Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum)

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N‐linked glycosylation sites, a potential N‐terminal signal peptide and a potential C‐terminal trans‐membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N‐terminal signal peptide sequences and C‐terminal trans‐membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae. Mol. Reprod. Dev. 52:174–182, 1999. © 1999 Wiley‐Liss, Inc.     

Cloning and characterisation of a zona pellucida 3 cDNA from a marsupial, the brushtail possum Trichosurus vulpecula

The mammalian zona pellucida (ZP) is an extracellular glycoprotein coat that plays vital roles throughout fertilisation and preimplantation development. Like that of eutherian mammals the brushtail possum ZP is composed of three glycosylated proteins of 137 kDa, 92 kDa and 62 kDa. The 62 kDa protein is a ZP3 orthologue based on its nucleotide and deduced amino acid sequence. The brushtail possum ZP3 cDNA isolated in this study is 1305 nucleotides with an open reading frame encoding a 422 amino acid peptide of 45.7 kDa. Possum ZP3 has a 46% amino acid identity with eutherian ZP3 and shares similar structural characteristics including 12 conserved cysteine residues, N-linked glycosylation sites and hydrophobic regions. Like human and rabbit ZP1 an altered furin cleavage site upstream of the C-terminal hydrophobic domain also occurs in possum ZP3 (S-R-K-R), suggestive of processing by a furin-related endoprotease. Expression of brushtail possum ZP3 is limited to the ovary. Characterisation of brushtail possum ZP3 will enable examination of its functional role in marsupial fertilisation and its effectiveness as an immunocontraceptive agent.     

Molecular cloning of bovine zona pellucida glycoproteins ZPA and ZPB and analysis for sperm-binding component of the zona.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, comprises three glycoproteins, ZPA, ZPB and ZPC, and plays important roles in fertilization. We have previously reported that apparent relative molecular masses of bovine zona glycoproteins on SDS/PAGE under nonreducing conditions after removal of poly N-acetyllactosamine at the nonreducing portion of sugar chains with endo-beta-galactosidase are 72 000, 58 000 and 45 000 [Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994) Biochim. Biophys. Acta 1201, 7-14]. The N-terminal amino-acid sequences and crossreactivity to antibodies specific to each porcine zona component show that the bovine components correspond to porcine ZPA, ZPB and ZPC, respectively. In this study, we deduced amino-acid sequences of bovine ZPA and ZPB by cDNA cloning and sequencing. Identities in amino-acid sequences between bovine and porcine counterparts were 77% for ZPA and 75% for ZPB, whereas between bovine and murine counterparts identities were 57% for ZPA and 37% for ZPB. The positions of Cys were completely conserved in bovine ZPA and ZPB compared with counterparts of other mammalian species. Bovine ZPA was processed between Ala and Asp on fertilization, suggesting that the consensus motif for the processing is Ala-Asp-Asp/Glu. We purified bovine zona components and examined their sperm-binding activity with an in vitro competition assay and sperm-bead-binding assay. As a result, ZPB showed the strongest sperm-binding activity among the components. ZPC also showed sperm-binding activity and the activity per molecule was about one-sixth that of ZPB according to the result of the sperm-bead-binding assay. We could not determine if ZPA has significant sperm-binding activity, but the activity may be much lower than that of ZPB even if ZPA has significant activity. Thus, ZPB may play a major role in sperm binding in bovine zona.     

Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins

The zona pellucida is a unique, oocyte-specific matrix that coats the surface of all mammalian eggs. Composed of three sulfated glycoproteins in the mouse (ZP1, ZP2, and ZP3), the zona pellucida facilitates early events in fertilization and protects the embryo during preimplantation development. Using DNA isolated from hamster-mouse somatic cell hybrids and from C57BL/6J X Mus spretus interspecific backcross progeny, Zp-2 was located on chromosome 7, 11.3 +/- 3.2 cM distal to Tyr, and Zp-3 was located on chromosome 5, 9.2 +/- 2.9 cM distal to Gus.     

Isolation of two novel metalloproteinase-disintegrin (ADAM) cDNAs that show testis-specific gene expression.

Metalloproteinase-disintegrins (ADAMs) are type 1 transmembrane proteins that contain a unique domain structure including a zinc-binding metalloproteinase domain. We have isolated cDNAs encoding two novel members of this family, ADAM29 and ADAM30 which show testis-specific expression. Three forms of ADAM29 were found that encode proteins of 820, 786 and 767 amino acids. All of the amino acid differences are located in the cytoplasmic domain. Two forms of ADAM30 were isolated that encode proteins of 790 and 781 amino acids, with the difference in the coding region occurring in the cytoplasmic domain. ADAM29 and ADAM30 map to human chromosome 4q34 and 1p11-13, respectively. An ancestral analysis of all known mammalian ADAMs indicates that the zinc-binding motif in the catalytic domain arose once in a common ancestor and was subsequently lost by those members lacking this motif.     

Calmodulin cDNAs from two species of Tetrahymena.

We describe the isolation and characterization of cDNAs encoding calmodulins of Tetrahymena thermophila and Tetrahymena pyriformis. It reveals that the deduced amino acid sequences of both calmodulins are precisely the same.     

