpolyacrylamide gel. The polyclonal antibody against CS-I specifically recognized the 33-kDa reCS-1 protein on Western blots of in vitro translated proteins, suggesting the authenticity of the eDNA clone. Besides enabling us to understand the molecular basis of signal transduction pathway(s) underlying fertilization and early cleavage, reCS-1 may find applications in the development of a contraceptive vaccine, and in the diagnosis and treatment of immune-infertility in humans.

INTRODUCTION

Antispermatozoal antibodies have been demonstrated to cause antifertility effects by inhibition of the fertilization process as well as postfertilization early embryonic devel-

Correspondence to: Dr. A.A. Javed, Reproductive Genetics, Ullman 813, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (U.S.A.) Tel. (212)430-4190; Fax (212)824-8919. Abbreviations: aa, amino acid(s); BCIP, 5-bromo-4-chloro-3-indolyl phosphate; BMV, brome mosaic virus; bp, base pair(s); BSA, bovine

opment (Menge and Naz, 1988; Naz, 1990). Mammalian fertilization involves the fusion of sperm and egg surface membrane(s) resulting in resumption of meiosis. Sperm surface molecules have been shown to be transferred to the egg membrane during fertilization (Gabel et al., 1979;

serum albumin; CS-1, cleavage signal-1 protein; CS-I, gene (DNA) encoding CS-I; GCG, Genetics Computer Group, Madison, WI; kb, kilobase(s) or 1000 bp; NBT, nitro blue tetrazolium; nt, nucleotide(s); oligo, oligoOeoxyribonucleotide; ORF, open reading frame(s); PAGE, polyacryiamide-gel electrophoresis; pfu, plaque forming unit(s); re, recombinant; SDS, sodium dodeeyl sulfate; tsp, transcription start point(s).

206 ions required for oocyte activation (Menge and N a z , 1988, Yanagimachi, 1981). The sperm surface antigen comprised of a doublet o f approx. 1 4 - k Da a n d approx. 1 8 - k Da proteins is involved in some step of early cleavage o f the fertilized oocyte: antibodies to it inhibit early cleavage of the oocyte without affecting pronuclear formation ( N a z , 1992). This extranuclear cleavage signal-1 protein (CS-1) seems to be evolutionarily conserved among various m a m m a l i a n species including h u m a n s . Further studies elucidating its exact

G a u n t , 1983). Thus, the sperm cell can share or impart antigenic specificities to fertilized ova and cleaving embryos by incorporating its surface antigens into the egg during fertilization or by cross-reacting with the antigens developed during early embryonic development. It h a s been proposed that the molecules that are incorporated into the oocyte m a y provide an extranuclear signal to the oocyte to cleave via their involvement in some step of the signal transduction pathway required for early embryonic development, or by providing a channel for physiologic flux of

