Rory M. Hope

Evolution and Nomenclature of the Zona Pellucida Gene Family

Abstract Three subfamilies of genes are acknowledged within the zona pellucida (ZP) gene family. At present, these subfamilies each have two names that are used interchangeably: ZPA or ZP2, ZPB or ZP1, and ZPC or ZP3. The ZPA genes encode the longest protein sequences and the ZPC genes the shortest. Recently, several sequences, which have no clear relationship to the three subfamilies, have been identified. These sequences include two paralogous ZP genes from Xenopus laevis and a single gene from the fish Oryzias latipes. We have conducted extensive phylogenetic analyses of the known ZP genes. As well as establishing the evolutionary relationships among these genes, the analyses make it clear that the dual nomenclature system is no longer feasible, because major paralogous groups are present in the ZPB (ZP1) family of genes of amniotes. We propose a unified system of nomenclature for the ZP gene family that removes the existing ambiguities.

Expression of three spalt ( sal ) gene homologues in zebrafish embryos

Three homologues of the Drosophila region-specific homeotic gene spalt (sal) have been isolated in zebrafish, sall1a, sall1b and sall3. Phylogenetic analysis of these genes against known sal DNA sequences showed zebrafish sall1a and sall1b to be orthologous to other vertebrate sal-1 genes and zebrafish sall3 to be orthologous to other vertebrate sal-3 genes, except Xenopus sall3. Phylogenetic reconstruction suggests that zebrafish sall1a and sall1b resulted from a gene duplication event occurring prior to the divergence of the ray-finned and lobe-finned fish lineages. Analysis of the expression pattern of the zebrafish sal genes shows that sall1a and sall3 share expression domains with both orthologous and non-orthologous vertebrate sal genes. Both are expressed in various regions of the CNS, including in primary motor neurons. Outside of the CNS, sall1a expression is observed in the otic vesicle (ear), heart and in a discrete region of the pronephric ducts. These analyses indicate that orthologies between zebrafish sal genes and other vertebrate sal genes do not imply equivalence of expression pattern and, therefore, that biological functions are not entirely conserved. However we suggest that, like other vertebrate sal genes, zebrafish sal genes have a role in neural development. Also, expression of zebrafish sall1a in the otic vesicle, heart sac and the pronephric ducts of zebrafish embryos is possibly consistent with some of the abnormalities seen in Sall1-deficient mice and in Townes-Brocks Syndrome, a human disorder which is caused by mutations in the human spalt gene SALL1.

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.

Isolation and characterisation of zona pellucida A (ZPA) cDNAs from two species of marsupial: regulated oocyte-specific expression of ZPA transcripts.

The zona pellucida (ZP) is an extracellular glycoprotein coat that is deposited around the oocyte during folliculogenesis and performs several functions that relate to fertilisation and preimplantion development. In eutherian mammals it consists of three major glycoproteins--ZPA, ZPB, and ZPC--but little is known about its molecular constitution in marsupials. We have isolated the cDNA encoding the ZPA homologue in two distantly related marsupial series: the possum, Trichosurus vulpecula (a phalangerid) and the dunnart Sminthopsis crassicaudata (a dasyurid). The two cDNA sequences were 86% identical and showed extensive regions of homology to eutherian ZPA proteins, particularly in the central region of the molecule. Many other features of the ZPA protein, except the positioning of the N-linked glycosylation sites, were also conserved between marsupials and eutherians. ZPA expression was shown to occur maximally in the cytoplasm of the oocyte primary follicles with a little, but significant, expression in oocytes of both primordial follicles and in the cytoplasm of the oocyte in follicles with an antral cavity. No expression was seen in surrounding follicle or granulosa cells.

6-phosphogluconate dehydrogenase polymorphism in the marsupial mouse, Sminthopsis crassicaudata.

The fat-tailed marsupial mouse, Smin thops i s crassicaudata, is a promising small marsupial for the laboratory (Martin, 1965; Ewer, 1968; Crowcroft and Godfrey, 1968; Godfrey, 1969). In particular, it has potential for genetic work, since it breeds in captivity, has a litter size of from two to ten, and a haploid chromosome number of seven. In order to obtain genetic markers for the species, a survey is being undertaken to detect naturally occurring electrophoretic variants of blood enzymes and proteins. Hope and Godfrey (1968) have described such variation in serum transferrin. Finnegan and Hope (1970) have described serum amylase variation whose mode of inheritance is still to be determined. In this report we describe a second autosomal blood marker, variation in erythrocytic 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase; EC 1.1.1.44; 6PGD). The variants are detected by vertical starch gel electrophoresis (Smithies, 1959). Animals were either raised in the laboratory or caught wild in several parts of South Australia and southwest Queensland. Blood samples of about 0.2 ml were obtained from lightly etherized animals by puncture of the orbital sinus. All other methods were as in Cooper et al. (1969a, b). Nearly all samples gave one of three 6PGD patterns upon electrophoresis. These patterns are called PGD 1-1, PGD 2-1, and PGD 2-2 (Fig. 1). As the designation suggests, 1-1 and 2-2 are homozygous for alleles P G D 1 and P G D 2, respectively, while 2-1 is the heterozygote for them. Family data support this hypothesis (Table I). The heterozygote possessed the usual intermediate hybrid band which is found in all heterozygotes for 6PGD electrophoretic variants, e.g., that in Drosophi la (Kazazian et al., 1965), various fish (Bender and Ohno, 1968), and doves (Cooper et al., 1969a). This

