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Primary Structure of the Mouse Sperm Receptor Polypeptide Determined by Genomic Cloning Ross A

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Primary Structure of the Mouse Sperm Receptor Polypeptide Determined by Genomic Cloning Ross A Proc. Nat!. Acad. Sci. USA Vol. 85, pp. 6409-6413, September 1988 Developmental Biology Primary structure of the mouse sperm receptor polypeptide determined by genomic cloning Ross A. KINLOCH, RICHARD J. ROLLER*, CAROLYN M. FIMIANI, DAVID A. WASSARMANt, AND PAUL M. WASSARMAN Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110 Communicated by H. Ronald Kaback, May 11, 1988 ABSTRACT The mouse sperm receptor, a glycoprotein MATERIALS AND METHODS It called ZP3, is synthesized and secreted by growing oocytes. Construction and Screening of Mouse Genomic Libraries. is present in more than a billion copies in the unfertilized egg's Mouse liver DNA was partially digested with Sau3A or extracellular coat, or zona pellucida. We have cloned and EcoPJ, and fragments were inserted between the BamHI characterized a region of the mouse (CD-1) genome that spans sites of A Charon 35 (15) or the EcoPJ sites of A DASH 10 kilobases of the ZP3 locus. The genomic clones described (Stratagene), respectively, as described by Maniatis et al. encompass the entire ZP3 coding region, which contains eight (16). Libraries were amplified on Escherichia coli LE392 exons. The exons were identified, mapped, and sequenced, and screened (2 x 106 plaques) by using an end-labeled, yielding the entire primary structure of the ZP3 polypeptide synthetic oligonucleotide complementary to ZP3 (60-mer; chain (424 amino acids; Mr, 46,300), which includes a 22-amino ref. 14). Two genomic clones were isolated and character- acid signal sequence. In addition, sequencing ofgenomic clones ized, ZP3-G5 from the Sau3A library and ZP3-G9 from the has revealed some unusual features of ZP3 mRNA and a region EcoRP library. just downstream of the ZP3 gene. Subcloning and Sequencing of Genomic DNA Fragments. The lO-kilobase (kb) EcoRI fragment of ZP3-G9 was isolated All mammalian eggs are surrounded by a relatively thick and subcloned into the EcoRI site of pGEM-3blue (Promega extracellular coat, called the zona pellucida, that participates Biotec, Madison, WI), producing plasmid pGEM-G9/R-A. at several steps in the fertilization pathway (1, 2). The mouse Three subclones, which together span the 10-kb EcoRI frag- egg zona pellucida is =7 zm thick, contains z3 ng ofprotein, ment, were generated as follows: The 4-kb and 1.5-kb Bgl II and is composed of three glycoproteins, called ZP1 (Mr, fragments of pGEM-G9/R-A were isolated and cloned into =200,000), ZP2 (Mr, =120,000), and ZP3 (Mr, =83,000), that the BamHI site of pGEM-3Z to produce plasmids pGEM- associate with one another through noncovalent interactions G9/Bg-2B and -G9/Bg-2D, respectively. The 5'-terminal 4.7- of crosslinked filaments (1-4). kb EcoRI fragment ofZP3-G5 was cloned into the EcoRI site to form an insoluble network of pGEM-3blue, to produce pGEM-G5/R1-A. Further sub- During fertilization of ovulated eggs, ZP3, one of the glyco- cloning of these three plasmids was carried out by isolating proteins, serves as both sperm receptor and inducer of the the desired fragments and cloning them into appropriately acrosome reaction (3, 5-7). The sperm receptor activity of digested pGEM vectors (Promega Biotec). Sequencing ofthe ZP3 is attributable solely to certain of its 0-linked oligosac- pGEM-derived plasmids was carried out by using dideoxy charides (3, 8), whereas the acrosome-reaction-inducing chain-termination (17) as described by Promega Biotec. activity of ZP3 is attributable to its polypeptide chain as well Purification of Oocyte RNA. Fully grown oocytes were (3, 9). Thus, ZP3 plays structural and functional roles in the isolated from 21- to 28-day-old, female, Swiss albino mice zona pellucida. (CD-1; Charles River Breeding Laboratories) by poking ZP1, ZP2, and ZP3 are synthesized and secreted exclu- excised ovaries with fine steel needles under a dissecting sively by growing and fully grown mouse oocytes (3, 10-13). microscope. All oocytes were freed of adhering follicle cells ZP3 is synthesized as a Mr 44,000 polypeptide chain to which by repeated pipetting. Isolation of oocytes was carried out in either three or four high mannose.type, N-linked oligosac- Earle's modified medium 199 (GIBCO) supplemented with charides are added cotranslationally, giving rise to precursors bovine serum albumin (3 mg/ml), sodium pyruvate (30 tkg/ with molecular weights of 53,000 and 56,000, respectively ml) and containing dibutyryl cAMP (100 ug/ml). Isolated (12). The oligosaccharides are then processed to complex- oocytes were washed in isotonic phosphate-buffered saline to type, presumably in the Golgi, and 0-linked oligosaccharides remove bovine serum albumin, transferred in a minimal are added, giving rise to the mature, M, 83,000 form of ZP3 volume to 30 Mal of extraction buffer (50 mM Tris HCl, pH that is secreted by oocytes. 