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Fig. 1, Immunological screening blots, CS.! insert size, organization of the human CS.! eDNA, and sequencing strategy. (Panel A) Duplicate replica filters, with positive signals for C$.i clone upon immunological screening (second round) and color development. The replica filters represent nearly 4000 pfu of which approx. 20 plaques are expressing CS-I protein as judged by CS-l-specific antibody binding. (Panel B)agarose gel electrophoresis of EcoRl restriction digestion; lanes: 2, CS-I positive clone; 3, a control recombinant clone picked arbitrarily; 1, Mr markers. The CS-I insert in lane 2 is approx. 1.8 kb, (Panel C) the upper line represents the eDNA and the scale in kb. The lower line represents the eDNA with various restriction sites, the heavy line shows the sequence encoding the mature CS-I protein. Arrows below the line show the various primers used in the total sequencing of both strands of the cDNA; sequencing data from each primer proceeded in the direction of the arrow overlapping the next primer (arrow) whose sequence was based on the data obtained from the previous primer generated sequence. Sequencing of M 13mp 18 and mp19 subclones of C$-1 was initiated using M 13 universal primers. A partial computer generated restriction map shows: B, BamHI site at nt 869; D, Ddel sites at 42, 364, 460, 58'~, 687, 828 and 1028; P, Pstl site at 123; and X, Xbal site at 542 and 1593. Methods. A human testis eDNA library obtained in ~gtl 1 (Clontech) was cm~structed using mRNA from normal, healthy 55 year old human testicle; the mRNA was primed with oligo dT (12-18) for reverse transcription and subsequently cloned in ~gtl 1 using EcoR[ linkers. Immunological screening was performed according to standard methods (Huynh et al., 1985). Approx. 80000 recombinants were plated with nearly 4000 pfu per plate. Expression of the cloned insert was induced and blotted onto nitrocellulose membrane by overlaying plates with dry membrane saturated with 10 mm IPTG; two such nitrocellulose replicas were obtained for each plate. These blots were incubated with CS-I polyclonal antibody (primary antibody, i:400 diluted, sera from rabbits immunized against CS-I protein) followed by washing and incubation in alkaline phosphatase conjugated secondary antibody (1:7500 diluted, alkaline phosphatase conjugated affinity purified goat anti-rabbit IgG). The blots inc~lbated with proimmune sera from the same rabbits or from rabbits injected with only Freund's adjuvant served as controls. The specificity and bioactivity of these sera have been established (Naz, 1992). Alkaline phosphatase based color assay was initiated by incubating the washed membranes in phosphatase substrates NBT and BCIP (Promega, Madison, WI, 1989; Blake et al., 1984). Plaques giving positive signal upon color development in duplicate filters were cored out for subsequent rounds of screening with 50-100 pfu/plate; this low density screening ensured isolated plaque formation for subsequent plaque purification. A positive clone was considered plaque purified when all plaques gave positive signal upon color development. The insert from t~e clone was excised by EcoRl and resolved by agarose gel electrophoresis (Sambrook et al., 1989).

207 G GGG CTG ACG CAG CAT TGC CAA TTC TAA ATC CAT CAT TTG ACT GAG GAG GAG AGG TTT

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ATG CCT TCA CCC TTC CGC TCC TCC GCA CTC ATG GGA ATG TGT GGC AGT AGA AGC ACT 229 g P S P F R S S A L H G N C G S R S T 44 GAT AAC TTG TCA TGC CCT TCT CCA TTG AAT GTA ATG GAACCA GTC ACT GAA CTG ATG 286 D N L S C P S P L N V N E P V T E L N 63 CAG GAG CAG TCA TAC CTG /LAG TCT GAA TTG GGC CTG GGA CTT GGA GAP, ATG GGA TTT 343 O E Q S Y L K S E L G L G L G E N G F 82

mechanism of action require a well characterized CS-1 molecule. This paper reports the complete eDNA and the deduced aa sequence of human testis CS-1 protein. It further describes that the CS-1 molecule is a unique protein without any complete homology with any known sequence in the GenBank database, and develops as an approx. 33-kDa molecule in the testis during spermatogenesis in humans.

RESULTS AND DISCUSSION

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TCT TCT ATA TGT TCT GGT CCC TCT CAT GCT AAC AGA AGA ACT GGA fiTA CCT TCT ACT 457 S S I C S G P S H A N R R T G V P S T 120 GCC TCA GTG GGC AAA TCC AAA ACC CCA TTA GTG GCA AGG AAG AAA GTG TTC CGA GCA 514 A

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TCG GTG GCT CTA ACG CCA ACA GCT CCT TCT AGA ACA GGC TCT GTG CAG ACA CCT CCA 511 S V A L T P T A P S R T G S V 0 T P P 158 GAT TTG GAA AGT TCT GAG GAA GTT GAT GCA GCT GAA GGA GCC CCA GAA GTT GTA GGA 628 D L E S S E E V O A A E G A P I: V V G 196 CCT AAA TCT GAA TCT GAA GTG GAA GAA GGG CAT GGA AAA CTC CCA TCA ATG CCA GCT 685 P

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GCT GAG GAA ATG CAT AAA AAT GTG GAG CAA GAT GAG TTG CAG CAA GTC ATA CGG GAG 742 A

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ATT AAA GAG TCT ATT GTT GGG GAA ATC AGA CGG GAA ATT GTA AGT GGA CTT TTG GCA 799

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ACAGAAATTA TAGGTTGGCA TGGATCCTAT TAGCTGTGTA ATACTGGAAT TATCAATGAT

(a) Cloning of cleavage signal 1 protein (CS-I) Immunological screening of a human testis ~gt 11 eDNA library using polyclonal antibodies against CS-1 gave seven positive signals on duplicate filters in the first round of screening nearly 80000 recombinant clones. These seven isolates were subjected to further screening. Upon subsequent screening, one clone was isolated which gave intense positive signal on rep=titive immunological analysis. The isolated positive CS-1 clone was plaque purified; the ~. phage DNA was isolated and subjected to restriction digestion with EcoRl to excise the insert. Agarose gel electrophoretic analysis with appropriate molecular weight markers established the size of the insert to be approx. 1.8 kb with no internal EcoRI sites (Fig. 1).