Blood O2 transport in newborn and adult of a very small marsupial (Sminthopsis crassicaudata)

Blood O2 transport and hemoglobin types have been studied in a Dasyurid marsupial (Sminthopsis crassicaudata) at the neonatal (10-20 mg) and adult (16 g) stages, and in part of the transition period. In neonates the blood was embryonic in type with erythrocytes nucleated and containing two Hb types both different from adult Hb. The oxygen equilibrium curves (OECs) at day 1 had a P50 of 38 mmHg at 36 degrees C and PCO2 = 43 mmHg. This is lower than in other neonatal marsupials, but higher than in fetal or neonatal eutherian mammals. Adult P50 under the same conditions were higher (59 mmHg), the normal relationship in viviparous animals. Hill plots of neonatal OECs showed a sharp upward bend at about 50% saturation. As in other embryonic and neonatal marsupials, in the upper part of the plot nH was greater than 4. This indicates aggregation of Hb tetramers. The Bohr effect of neonatal blood at higher PCO2 values (43-71 mmHg PCO2) was zero. The special features of neonatal blood had largely disappeared by day 6.

Expression of PGK-A in the Australian brush-tailed possum, Trichosurus vulpecula (Kerr), consistent with paternal X inactivation

An extensive survey of erythrocytes of marsupials other than kangaroos for electrophoretic variation in X-linked enzymes revealed two rare PGK-A phenotypes in the phalangerid Trichosurus vulpecula and one in Trichosurus caninus. Four putatively heterozygous females expressed only the variant allelic isozyme in some tissues but expressed a trace of the normal isozyme in others. A putatively hemizygous male expressed only the variant isozyme in all tissues. The phenotypic patterns were consistent with those observed in kangaroos known to exhibit partial or complete paternal X inactivation in cells of females. Two of the T. vulpecula were a mother and her female pouch young, further suggesting that paternal X inactivation occurs in T. vulpecula. This peculiar mechanism of dosage compensation may not be restricted to kangaroos.

Structure, sequence and function of a marsupial LIF gene: Conservation of IL-6 family cytokines

Leukaemia Inhibitory Factor (LIF) is a multifunctional cytokine with an obligate role in the mouse in embryonic implantation. In this paper we demonstrate the existence of a functional LIF gene in the marsupial Sminthopsis crassicaudata, and the presence of LIF-related sequences in the monotreme Tachyglossus aculeatus (Australian echidna). Isolation of genomic and cDNA clones from S. crassicaudata, indicated that the LIF gene is highly conserved between marsupials and monotremes in terms of sequence and genomic organisation. Critical functional residues within the LIF sequence were also conserved including residues implicated in intracellular LIF activity, and in interaction with the receptor subunits LIFR and gp130. These findings suggest that the structure and biochemical function of the protein is likely to be conserved. Consistent with this, purified recombinant S. crassicaudata LIF interacted functionally with mouse receptor components and was sufficient for maintenance of mouse embryonic stem (ES) cells in the undifferentiated state. Conservation of LIF outside eutherians is intriguing given the markedly divergent reproductive strategies which include, for some marsupial species, embryonic diapause, and in monotremes, the absence of implantation. The availability of marsupial LIF probes provides an opportunity to investigate conservation of expression and function in these mammals.

EMBRYONIC GLOBINS OF THE MARSUPIAL THE TAMMAR WALLABY (MACROPUS EUGENII): BIRD LIKE AND MAMMAL LIKE

1. Tammar Wallaby embryonic blood has been shown to have three α‐like and two β‐like globin chains in its four haemoglobin components and partial sequences of several chains have been determined.

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the α-globin gene family in mammals by isolating and characterizing α-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar α-globin family consists of at least four genes including a single adult-expressed gene (α), two embryonic/neonatally expressed genes (ζ and ζ′), and θ-globin, each orthologous to the respective α-, ζ-, and θ-globin genes of eutherian mammals. The results suggest that the θ-globin lineage arose by duplication of an ancestral adult α-globin gene and had already evolved an unusual promoter region, atypical of all known α-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that θ-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human θ-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three α-like and one β-like globin genes linked in the order 5′–ζ–α–θ–ω–3′ existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar ζ-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.