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a Section of Molecular & Cellular Biology, University of California, Davis, California 95616
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In an earlier study by Hardy and Hedrick [10], the protease responsible for the gp43-to-gp41 conversion in Xenopus egg envelopes was purified from the pars recta secretory granules and was termed oviductin due to its origin. They showed oviductin to be a 66-kDa protein, as determined by both reducing and nonreducing SDS-PAGE, with characteristics of a serine active site protease with trypsin-like specificity, as determined by specific inhibitor and substrate studies. When isolated envelopes were treated with purified oviductin, gp43 was converted to gp41, and the physical changes associated with oviductal transit were observed. We have further investigated the structure/function relations of oviductin by cloning oviductin cDNA, determining the domain structure of the protein, and examining changes in sperm binding to CEs treated with purified oviductin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning and Sequencing of Oviductin cDNA
The pars recta portions of oviducts were removed from anesthetized females hormonally stimulated four days before with 35 IU of eCG (Sigma, St. Louis, MO) according to the method of Hedrick and Hardy [11]. Messenger RNA was isolated directly from the tissue using a FastTrack kit from Invitrogen (Carlsbad, CA). Complementary DNA was generated by reverse transcription using 5 µg mRNA, Moloney murine leukemia virus (MMLV) reverse transcriptase, and random hexamers. The DNA was purified using a Centricon 10 cartridge (Amicon Inc., Beverly, MA). For amplification of the N-terminal region of oviductin cDNA, degenerate polymerase chain reaction (PCR) primers were designed on the basis of the oviductin N-terminal amino acid sequence KGQHPWT (amino acids 9–15) determined by Edman sequencing of the isolated protease [10], 5'-AARGGNCARCAYCCNTGGAC-3' (sense), and the conserved amino acid sequence flanking the Ser of serine active site proteases (GDSGGP), 5'-TTCCTGCAGGRGGK-CCNCCRGARTCWCC-3' (antisense). The primers were synthesized by the Protein Structure Laboratory at the University of California, Davis. The PCR was performed using Taq DNA polymerase from Promega (Madison, WI), with an initial denaturation at 95°C for 7 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 2 min, and extending at 72°C for 3 min, with a final extension at 72°C for 10 min. The PCR product (~540 base pairs [bp]) was separated on an agarose gel, and the band was excised and purified using GeneClean II (Bio 101, Inc., La Jolla, CA). The purified product was inserted into a pGEM-T PCR vector (Promega) for DNA sequencing.
On the basis of the cDNA sequence obtained above, perfect-match PCR primers were designed for use in synthesizing a ~470-bp probe for screening a pars recta cDNA library, 5'-GGACAGTATCATTGAAACGG-3' (sense), and 5'-ATACGAAGACACAACGGTTC-3' (antisense). The library was made using a Stratagene (La Jolla, CA) ZAP-cDNA synthesis kit, starting with pars recta mRNA isolated using Stratagene kits (RNA isolation kit and Poly(A) Quik mRNA isolation kit). Complementary DNA was generated using 5 µg mRNA primed with a XhoI-oligo(T) linker/primer, and MMLV reverse transcriptase. After second-strand cDNA synthesis using DNA polymerase I, EcoRI adapters were ligated to both ends, and the products were modified by XhoI enzyme digestion to generate an XhoI overhanging arm at the 3' end. This was followed by unidirectional insertion into a Uni-ZAP XR vector (Stratagene; precut by XhoI and EcoRI), packaging into viral coats using Gigapack II Gold packaging extracts (Stratagene), and amplification using XL1-Blue MRF' cells. The library was screened using the PCR product described above, and positive clones were selected and transformed into XL1-Blue MRF' cells for plasmid preparation. Two independent clones were selected after secondary and tertiary screening, and sequenced in both directions.
Since the initial cDNA clones did not possess a start Met sequence, 5' rapid amplification of cDNA ends (RACE) [12] was performed using a kit from Gibco BRL (Grand Island, NY). The starting material was total RNA isolated from pars recta oviduct using an RNA isolation kit from Stratagene. The PCR product that was obtained was purified using a GlassMAX spin cartridge (Gibco BRL) and sequenced directly. A start Met was found, but then efforts were made to obtain a more complete 5' untranslated region sequence, by using a different RACE kit (Clontech Labs., Palo Alto, CA), and by increasing the temperature of the reverse transcription step from 42°C to up to 55°C. However, a product of the same size was observed for all efforts.