907

ATGCACTGGT GGAGGTGTTA TTTGTGCTTT AGAAGATACT TGCTGTTGAGCTGGGCTACT 9 6 7 GTATACAGTG TACAATGTGT ATTTCTTCAA CCATATATTT TAAAAAGACG TACATAGAAA 1027 CTTAGGCACT TTGCTATTTC TTTTCTAAAC TATCAAAAAC TCTAGCAGTT TGAAAAGCCT 1087 AATATTTATT TGTATGTCAA TATTTTTCA1 TTGATTCCCT ATTAGAATTA ATTTTAAAAC 1147 TTGAAGACTT CCAGACTTAT CCAACTTATA AATAACATAT TTCTTCAGAC TAACATCTTA 1207 AAACACTGAC CTCTATGAGGTATTTACTGT GCAATAACTG ATTCATTTTT TTCAGAGCTT 1267 GAAGCATCCA ATGATTTTTC CCTCCACTGC TGTTAATTAA TGTCACTTCCAAGAAGAAAA 1327 ACTGTTCTGT TGTAAAAAAT ATAATTGCTC TTAATTCTTG GGGAGGTTACTAATAGCAGT 1387 AGGATAGAAT TTTATGAGGT TACCTACAAC TACTTAATGT ACTTACACTG TAAGCCTTGT 1447 TGCTTTACCC AAGACAAATG TAATTTTATC ATTGCTTATG TAGTATTTTT CTTTTGGAAA 1507 TGTGCCTTAT GTTAAACACT ATGTACTTTT ACTTTTTGCA TTGTCCAGAC TTCTTTATTA 1567 GATGGAGATG TTTCTTTTTC TGTCTTCTAG ACTAAATAGA GTATCATCCA AATAATGGGG 1627 CCTATGACTT GAATGAATAG AAATGAATAA GCTGGTGTTT GTTTTTTCAA AATGGAAGTA 1687 ATTTAGATTT GTTCTCCTCA TACATAAAAT GATTTTAGTT CAGTTTTAAC CAGTGAJULAC 1747 TTTGTTTTTA TGAAAAAAAA GGAAAATGGT TTCCCATTTG GTTTTATATG TGTTAAATAA 1807 ATGTGTAAAG TAACCACCCC C 1829

Fig. 2. Sequence of eDNA and the deduced aa sequence of CS-I. A strong potential N.glycosylation site (NLS) at nt 233-241 corresponding to aa 46-48 is underlined. The potential polyadenylation signal sequence is underlined at nt 1803-1808. See Fig. 1 panel C for positions of various sequencing primers used for complete sequence determination. The cDNA sequence data reported in this paper has been submitted to GenBank and assigned the accession No. M61199. Methods. The 1.8-kb CS-1 cDNA insert was gel purified by electro-elution and subcloned in Ml3mpl8 and mpl9 (Messing, 1983). Various subclones were characterized by partial dideoxynucleotide chain-termination sequencing (Sanger et al., 1977; Tabor and Richardson, 1987) using a Sequenase DNA sequencing kit. Two subclones, one each of M13mpl8 and mpl9 corresponding to the two strands of the CS-1 cDNA insert were selected for total single-stranded DNA sequencing of both strands. Sequencing was

