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.

Cloning of leptin cDNA and assignment to the long arm of chromosome 5 in the marsupial Sminthopsis crassicaudata.

We have isolated and sequenced full-length cDNA clones for leptin in the dasyurid marsupial Sminthopsis crassicaudata (fat-tailed dunnart). Southern and in situ hybridisation data indicated a single leptin gene in the S. crassicauda- ta genome, localised to arbitrary chromosome bands 5q24--> q31 on the long arm of chromosome 5, the short-arm terminus of which bears the only nucleolar organising region. The nucleotide sequence of the cDNAs revealed that the primary translation product of S. crassicaudata leptin is composed of 167 amino acid residues, with a potential signal peptide of 21 residues. The mature protein of 146 amino acids is 82% similar to both the mouse and human proteins and is predicted to have a molecular weight of 16.26 kDa. Northern blot analysis revealed that the corresponding mRNA is approximately 3.9 kb in size and is expressed only in white adipose tissue of this marsupial species. Evolutionary analyses indicate that S. crassicaudata leptin cDNA has evolved at a significantly faster rate than cDNAs from eutherian mammals.

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.

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.

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 alpha-like and two beta-like globin chains in its four haemoglobin components and partial sequences of several chains have been determined. 2. The major embryonic beta-like chain (epsilon) is similar to other mammalian embryonic beta-like chains on the basis of sequencing its first 60 amino acids. 3. There is another embryonic beta-like chain present in one haemoglobin component. It was designated omega and, in its first 54 amino acids, it has features that are more like avian globins than mammalian globins. 4. The one alpha-like embryonic globin sequenced has mammalian rather than avian characteristics. 5. A provisional phylogenetic tree of beta-like globins has been determined. The Tammar epsilon-globin forms a monophyletic group with marsupial and other mammalian embryonic globins; the omega-globin forms a monophyletic group with bird adult and embryonic globins.

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata.

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes-adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic-expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.

Human sex determination using multiplex polymerase chain reaction (PCR).

A sex determination method has been developed using the polymerase chain reaction (PCR) which involved the amplification of the sex-determining region Y (SRY) gene and the amplification of the HLA-DQa gene as an internal control. This method, which can be applied to old and degraded DNA samples, allowed confident sexing of biological samples producing readily identifiable PCR products using two sets of primers in the one PCR reaction. The PCR products were short DNA sequences of 139bp and 239/242bp for the SRY and HLA-DQa regions respectively. The application of this method in forensic science will allow determination of the gender of perpetrators of crimes involving biological materials.

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.

Evolution and expression of a beta-like globin gene of the Australian marsupial Sminthopsis crassicaudata.

A beta-like globin gene was isolated from the Australian dasyurid marsupial Sminthopsis crassicaudata. Nucleotide-sequence analysis of promoter and coding regions of the gene revealed that it was orthologous to eutherian early-expressed (epsilon, gamma, eta) beta-like globin genes. Comparison of the conceptually translated sequence of the gene with a partial amino acid sequence of the adult beta-globin chain from S. crassicaudata provided evidence that the gene was not expressed in adult tissues. In addition, Northern analysis of RNA isolated from an embryo, pouch young, and adult bone marrow indicated that the gene was expressed predominantly in embryonic tissues and that there was a significant reduction in the expression of the gene within a day of birth. These results provide strong support for the hypothesis of Koop and Goodman [Koop, B. F. & Goodman, M. (1988) Proc. Natl. Acad. Sci. USA 85, 3893-3897] that an embryonic beta-like globin gene existed prior to the divergence of the eutherian and marsupial lineages and that this gene was already differentiated with respect to its promoter regions and developmental expression. The observation that epsilon-globin mRNA was present at least until day 4 postpartum suggests that the epsilon-globin chain may play some role in influencing the physiological properties of hemoglobin in S. crassicaudata neonates.

Selected features of marsupial genetics.

As a consequence of the ancient separation of the marsupial and eutherian lineages, comparative genetical studies of these two mammalian taxa can be particularly informative. The potential for marsupial genetical research has been enhanced by the development of laboratory colonies of three 'model' species--Macropus eugenii, Monodelphis domestica and Sminthopsis crassicaudata. In this paper two selected aspects of marsupial genetics are reviewed, one involving cytogenetics and the other linkage. Marsupials provide a spectacular example of karyotypic conservation. The so-called 'basic karyotype' (2n = 14) is probably ancestral in all extant marsupials. Karyotypes that do not conform to this basic arrangement are thought to have been derived from it. A notable feature of the basic karyotype is that it has been retained, possibly for as long as 150 million years, in morphologically, behaviourally and ecologically diverse species from at least five Australian and two American families; this suggests that selective forces, presently unknown, have acted to conserve the basic chromosome form and number in these species. With respect to genetic linkage, family studies in S. crassicaudata and more recently M. domestica have indicated extreme differences between the sexes with the recombination frequencies for linked loci being very much greater in males than in females, a situation that is strikingly different from that in eutherian mammals. These differences in linkage values are paralleled by differences in the number and distribution of chiasmata during male and female meiosis. Prospects for further research in marsupials, particularly research that builds upon the observations of karyotypic conservation and genetic linkage, are noted.