Sequencing of DNA was performed on the University of California, Davis campus by either the Division of Biological Sciences or the Advanced Plant Molecular Genetics DNA sequencing facilities. Sequences were analyzed by the Genetics Computer Group (GCG; Madison, WI) sequence analysis software, using default parameters. The signal sequence was predicted using the PSORT program [13]. Possible O-linked glycosylation sites were predicted using the NetOglyc program [14]. For the CUB domain alignment, sequences were adjusted by hand following Bork and Beckmann [15]. Genbank and Swiss-Prot accession numbers for proteins used for comparison were human chymotrypsinogen b, P17538; mouse elastase 2, P05208; human hepsin, X07732; Drosophila tolloid, P25723; hamster calcium-dependent serine protease, P15156; human bone morphogenetic protein 1, P13497; and pig spermadhesin AQN-1, P26322.
Northern Blot Analysis and mRNA Tissue Expression
A Northern blot analysis was performed to determine the message size for oviductin. A probe (~600 bp) was synthesized by PCR using the primers 5'-TCAGGGTTATCGAAATCTTC-3' (sense) and 5'-TGCACCGCTCATTGTTCTTG-3' (antisense), with 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, using Taq polymerase. The probe was purified using a PCR clean-up kit from Qiagen (Chatsworth, CA), and then random primer-labeled using a Redi-prime kit (Amersham, Arlington Heights, IL) and [32P]dCTP according to the manufacturer's instructions. Twenty micrograms of pars recta oviduct RNA (isolated using a Stratagene kit) was separated on a 1.0% agarose, 0.22 M formaldehyde, 10 mM HEPES, 1 mM EDTA gel; transferred to Hybond-N (Amersham) by capillary action [16] using 20-strength SSC (single-strength SSC = 0.15 M sodium chloride, 0.015 M sodium citrate; Gibco BRL); and then UV-cross-linked. The membrane was prehybridized in Rapid Hyb solution (Amersham) for 30 min; this was followed by hybridization with probe at 65°C for 5 h, and washing twice in double-strength SSC, 0.1% SDS at 65°C for 15 min, then twice in 0.2-strength SSC, 0.1% SDS at 65°C for 15 min. Blots were exposed to film for 9 h. The size of the RNA was determined by comparison with 1-kilobase (kb) RNA standards (Gibco BRL).
For detection of oviductin mRNA in various tissues by reverse transcription (RT) followed by PCR amplification (RT-PCR), a Titan One Tube RT-PCR kit from Boehringer Mannheim (Indianapolis, IN) was used with 1 µg total RNA (isolated using a Stratagene kit) for each tissue sample. Tissues included pars recta oviduct, ovary, lung, liver, and skeletal muscle from an eCG-stimulated animal (three days post-injection). For amplification of oviductin cDNA, the same PCR primers were used as above for the Northern blot. As a control, ß-tubulin cDNA was amplified using the primers 5'-GGGCTTCCAGCTGACTCACT-3' (sense) and 5'-GCGCTTGAACAGCTCCTGGAT-3' (antisense). After the initial RT step (30 min at 50°C), the PCR program consisted of 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C for 2 min. Ten microliters of each sample was analyzed by agarose gel electrophoresis.
Isolation of Oviductin Protease
Pars recta oviducts, isolated as described above, were homogenized, and the secretory granules were extracted as described previously [10, 17]. Oviductin was purified from the extract using a p-aminobenzamidine-agarose affinity column followed by hydroxyapatite chromatography as described by Hardy and Hedrick [10]. Enzymatic activity was monitored using the fluorogenic substrate N-tert-butoxycarbonyl-Phe-Ser-Arg-7-methylcoumaryl-4-amide (Boc-Phe-Ser-Arg-MCA, from Sigma), with a typical assay consisting of 395 µl assay buffer (200 mM NaCl, 1 mM CaCl2, 10 mM Tris pH 8), 5-µl sample, and 1-µl substrate (5 mM in dimethyl sulfoxide).