7.5/300 mM NaCl/5 mM EDTA/0.4% NaDodSO4) contain- A cDNA encoding a 20-amino acid stretch of ZP3 poly- ing carrier calf-liver tRNA (15 ,ug), and frozen on dry ice. peptide chain was isolated and partially characterized (14). After thawing, 20 ,ul of phenol was added, and the mixture Based on the reported sequence of the ZP3 cDNA fragment, was thoroughly Vortex mixed. Twenty microliters of chlo- we prepared a synthetic oligonucleotide probe, used it to roform/isoamyl alcohol, 24:1 (vol/vol), was then added, and screen CD-i mouse genomic libraries, and partially charac- the mixture was thoroughly Vortex mixed again. Aqueous terized two overlapping clones.t Results of experiments presented here provide the entire primary structure of the Abbreviation: nt, Nucleotide(s). ZP3 polypeptide chain, as well as information about ZP3 gene *Present address: Department of Molecular Genetics and Cell organization and some unusual features of ZP3 mRNA. Biology, University of Chicago, Chicago, IL 60637. tPresent address: Department of Molecular Biophysics and Bio- chemistry, Yale University, New Haven, CT 06511. The publication costs ofthis article were defrayed in part by page charge MJhe sequence reported in this paper is being deposited in the payment. This article must therefore be hereby marked "advertisement" EMBL/GenBank data base (IntelliGenetics, Mountain View, CA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03851). 6409 Downloaded by guest on September 23, 2021 6410 Developmental Biology: Kinloch et al. Proc. NatL Acad Sci. USA 85 (1988) and organic phases were separated by centrifugation, and A B c D RNA was precipitated from the aqueous phase by addition of a bc a b c a b c a b c ethanol (100 1ul) and stored at - 200C overnight. Preparation of RNA Probes. High-specific-activity RNA 52907 529 probes were transcribed with T7 or SP6 RNA polymerase -521 501 501- from template sequences cloned into pGEM vectors as 404--- -52951 --404 -404 404- described by Promega Biotec. 327-- Ribonuclease Protection Assays. RNA preparations to be -327 examined were combined with 2 x 106 dpm of probe RNA, 327*- _ --327 3274 242-- -242 lyophilized, and dissolved in 20 .14 of hybridization buffer 2190 containing 80%6 (vol/vol) formamide, 40 mM Mops (pH 6.6), t --242 242- 0.4 M NaCl, and 1 mM EDTA. RNA was denatured by 190-- incubation at 850C for 5 min and hybridized by incubation at - - 183 450C overnight. Then, unhybridized RNA was digested by -147 --190 adding 200 p.l of a mixture containing 10 mM Tris HCl (pH 147- 137h 132 Igo_ - . 7.5), 300 mM NaCl, 5 mM EDTA, RNase A (40 ,ug/ml), and 132-- g 114>- RNase T1 (2 pug/ml) and incubating for 1 hr at 370C. Digestion -110 was terminated by adding and -=147 NaDodSO4 (0.7%) proteinase 147= K (150 ,ug/ml) and incubating for 15 min at 37°C. Protected 120p RNA was purified by extraction with phenol/chloroform/ 132- isoamyl alcohol, 25:24:1 (vol/vol), and then by ethanol * -?14 precipitation in the presence ofcarrier tRNA (15 ,ug). Purified 110- RNA was analyzed on 5% or 6% polyacrylamide/8 M urea gels. Bands were visualized by autoradiography ofdried gels. 67--. t_-67 RESULTS 67- Genomic Map of ZP3 Clones. Two overlapping genomic clones were isolated. was isolated from a One, ZP3-G5, FIG. 2. Identification ofZP3 exons by RNase protection assays. Sau3A genomic library and contains a 12.5-kb insert. An- Antisense RNA, transcribed from pGEM-G9/Bg-2B (A), pGEM- other, ZP3-G9, was isolated from an EcoRI library and G9/Bg-2D (B), pGEM-G5/R1-A (C), and pGEM-G5/B (D) (see Fig. contains a 19-kb insert. The relationship of these clones to 1B), was protected with RNA from 250 isolated oocytes (A, lane a; one another is shown in Fig. LA. Southern hybridization B, lane b; C, lane b; D, lane c), liver RNA (A, lane b; B, lane c), or analysis on blots containing restriction endonuclease digests E. coli rRNA (C, lane a; D, lane b). Protected fragments and ofZP3-G5 and -G9 mapped an oligonucleotide probe (60-mer; molecular weight standards (Msp I-digested p17-2) (A, lane c; B, lane ref. 14) used to screen the libraries to 5'-terminal EcoRI a; C, lane c; D, lane a) were resolved on 5% polyacrylamide/ureagels fragments. Accordingly, the 5'-terminal EcoRI fragment (10 and visualized by autoradiography. Lengths (in nucleotides) of kb) ofZP3-G9 was isolated, subcloned, and characterized by protected fragments (arrowheads) and molecular size standards sequence analysis ofboth strands. The location of subcloned (hash marks) are indicated. o, Origin. fragments is shown in Fig. 1B. A partial restriction map for sites employed in subcloning and exon mapping is presented data).
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