Transfection of genes for human cell surface antigens identified by monoclonal antibodies.

Six murine L cell lines expressing five different human cell surface antigens have been prepared by DNA-mediated gene transfer. Ltk- cells were transfected with calcium phosphate co-precipitates of human genomic DNA and a plasmid containing the Herpes simplex thymidine kinase gene. After HAT selection, transfectants expressing specific cell surface antigens were identified by in situ immune rosetting using monoclonal antibodies. In this way, transfected cell lines expressing the CD9 antigen, the CD31 antigen (two lines), a unique platelet antigen, an X-linked antigen (R1), and a previously unreported monocyte antigen 11D1 were prepared. These cell lines have proved useful in the definitive assignment of monoclonal antibodies to specific CD groups. In addition, they provide a source of mRNA for use in expression cloning of the genes for these antigens.

Novel sex differences in linkage values and meiotic chromosome behaviour in a marsupial.

There have been few reported family studies of gene segregation in marsupials and no report on genetic linkage in a marsupial species. The paucity of such genetic data probably stems from the lack of marsupial species sufficiently well adapted to a laboratory environment to give the abundant progeny from controlled crosses which are required in such work. A colony of the fat-tailed insectivore Sminthopsis crassicaudata (Marsupialia: Dasyuridae), which was established in the R. A. Fisher Laboratories, University of Adelaide, more than 20 years ago, has now provided unusual linkage data. These data indicate that the linkage situation in this marsupial species differs markedly from that in eutherian mammals. Extremely large differences exist between the sexes in the values of the recombination frequencies, with much closer linkage occurring in females. Cytological examination of meiosis in males and females has revealed a major difference between the sexes in chromosome behaviour.

Genetic variation in natural and laboratory populations of the marsupial Sminthopsis crassicaudata.

Gel electrophoresis of blood proteins has detected allelic variation at five loci (TRF, PGD, SOD, ADA, GPI) in a laboratory colony of the dasyurid marsupial Sminthopsis crassicaudata. Family data show no significant departures from Mendelian expectations. Analysis of blood from wild-caught progenitors of the colony revealed significant differences in gene frequency between groups of animals captured from different parts of southern and central Australia and showed that there are two major population clusters. These interpopulation differences are particularly marked at the TRF locus and indicate that the river Murray is a barrier for this species.

Cross-reaction of a monoclonal antibody with fibroblasts from marsupials of the Macropodidae and Tarsipedidae families.

A monoclonal antibody, GA-1, was prepared against an M. rufogriseus cell surface antigen on an M. rufogriseus (red necked wallaby)--mouse somatic cell hybrid. Fibroblasts from a number of marsupial species were tested for reaction with GA-1, and only those from the Macropodidae species and the sole member of the family Tarsipedidae reacted, indicating that the Tarsipedidae is possibly more closely related to the Macropodidae than to any other group of marsupials examined.

Provisional mapping of the gene for a cell surface marker, GA-1, in the red-necked wallaby Macropus rufogriseus.

A series of M. rufogriseus-mouse somatic cell hybrids was constructed and analysed cytologically, enzymatically and immunologically. A monoclonal antibody, GA-1, was prepared against an M. rufogriseus cell surface antigen on an M. rufogriseus-mouse somatic cell hybrid. A gene determining the expression of this antigen was provisionally assigned to the long arm of the M. rufogriseus chromosome 3. The monoclonal antibody also reacted with an M. rufus (red kangaroo)-mouse somatic cell hybrid containing only the M. rufus chromosome 5, the G-banded chromosome identical to M. rufogriseus 3q. The results also suggest synteny of the genes for the marsupial enzymes hypoxanthine phosphoribosyltransferase and phosphoglycerate kinase-A.

Mapping a marsupial X chromosome using kangaroo-mouse somatic cell hybrids.

A series of marsupial-eutherian somatic cell hybrids was produced by fusion between lymphocytes from the red kangaroo (Macropus rufus) and HPRT-deficient mouse cells. The hybrids lost marsupial chromosomes and could therefore be used to map marsupial genes. Several of the hybrids contained a complete red kangaroo X chromosome and expressed the kangaroo form of the enzymes HPRT, G6PD, and PGKA. A number of HPRT-deficient revertant cell lines were derived from the hybrids. These possessed a variety of partially deleted X chromosomes. With these cell lines, it has been possible to establish the X-linkage of the genes for HPRT, G6PD, and PGKA in this marsupial and to localize these three genes to the terminal portion of the euchromatic arm of the red kangaroo X chromosome.

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 if X-linked enzymes revealed two rare PGK-A phenotypes in the phalangerid Trichosurus vupecula 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 parternal X inactivation in cells of females. Tow 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.

The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens.

Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).