Egg Envelope Isolation and Treatment
Oviposited eggs were obtained from female frogs after an injection of 35 IU of eCG followed 4 days later by injection of 1000 IU of hCG (Scripps Laboratories, San Diego, CA) [11]. Eight to 12 h after the final injection, eggs were stripped from females and dejellied using mercaptoethanol as described previously [18]. Coelomic eggs were obtained from the body cavities of females whose oviducts had been ligated before hormonal stimulation [11, 19]; eggs were collected 10–12 h post-hCG. Both CEs and VEs were isolated from eggs and stored in a high-salt solution at 4°C as described previously [19]. Before use, the envelopes were washed extensively with distilled water by repeated centrifugation.
To selectively convert gp43 to gp41, pelleted CEs (20 µl) were resuspended in 500 µl of protease assay buffer containing 2.5 mIU/ml purified oviductin, and incubated for 30 min at room temperature [10]. Trypsin (type II; Sigma) was also used under identical conditions. At the end of the incubation period, the envelopes were washed by centrifugation 3 times each in 1.5 ml distilled water. Portions of the pelleted envelopes were then used in sperm binding studies or analyzed by SDS-PAGE [20].
Sperm Binding Assays
Sperm were collected by macerating a testis in 1 ml of one-third-strength DeBoer's solution (DB; single-strength DB = 110 mM NaCl, 1.3 mM CaCl2, 1.3 mM KCl, to pH 7.2 with NaHCO3). The large testicular tissue fragments were allowed to settle; then the sperm suspension was added to an equal volume of isolated egg envelopes in one-third-strength DB. A 5-min incubation period was used to allow for sperm-envelope binding, and then the mixture was passed over a 105-µm nylon mesh screen folded into a funnel shape, which retained the envelopes but allowed the unbound sperm to pass through as the screen was rinsed with one-third-strength DB. The envelopes were pipetted from the screen and added to an equal volume of 2.5% glutaraldehyde in one-third-strength DB and then transferred to a microscope slide. Bound sperm were counted on portions of the envelopes that were not folded over onto themselves.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The starting point for the cloning of Xenopus oviductin cDNA was PCR amplification of the N-terminal region of the protease molecule, using cDNA templates made by reverse transcription of oviduct (pars recta) mRNA, a sense primer representing the N-terminal amino acid sequence determined by Edman sequencing of purified, proteolytically active oviductin, and an antisense primer based on the conserved sequence flanking the Ser of serine active site proteases. The ~540-bp cDNA sequence that was obtained was then used to make a probe for screening a pars recta cDNA library. Out of 29 positive clones, two were selected for sequencing in both directions. A 5' RACE reaction was necessary to complete the sequence. The composite cDNA was 3689 bp long, had a single open reading frame of 3012 bp, an ATG start site consistent with Kozak's rules, a rather short 5' untranslated region of 6 bp, and a 3' untranslated region of 652 bp, plus a poly(A) signal sequence followed by the poly(A) tail. The sequence was entered into the Genbank database with an accession number of U81291.
Northern hybridization showed that the pars recta mRNA was approximately 5.1 kb in size (Fig. 1A). This size was larger than the cDNA sequence that was obtained, but a reasonable match considering that only a very short portion of the untranslated 5' region was sequenced. Efforts were made to obtain more of the 5' untranslated sequence by increasing the temperature of the reverse transcription step to overcome blockage of the enzyme by secondary RNA structure. However, the problem persisted, and therefore a complete mRNA sequence for oviductin was not obtained, although the open reading frame appeared complete (see the sequence analysis below).
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An RT-PCR approach was used to determine the tissue specificity of oviductin expression. As shown in Figure 1B, the message for oviductin appeared to be specific to pars recta oviduct, with no detection in ovary, liver, lung, or skeletal muscle from an eCG-stimulated animal.