(b) Sequencing of cDNA and primary structure of the CSI gene The nt sequence of human testis CS-1 eDNA was established by total sequencing of both strands. Various primers were used and each segment was sequenced several times; alignment of sequences yielded a 1828-bp eDNA. Computer generated translation of the cDNA in all reading frames resulted in the identification of the longest ORF of 249 aa. Taking it as the ORF encoding the CS-1 protein, the first ATG, Met start codon is at nt 98 and the stop codon TAA is at nt 845. The eDNA thus has a 97bp 5' and a 983-bp 3' non-coding region and a potential polyadenylation signal (AATAAA) starting at nt 1803. The deduced aa sequence is shown below the eDNA sequence in Fig. 2. Computer-generated analysis (GCG, 1991; Devereux et al., 1984) indicates the CS-I translated protein to hav~ a calculated Mr of 26819 and an isoelectric pH of 4.8;

initiated using universal primer and further sequenciv~ primers were c o n structed based on the sequencing data obtained til' :otal sequencing of the insert was achieved. The sequencing strategy ,~'ith the various primers is depicted in Fig. 1. Each segment of the clon~ was sequenced several times in both directions. All synthetic oligo pr~'~nersafter cleavage, deprotection, vacuum drying and ethanol precipit~ion were used without further puri-

fication.

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Fig. 3. Restriction digestion and in vitro transcription of pGEMCS-1.1 and pGEMCS-!.9. (Panel A) Agarosc (0.8%) gel electrophoretic analysis of restriction digests: lanes: 1, M, markers; 2, pGEMCS-I.I undigested; 3, pGEMCS-I, i Xhol; 4, pGEMCS-I. ! Sacl (incomplete digestion); 5, pGEMCS1.9 Xhoi; 6, pGEMCS-I.9 Sacl; 7, pGEMCS-I.9 EcoRl. (Panel B) i.5% agarosc-2.2 M formaldehyde gel dectrophorctic analysis of linear pGEMCS!.9 Xhol and Sacl digested in vitro run-off transcription product. Lanes: 1, RNA Mr markers; 2, pGEMCS-I.9 Xhol digested T7 RNA polymerase transcript without cap; 3, like 2, but wkh cap; 4, pGEMCS-I.9 $,ci digested SP6 RNA polymerase transcript without cap; 5, like 4, but with cap. (Map C) pGEMCS. 1.9 clone showing the position of some of the restriction enzyme site in relation to the C$-i insert, and the SP6 and T7 RNA polymerase promoters, i)The, clone upon linearization by Xhol digestion, followed by T7 RNA polymerase transcription yields a CS-! sense strand transcript. ii) Linearization of the clone by digestion with Sacl followed by SP6 RNA polymcrasc transcription yields a C$-! anti-sense strand transcript. Methods. Transcription vectors pGEM-1 IZf ( + / - ) (Promega, Madison, WI) are cloning vectors containing the SP6 and T7 RNA polymerase promoters on either strand for in vitro RNA transcription (Promega, 1989; Melton et al., 1984). For cloning, the pGEM vectors were digested to completion with EcoRl followed by dephosphorylation using calf intestinal alkaline phosphatasc. The ligation of 250 ng of gel purified electro-eluted C$-i insert with 100 ng of dephosphorylated pGEM vectors was performed in a 20 pl reaction volume. The ligation mixture (2 pl) was used in a mini-transformation procedure using 20/~1 of DH5a competent cell following the manufacturer's protocol. Recombinant colonies were selected on the basis of the presence of insert upon digestion with EcoRl and CS-! insert subsequently confirmed by double-stranded dideoxy sequencing. Recombinant clones were characterized by restriction mapping to identify clones containing insert in opposite orientation with respect to the T7 and SP6 promoter sequences present in the vector. The pGEMCS-1 clones were digested with Sacl, Xbal and Xhol to linearize the plasmids (yielding a unique fragment) for subsequent run-off transcription using T7 RNA polymerase and SP6 RNA polymerase (Promega, 1989). The sense strand RNA transcript was identified by hybridization of duplicate dot blots and denaturing gel dectrophoresis blots of all transcripts with a2p 5' end labeled (Sambrook et al., 1989), probes CSF (5'-GCCCACTGAGGCAGTAGAAG) and CST (5'-GGGGAGGTTACTAATAGCAG) corresponding to the anti-sense and sense strand sequence. Transcription of lincarized pGEMCS-I clones were performed using Riboprobc in vitro transcription kit from Promega following the manufacturer's protocol for standard reaction. Run off transcription of Xhol and Sac I digested pGEMCS-1 clones were performed with T7 RNA polymerase and SP6 RNA polymerase in the absence and presence of cap analog m7-G(5')ppp(5')G (Pharmacia, Piscataway, NJ)(Melton et al., 1984; Kreig and Melton, 1984).Template DNA from the transcription reaction were removed by incubation with RQI RNase-frec DNase at 1 unit/ltg template DNA for 15 min at 37°C followed by phenol extraction and ethanol precipitation. The transcribed RNA was dissolved in diethyl pyrocarbonate-treated water and used for in vitro translation (Promega, 1989) RNA gels and dot blots (Sambrook et al., 1989). a m o n g the charged group aa, there axe 14 Arg, 12 Lys and 4 His representing the positively charged aa, and 2 Tyr, 2 Cys, 3 2 G l u and 6 A s p as deprotonated negatively