Sequence and analysis of zona pellucida 2 cDNA (ZP2) from a marsupial,the brushtail possum,Trichosurus vulpecula

All mammalian eggs are surrounded by the zona pellucida, an extracellular coat involved in vital functions during fertilization and early development. The zona pellucida glycoproteins are promising antigenic targets for development of contraceptive vaccines to control pest populations of marsupials in Australia and New Zealand. Our current understanding of the function of the zona pellucida glycoproteins is based almost entirely on the mouse and may not be representative of gamete interactions in all eutherian or marsupial mammals. This study reports the isolation and characterization of the ZP2 gene from the brushtail possum (Trichosurus vulpecula). The brushtail possum ZP2 mRNA is 2,182 nucleotides long with an open reading frame coding for a polypeptide chain of 712 amino acids with a molecular mass of 79,542 d. The deduced amino acid sequence of possum ZP2 is 48 to 55% identical to that of eutherian mammals. It shares several structural characteristics including N-linked glycosylation sites, location and number of cysteine residues, and hydropathy profile. The brushtail possum ZP2gene is expressed exclusively in the ovary. Further studies are planned to elucidate the specific site of ZP2 expression within the ovary and its function during fertilization in marsupials. Mol. Reprod. Dev. 51:322–329, 1998. © 1998 Wiley-Liss, Inc.     

Isolation and expression pattern of two putative acyl-ACP desaturase cDNAs from Bassia scoparia

The seed lipids of some higher plants contain unusual fatty acids with potentially valuable non-food uses. Seeds of Bassia scoparia contain one such monounsaturated fatty acid, 16:1Delta5. This fatty acid can be used for the production of an insect oviposition pheromone, which is potentially valuable in the control of the mosquito Culex quinquefasciatus, a vector of West Nile virus. Previous work has established that a number of unusual monounsaturated fatty acids are produced by variant forms of the ubiquitous acyl-ACP desaturases. The isolation and initial characterization of two putative acyl-ACP desaturases from B. scoparia, one of which is seed-specific, suggests that such a variant enzyme occurs in this species.     

Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes

The isolation of the porcine zona pellucida, the glycoprotein envelope surrounding the mammalian oocyte, and the purification of its glycoprotein families is described. The zona pellucida was prepared from oocytes isolated from pig ovaries using a razor blade device and sieving procedures with Teflon or nylon screens. In 6-7 man h, the zona pellucida from 5 X 10(5) oocytes was obtained yielding 12 mg protein and 2.2 mg carbohydrate. The absorptivity of heat solubilized and filtered zona pellucida was A1%280 nm, 1 cm = 10.8. The four glycoprotein families of the zona pellucida were purified by two-dimensional polyacrylamide gel electrophoresis and electrophoretic elution. The electrophoretic purity of these families was greater than 90%. The protein and carbohydrate content and the amino acid and monosaccharide compositions of each of the glycoprotein families were determined.     

Isolation of cDNAs encoding two purine biosynthetic enzymes of soybean and expression of the corresponding transcripts in roots and root nodules

Soybean nodule cDNA clones encoding glycinamide ribonucleotide (GAR) synthetase (GMpurD) and GAR transformylase (GMpurN) were isolated by complementation of corresponding Escherichia coli mutants. GAR synthetase and GAR transformylase catalyse the second and the third steps in the de novo purine biosynthesis pathway, respectively. One class of GAR synthetase and three classes of GAR transformylase cDNA clones were identified. Northern blot analysis clearly shows that these purine biosynthetic genes are highly expressed in young and mature nodules but weakly expressed in roots and leaves. Expression levels of GMpurD and GMpurN mRNAs were not enhanced when ammonia was provided to non-nodulated roots.     

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731 bp) and SasesquiTPS1 (1680 bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-α-terpineol and (−)-limonene, along with small quantities of linalool, myrcene, (−)-α-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were α-bulnesene, γ-muurolene, α- and β-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.     

Isolation of two cDNAs encoding E2 ubiquitin-conjugating enzymes from Pavlova lutheri (Haptophyceae)

Two novel cDNAs, Plubc1 and Plubc2, encoding ubiquitin-conjugatingenzyme E2, were isolated from a Pavlova lutheri cDNA library. They areeach encoded by single copy genes in thealgae genome. Sequence comparison withplant, yeast and algal E2 sequences showedthat PlUBC1 and PlUBC2 are members of newE2 subfamilies. Time-course expressionanalysis of the two cDNAs revealed thatPlubc1 is transitionallyover-expressed at the end of theexponential phase of growth of the culture,while Plubc2 is constitutivelyexpressed at the same level throughout thecell growth. The phylogenetic study and thedifferent expression patterns suggest thatthese two enzymes could exhibit differentphysiological functions in P. lutheri.The partial sequence of the 18S rRNA geneand the full-length cDNA sequence of Plubc1 and Plubc2 reported in thispaper will appear in the Genbank databaseunder the accession numbers AY135218,AY135219 and AY135220 respectively.     

Isolation and mapping the chicken zona pellucida genes: an insight into the evolution of orthologous genes in different species

The avian oocyte is surrounded by a specialized extracellular glycoproteinaceous matrix, the perivitelline membrane, which is equivalent to the zona pellucida (ZP) in mammals and the chorion in teleosts. A number of related ZP genes encode the proteins that make up this matrix. These proteins play an important role in the sperm/egg interaction and may be involved in speciation. The human genome is known to contain ZP1, ZP2, ZP3, and ZPB genes, while a ZPAX gene has also been identified in Xenopus. The rapid evolution of these genes has confused the nomenclature and thus orthologous relationships across species. In order to clarify these homologies, we have identified ZP1, ZP2, ZPC, ZPB, and ZPAX genes in the chicken and mapped them to chromosomes 5, 14, 10, 6, and 3, respectively, establishing conserved synteny with human and mouse. The amino acid sequences of these genes were compared to the orthologous genes in human, mouse, and Xenopus, and have given us an insight into the evolution of these genes in a variety of different species. The presence of the ZPAX gene in the chicken has highlighted a pattern of probable gene loss by deletion in mouse and gene inactivation by deletion, and base substitution in human.     

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula)

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.     

Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains.

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.