Sequence Analysis
The open reading frame of the oviductin cDNA coded for a protein of 1004 amino acids corresponding to a molecular mass of 110.6 kDa, much larger than the mature oviductin protease of 66 kDa. There were a total of five possible N-glycosylation sites on the complete cDNA sequence (Fig. 2, open squares), and two O-linked glycosylation sites (Fig. 2, filled diamonds). The mature oviductin protease domain began at Ile46 of the translated cDNA sequence, identified by the N-terminal amino acid sequence as determined for the purified protein (Fig. 2, double underline). A PSORT analysis showed a hydrophobic signal sequence with a predicted cleavage site after Gly19. Thus, a pro-enzyme region constituted residues 20–45. The overall structure of the oviductin glycoprotein is that of a regulated secretory glycoprotein, consistent with its presence in secretory granules of the pars recta oviduct.
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The deduced oviductin amino acid sequence was compared to other proteins using FASTA and BLAST programs (GCG), which revealed that residues 46–287, containing the oviductin N-terminal amino acid sequence, represented a protease with up to 43% identity to human chymotrypsin b, mouse elastase 2, and human hepsin (Table 1, and Fig. 2). The database search also revealed an additional protease domain in the second half of the sequence spanning approximately residues 584–822 (Fig. 2). This second protease domain showed a lower value of 38% identity to the serine proteases found to be most similar to the first protease domain (Table 1); a database search found no better matches. When the two protease domains, designated oviductin-
and -ß, were compared to each other, they exhibited 32% identity, lower than when compared to proteases from other species and tissues.
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The deduced amino acid sequences of the oviductin- and -ß protease domains were aligned with those of chymotrypsin, elastase, and hepsin to identify conserved domains in the proteases (Fig. 3A). The alignment showed that the hydrophobic N-terminal Ile or Val of the mature proteases, which is required for proper folding to form the catalytic pocket in serine active site proteases (see [21]), was present in both the oviductin- and oviductin-ß protease domains. An Arg preceding the N-terminal Ile of oviductin- indicated that the inactive pro-oviductin- molecule would be activated by a trypsin-like protease. In contrast, the oviductin-ß protease domain possessed a Ser instead of an Arg preceding the N-terminal Ile. The protease domain alignment also revealed that the oviductin- domain possessed a trypsin-like protease binding pocket Asp (Fig. 3A, shaded area), consistent with the measured activity of the oviductin- protease [10]. The oviductin-ß protease domain also had this residue, indicating it would also have trypsin-like substrate specificity. However, several of the other conserved amino acids found in serine proteases were absent in oviductin-ß, particularly in the regions surrounding the active site His, Asp, and Ser. In fact, the active site His was replaced by a Ser in oviductin-ß, through a 2-bp codon change. These results together suggest that oviductin-ß would not possess proteolytic activity but may possess substrate binding activity.
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The other regions of the oviductin transcript, apart from the protease domains, were analyzed for similarity to other proteins using the BLAST program. It was found that these regions (Fig. 2) were related to CUB domains, which possess antiparallel ß-barrel topography and are common to many extracellular, developmentally regulated proteins [15]. In a multiple alignment of CUB domains from several other proteins (Fig. 3B), it was revealed that only one of the oviductin CUB domains, the second, possessed all of the four conserved Cys residues involved in the formation of two disulfide bonds for secondary structure [15].
Sperm Binding Assays
To study the mechanism of how purified, mature oviductin () protease might render envelopes penetrable by sperm, a sperm binding assay was developed using isolated egg envelopes. As shown in Figure 4 and Table 2, untreated CEs had few bound sperm, while CEs treated with purified oviductin showed a dramatic increase in sperm binding, to levels exceeding those observed with VEs. To test whether proteolysis of gp43 alone was sufficient to induce sperm binding, or whether oviductin's CUB domains or some other feature might mediate sperm binding, trypsin was used to treat isolated CEs. This experiment was based on an earlier finding that trypsin can mimic oviductin in selectively cleaving gp43 to gp41 [10]. We found that trypsin was just as effective as oviductin in increasing sperm binding to CEs (Fig. 4D, Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Upon cloning and sequencing of oviductin cDNA, it became apparent that the isolated transcript coded not only for the 66-kDa active oviductin protease that was originally purified from Xenopus oviduct (pars recta), but for a much larger protein, approximately twice the expected size. This indicated that extensive post-translational processing must occur to yield the mature oviductin protease. Analysis of the sequence revealed a secretion signal sequence at the N terminus which would be cleaved from the protein, followed by the oviductin protease domain containing the N-terminal amino acid sequence that had been determined for the mature enzyme; this domain was designated oviductin-. In addition, another protease domain was found in the second half of the molecule, and was designated oviductin-ß. Sequence analysis revealed that both oviductin- and -ß coded for proteases with trypsin-like substrate specificity, possessing an Asp in the substrate binding pockets for binding to basic residues. This matched the experimental evidence that active oviductin had a substrate specificity for the sequence Phe-Ser-Arg [10]. However, oviductin-ß was missing the required His of the active site triad; therefore, oviductin-ß would probably not possess proteolytic activity, but since it retained a structurally complete substrate binding pocket, it may possess binding activity.