charged aa. A strong potential glycosylation site is present at position aa 4 6 - 4 8 ( U W G C G , 1989). Extensive computer search o f the G e n B a n k d a t a b a s e with k n o w n se-

209

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Fig. 4, Analysis of in vitro translaUon products. (Panel A)Autoradiograph of an enhanced dried 0.1% SDS 16% PAGE gel of in vitro translation products of various run-off transcripts. Lanes: 1 and 4, protein Mr markers; 2, blank translation with no added RNA; 3, BMV control transcripts; 5, pGEMCS-I.9 Sacl digested SP6 RNA polymerase capped transcript; 6, as in 5, but uncapped transcript; 7, pGEMCS-I.9 Xhol digested T7 RNA polymerase capped transcript; 8, as in 7, but uncapped transcript. Lanes 5 and 6 have no labeled translation product representing the anti-sense strand run-off transcripts, whereas lanes 7 and 8 have a unique approx. 33 kDa translation product, both lanes representing translation of sense strand run-off transcripts. (Panel B) Immunological Western-blot analysis of in vitro translation products. Lanes: 1-3, incubated in CS-! primary antibody; 1, pGEMCS-I.9 Sacl digested SP6 transcript translation product; 2, pGEMCS-I.9 Xhol digested T7 transcript translation product; 3, blank translation with no added RNA. Lanes 4-5 incubated in pre-immune sera, they represent same samples as in lane 1-3. The CS-I translated product present in lane 2 is indicated by an arrow. Methods. Rabbit reticulocyte lysate nuclease treated, aa mixture minus leucine and plus [ m4C]leucine(308 mCi/mmoi, 50/~Ci/ml) were used for in vitro translation (Promega, 1989). Run-off transcripts, approx. 250 ng were ~ranslated in a total volume of 50/~! containing 35/~l rabbit reticulocyte lysate. Prior to translation, 5 #! (25nCi) [ ~4C]leucine in 2% ethanol was transferred to a 1.5 ml tube and evaporated to dryness, all components required for translation were then added to the tube following the standard protocol provided by the supplier. Translated products were characterized by withdrawing a 5 ttl aliquot from each reaction and adding to tubes containing 20/~l protein loading buffer (62.5 mM Tris. HCI pH 6.8/10% glycerol/2% SDS/5% ~-mercaptoethanol/0.05% bromophenol blue) and 1/~l/1-mercaptoethanol. The above mixture was kept in a boiling water bath for 2 min followed by application of 10 #l aliquots to a 16% polyacrylamide-0.1% SDS gel for electrophoretic separation (Promega, 1989; Laemmli, 1970). Following electrophoresis the gel was stained, destained and dried; the dried gel was lightly and evenly sprayed with EN3HANCE autoradiography enhancer spray (NEN Dupont), and kept for autoradiography at -70°C for 72 h. In vitro translation products were resolved ill 16% polyacrylamide-0.1% SDS gel (Laemmli, 1970). Following electrophoresis the gel was equilibrated in Dunn carbonate transfer buffer (10 mM NaHCO3/3 mM Na,CO3/20% methanol) (Dunn, 1986) for semi-dry electro-blotting onto nitrocellulose membranes (Towbin et ai., 1979). Electro-blotting was done for 35 min at 12 volts using a Trans-BIot SD (BioRad) semi-dry electrophoretic transfer cell. Two sheets of nitrocellulose equilibrated in the transfer buffer were placed to monitor the possibility of proteins passing through the first membrane. The Western blots were washed in TBS (50 mM Tris. HC! pH 7.9/150 mM NaCl), blocked with 3% BSA and cut into strips. These strips were incubated either in CS-I specific polyclonal antibody (1:400 diluted in TBS containing 0.01% sodium azide/0.1% BSA/1 ~ E. coil extract for background reduction), or, in pre-immune rabbit sera (1:400 diluted in TBS, all other components being the same as for CS-I antibody) for 14-16 h shaking at room temperature. Following primary antibody incubation, the membranes were washed three times in TBST (TBS plus

quences did not result in the identification of any nt sequence having considerable homology with the CS-I eDNA. (c) In vitro transcription The CS-1 cDNA insert obtained from the ~,gtl I clone was cloned in pGEM-11Zf( + ) and ( - ) for transcription. Recombinant clones containing the insert were purified, two such clones pGEMCS-I.1 and pGEMCS-1.9 were processed for further characterization. Various restriction enzymes were used which cleave only at the multiple cloning sites of the vector to yield a unique fragment. Digestion with XhoI and Sacl yielded unique fragments. Fig. 3A shows restriction map of pGEM-CS clones with either Xhol, Sacl or EcoRI. Lane 7 shows EcoRI digestion yielding the 1.82-kb CS-I insert and 3.23-kb vector fragment. Lanes 3-6 show unique fragments of 5.05-kb indicating linearization of the clone with no internal sites in the CS1 insert. Double-stranded DNA sequencing of pGEM-CS clones established the orientatio~ of ~he g~,NA-like strand insert sequence with reference to either the T7 or SP6 RNA polymerase promoter sequences flanking the insert sequences. Digestion of pGEMCS- 1.9 with Xhol produced a unique fragment containing the vector sequence at one end of the T7 promoter and the CS-1 sequence at the other end of the promoter (see Fig. 3C). Similarly, SacI digestion of pGEMCS-I.9 gave a fragment with SP6 promoter sequence flanked by CS-I insert and vector sequence on either side. Transcription of pGEMCS-I.9 using T7 and SP6 RNA polymerase produced an authentic approx. 1.8kb RNA corresponding to the approx. 1.8-kb CS-I insert (see Fig. 3B); the 1.8-kb size was established by denaturing agarose gel electrophoresis with RNA markers. Transcription reactions were performed in the absence or presence of cap analog m7-G(5')ppp(5')G; upon denaturing agarose gel electrophoretic analysis differences were noticed in the transcription reactions containing no cap analog. These reactions produced extra bands in addition to the expected approx. 1.8-kb transcript. Fig. 3B, lane 2 contains an approx. 0.45-kb band and lane 4 contains two bands larger than the approx. 1.8-kb band. These extra transcripts upon translation do not seem to yield any product as judged from the autoradiogram (see Fig. 4A). Northern and dot blot analysis of T7 and SP6 RNA polymerase generated transcripts by hybridization with

0.05% Tween-20) and once in TBS. The washed membranes were then incubated in alkaline phosphatase conjugated-affinity purified goat antirabbit lgG (1:7500 diluted in TBS) for 90 rain shaking at room temperature. The membranes were again washed in TBST and TBS as above. Alkaline phosphatase specific color assay was developed using NBT and BCIP substrates (Promega, 1989; Blake et al., 1984) in alkaline phosphatase buffer (100 mM Tris. HCI pH 9.5/100 mM NaCI/5 mM MgCi,).

210

primers corresponding tO the sense and anti-sense sequence, established the sense and anti-sense transcripts. Sacl-digested pGEMCS-1.9 transcribed by SP6 RNA polymerase generates the anti-sense strand RNA, whereas, T7 RNA polymerase directs the transcription of the sense strand of reCS-1 using pGEMCS-1.9 DNA digested by XhoI. This inference was further proved by in vitro translation of both the transcripts.