Since the molecular mass of the mature oviductin protease (containing the protease domain) is 66 kDa, we conclude that the oviductin- and -ß protease domains are separated during post-translational processing. One definite processing site would be after Arg45 to give the N-terminal amino acid sequence determined for mature oviductin- protease (see Fig. 1). Cleavage at this site would require the action of a protease with trypsin-like specificity, possibly oviductin- itself through autocatalysis. As for processing on the C-terminal side of oviductin-, a logical position would be after Ser583 to reveal the conserved N-terminal sequence of the oviductin-ß protease domain (Fig. 3A). Cleavage after Ser583 would require a more unusual protease specificity, although cleavage of a Ser-Ile bond has been demonstrated for activation of human apolipoprotein a [22]. These proposed cleavage sites for active oviductin-, after Arg45 and Ser583, would yield a mature protein of 60 kDa, and along with N-glycosylation at any or all of the three possible sites, this value is very close to the molecular mass of 66 kDa determined for the purified protein by SDS-PAGE. If indeed there is a processing site after Ser 583, this would also yield a protein containing the oviductin-ß protease domain, with a molecular mass of 46 kDa or more depending on the extent of glycosylation at the two possible N-glycosylation sites and two predicted O-linked glycosylation sites within the deduced amino acid sequence. It must be kept in mind that the existence of the oviductin-ß-containing protein is speculative at this time since it has not been identified or characterized. It is possible that this portion of the mosaic oviductin glycoprotein is degraded within the secretory cells or degraded shortly after secretion.
The presence of two separate proteases within a single translation product is highly unusual, since the only known example in which several different protease molecules are produced through the processing of a single transcript are the polyproteins of viruses [23]. Angiotensin I-converting enzyme (ACE), a metallopeptidase found in several mammalian tissues, is known to possess two exoprotease domains, but these proteases are not separated after translation [24, 25]. The two protease domains of ACE appear to be a result of gene duplication, since the domains are very similar (68% identical for human kidney ACE). In contrast, the Xenopus oviductin protease domains were found to show only 32% identity, a lower value than when either protein was compared to other proteases from different species and tissues (see Table 1). This could indicate that the domains are spliced together from different genes, or that they are derived from a single gene but the domains have undergone differential evolution. The cotranslation of the gene products suggests that it is important that the proteins are expressed simultaneously. This is particularly intriguing considering that the oviductin-ß protease does not appear to have the potential to be proteolytically active. Although it is unknown whether intact oviductin-ß protease is actually released by the pars recta secretory cells, is it difficult to imagine that this molecule serves no function since it is cotranslated with oviductin- and is a distinct protein, i.e., not merely a degenerate form of oviductin-, and may have a functional binding site.
CUB Domains
In addition to the two protease domains present in the translated oviductin transcript, three CUB domains were found. According to the processing sites for oviductin proposed above, the active oviductin- protease domain would be coupled to the first two CUB domains, while oviductin-ß would be coupled to the third, and in both instances the CUB domains would be C-terminal to the protease domains. These domains, named for the first three proteins found to possess them—Complement C1r/C1s, Uegf (embryonic sea urchin protein fibropellin), and Bmp1 (bone morphogenetic protein 1)—consist of a stretch of approximately 110 amino acids in an antiparallel ß-barrel conformation and are found as an extracellular domain in a wide range of proteins [15]. Such proteins include proteases such as the astacin metalloendopeptidases Bmp1, tolloid [26], and C. elegans hatching enzyme HCH-1 [27], as well as serine proteases such as the human complement subcomponents C1s and C1r, and complement-like hamster Casp [28]. It should be noted that only the second oviductin CUB domain possessed all of the four conserved Cys characteristic of CUB domains, while the first and third CUB domains were each missing one of these Cys (see Fig. 3B). However, the CUB domains of the complement proteins are known to miss the first Cys. Thus, the presence of four Cys in the domain is not structurally required for classification as a CUB domain [15].
Although the majority of the proteins containing CUB domains are functionally involved in developmental processes, a specific function for CUB domains has not been determined [15]. An exception are the spermadhesin molecules, which have been implicated in mediating sperm-egg envelope interactions in mammals [29, 30]. Spermadhesins are composed of 109–133 amino acids, and thus are essentially a single CUB domain. They are derived from the male accessory glands, are deposited onto the sperm surface during ejaculation, and appear to be involved in the binding of sperm to carbohydrates on the egg envelope [31]. It is tempting to speculate that the CUB domains of the Xenopus oviductin proteins may have a function similar to that of the spermadhesins, tethering the proteins to the egg envelope after the egg has passed through the oviduct, and mediating sperm binding to the envelope. The possible function of CUB domains in oviductin- function was addressed in sperm-envelope binding assays, discussed below.
Oviductin- Function in Sperm Binding
Using a sperm-egg envelope binding assay, we showed that pretreatment of isolated CEs with purified oviductin- protease dramatically increased sperm binding. This is the first direct examination of the mechanism by which oviductin- might increase fertilizability of eggs. To test whether the protease action of oviductin- alone was responsible for the observed increase in sperm binding, or whether the CUB domains might be involved, we treated CEs with the protease trypsin. The basis for this experiment was the finding that trypsin can mimic the action of oviductin in selectively hydrolyzing gp43 to gp41 [10]. We found that trypsin treatment also increased sperm binding to CEs in a manner apparently identical to that of oviductin; the ability of trypsin to increase sperm binding to CEs has also been demonstrated in Bufo japonicus [32]. Recently, we also found that trypsin treatment of Xenopus coelomic eggs renders them fertilizable [33]. Therefore, it appears that processing of gp43 alone is responsible for increasing sperm binding to the envelope, and this increase in binding correlates with increased fertilizability. It must be kept in mind that the experiments were performed under artificial conditions, and the conditions in vivo may be such that CUB domains associated with oviductin- protease activity become important. It is interesting that all of the Xenopus egg proteases so far characterized and known to interact with the egg envelope contain CUB domains: hatching enzyme Uvs2, an astacin metalloprotease released by the embryo to break down the envelope [34]; ovochymase, a serine protease anchored in the extracellular matrix (perivitelline space) of the egg and released at egg activation ([35] and unpublished data: Genbank accession number U81290); and now the oviductin proteases. Since the majority of proteins identified as possessing CUB domains are extracellular, and in light of the results obtained from studies with the spermadhesins, it is possible that CUB domains function to target proteins to extracellular matrix glycoproteins. In the case of oviductin, binding to the egg envelope may be important in vivo to keep the protease in place as the egg travels down the oviduct, so that the protease has time to completely process gp43. In addition, the CUB domains may assist in binding of soluble enzymes to the particulate or insoluble envelope, thereby assisting in positioning the protease to catalytically act on its envelope substrate. However, this scenario does not fit for oviductin-ß, assuming that it is secreted and binds to the egg extracellular matrix. Since oviductin-ß would not possess protease activity, its presence on the envelope must have a different functional significance, e.g., enhancing sperm binding, or inducing the sperm acrosome reaction. To test these hypotheses it will be necessary to determine whether oviductin-ß is secreted as an intact protein, and to examine its possible role in the fertilization process. It would also be interesting to determine whether oviductin-, expressed without CUB domains, is as effective in altering the sperm binding properties of the egg envelope. Alternatively, CUB domains by themselves could be expressed, and their ability to bind to egg envelopes and enhance sperm-envelope interactions examined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: LeAnn Lindsay, Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616-8535. FAX: 530 752 3085; lllindsay{at}ucdavis.edu
Accepted: November 25, 1998.
Received: July 6, 1998.
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REFERENCES |
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