(d) In vitro translation Run-offtranscripts generated by T7 and SP6 RNA polymerase were translated in rabbit reticulocyte lysate system. The products were resolved by SDS-PAGE and subjected to either autoradiography or immunological analysis of Western blots. Autoradiography of the enhanced gels revealed the presence of a single approx. 33-kDa in vitro translated product of T7 RNA polymerase capped and uncapped transcript using pGEM-CS-1.9 DNA digested by XhoI (Fig. 4A, lanes 7-8). The intensity ofthe translated product on the autoradiograph was nearly equal as compared visually for both the capped and uncapped transcripts (Fig. 4A, lanes 7-8). Run-off anti-sense transcripts generated by SP6 RNA polymerase (Fig. 4A, lanes 5-6) did not yield any translated product either for capped or uncapped transcripts judged by the total absence of any radioactively labeled protein band on the autoradiograph. This is due to the fact that there are numerous stop codons in all reading frames. Translation of control BMV transcript as shown in Fig. 4A, lane 3 gave expected translated products. Immunological analysis of Western blots (Fig. 4B) yielded confirmatory results. The CS-1 polyclonal antibody recognized specifically a single approx. 33-kDa protein band: a translation product of T7 RNA polymerase transcript of pGEM-CS-1.9 DNA digested by Xhol (Fig. 4B, lane 2), The control antibody did not specifically bind to any protein band in the SP6 RNA polymerase anti-sense transcript (product of pGEM-CS-I.9 digested by Sacl) translation sample as well as in the blank translation (Fig. 4B, lanes 4-6). The Western blot showed many nonspecific bands. This can be attributed to the fact that the primary antibodies were obtained from rabbits and the in vitro translation system was rabbit reticulocyte lysate such that many host cross-reacting antibodies are expected to be present. This fact is substantiated by the same nonspecific bands being present in the blot incubated with the pre-immune serum as the primary antibody (Fig. 4B, lanes 4-6). The specific approx. 33-kDa band (shown by an arrow in Fig. 4B, lane 2) corresponding to reCS-1 protein was recognized only by the CS-l-specific antibody, In the absence of any aa sequence or nt sequence data, all characterization criteria were based on the specificity of the antibody. Identification and characterization beyond the

initial isolation by immunological screening of a clone producing a fused protein possibly of reCS-I could only be achieved by the expression of the eDNA insert to yield an unfused protein followed by more rigorous immunological analysis. This aim was achieved by in vitro transcription and in vitro translation of the transcript in a rabbit reticulocyte lysate system. The strong binding of CSol-specific antibody to the expressed fused reCS-I protein was not considered to be ample proof for an authentic CS-1 protein and in turn a CS-I eDNA clone. To further prove that the isolated clone was the reCS-I eDNA clone, the insert was excised and cloned in transcription vector pGEM 11Zf containing promoter sequences for the T7 and SP6 RNA polymerase. Clones in the transcriptiofi vector were selected to transcribe either the sense or anti-sense strand of the insert. These transcripts upon translation in the rabbit reticulocyte lysate system yielded unfused protein; the translated protein from one of the clones (pGEMCS-I.9) expected to yield the reCS-I protein did actually give a single radioactively labeled band which was specifically picked up by the CS-l-specific antibody. This confirmed the isolated clone to be of reCS-1.

(e) Conclusions (1) The isolation of CS-I eDNA clone and its identification are based on the specific binding of CS-1 polyclonal antibody. This antibody has previously been shown to be specific for CS-I and inhibit cleavage of fertilized eggs without apparently affecting pronuclear formation (Naz, 1992). (2) Total DNA sequencing of the 1828-bp insert and the subsequent computer generated translation indicates that the clone has an exceptionally long 3' non-coding sequence. The longest ORF frame codes for a 249-aa protein. (3) The molecular size of the reCS-I corresponds to the combined size of the doublet antigens (approx. 14 and 18 x 103). Since the reCS-1 in the present study is isolated from the testis and the doublet antigens in the previous study were isolated from the sperm cells (Naz, 1992), it appears that the doublet antigens develop as a single polypeptide of approx. 33-kDa in the testis, which is subsequently cleaved to form the two peptides of approx. 14 kDa and 18 kDa present on the mature sperm cells. (4) Large scale in vitro translation will enable us to work on the purification of the recombinant CS-1 protein to raise specific antibodies to investigate its effect on fertility and embryonic development. (5) Besides enabling us to understand the molecular events underlying fertilization and early embryonic development, the reCS-1 may find applications in the development of a contraceptive vaccine and in diagnosis and treatment ofimmunoinfertility in humans (Naz, 1987; Naz et al., 1984; 1986).

211 ACKNOWLEDGEMENTS S u p p o r t e d in p a r t by a g r a n t f r o m t h e N I H ( H D 2 4 4 2 5 ) to R.K.N.

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