P. G. Johnston

Controlling elements in the mouse. IV. Evidence of non-random x-inactivation.

The non-random X chromosome expression that has been observed with coat markers in female mice heterozygous for the Xce alleles, Xce a and Xce b , has now been investigated with the electrophoretic enzyme marker, Pgk-1 . Because the Xce status of the Pgk-1 a marked chromosome was not known, PGK expression was assessed in Pgk-1 a / Pgk-l b heterozygotes which carried either Xce a or Xce b on their Pgk-1 b chromosome. The PGK-1A allozyme was found to predominate in both genotypes but when Xce b was present on the Pgk-l b chromosome the expression of the two allo-zymes was less unequal. This effect was seen in both liver and kidney of adults and to at least the same degree in embryos aged 13·5 and 7·5 days. The results have been interpreted to mean that the non-random X expression derives from a primary non-randomness of the X inactivation process and that a new and more extreme Xce allele, designated Xce c , was present on the Pgr-1 a -marked X chromosome.

Mapping the distribution of the telomeric sequence (T2AG3)n in rock-wallabies, Petrogale (Marsupialia: Macropodidae), by fluorescence in situ hybridization. I. The penicillata complex

The eight Petrogale (rock-wallaby) species of the penicillata complex have a variable rate of karyotypic evolution, with species differing from the ancestral karyotype by two to six rearrangements. The distribution of the predominant vertebrate telomeric sequence (T2AG3)n was examined by fluorescence in situ hybridization (FISH) to determine if this sequence is retained during centric fusion events or is involved in other rearrangements. In all submetacentric chromosomes derived by centric fusions, the telomeric sequence was identified at or near the centromere, indicating that the (T2AG3)n sequence is consistently retained. In two acrocentric chromosomes, derived by centromeric transpositions from submetacentric fusion chromosomes, an interstitial signal was observed at the presumed site of the fusion. This represents the identification of a novel mechanism by which the (T2AG3)n sequence may become interstitial. Other interstitial telomeric signals were identified just below the centromere of chromosome 1 and interstitially on chromosome 4 in all eight species of the penicillata complex. These may be related to, respectively, the formation of euchromatic short arms on chromosome 1 and a more ancient rearrangement of chromosome 4 within marsupials.

Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia: Macropodidae)

Chromosomal rearrangements in the two currently recognised races of Petrogale godmani were examined using C- and G-banding. The nominate race P. godmani godmani (2n = 20) was found to possess an inverted chromosome 5 and an acrocentric 6-10 fusion, which can be derived from a 6-10 centric fusion by a centromeric transposition. The Cape York race (2n = 22) was found to retain the ancestral submetacentric chromosome 4 and the ancestral chromosome 5. Thus despite their genic similarity, the two races clearly have major chromosomal differences and should be regarded as separate species. Petrogale g. godmani shares two derived chromosomes with another Queensland taxon, the assimilis race of P. assimilis, indicating recent common ancestry. The Cape York race retains characteristics of an ancestral stock of Petrogale and its genic similarity with P. g. godmani could therefore be the result of extensive introgression.

Taxonomy of rock-wallabies, Petrogale (Marsupialia : Macropodidae). III. Molecular data confirms the species status of the purple-necked rock-wallaby (Petrogale purpureicollis Le Souef)

Mitochondrial DNA (mtDNA) analysis was undertaken to resolve the systematic uncertainties surrounding the morphologically distinct purple-necked rock-wallaby (P. lateralis purpureicollis) of north-west Queensland, Australia. A comparison of mtDNA sequence divergence using both whole mtDNA restriction site and control-region sequence analyses revealed that P. l. purpureicollis was as well differentiated from other P. lateralis (black-footed rock-wallaby) taxa as P. lateralis was from P. penicillata (brush-tailed rock-wallaby) or P. assimilis (allied rock-wallaby). Phylogenetic analysis of the sequence data suggests thatP. lateralis (sensu lato) is paraphyletic, with P. l. purpureicollis being more closely aligned to P. penicillataand P. assimilis than to P. lateralis (sensu stricto). Data are also presented that demonstrate significant differences in the distribution of the telomeric repeat sequence (TTAGGG)n between the chromosomes of P. l. purpureicollis and the karyotypically similar MacDonnell Ranges race of P. lateralis. In addition, meiosis appears to be severely disrupted in the majority (73%) of oocytes examined from two P. l. purpureicollis MacDonnell Ranges race hybrids. In light of these findings we recommend that the purple-necked rock-wallaby be reinstated as a full species, P. purpureicollis Le Souef 1924.

Sex-chromosome elimination in the bandicoot Isoodon macrourus using Y-linked markers

Cytogenetic studies have shown that the Y chromosome is eliminated from many somatic cell types of the bandicoot Isoodon macrourus, an Australian marsupial. Molecular techniques allow examination of a greater range of tissue types than that possible using cytogenetic techniques. The presence or absence of the Y chromosome was established using partial sequences of the Y-linked SRY and UBE1Y genes in I. macrourus, with the X-linked gene G6PD as a control. We show that a very small proportion of cells comprising hematopoietic tissues, and even fewer cells in peripheral blood, retain the Y chromosome. The Y chromosome is retained in most brain, liver, kidney, and lung cells and in cardiac and skeletal muscle. We also show that the bandicoot Y chromosome is retained in some cell types within tissues previously believed to completely eliminate the Y chromosome.

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme α-galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.

Karyotype and nuclear DNA content of the Australian lungfish, Neoceratodus forsteri (Ceratodidae: Dipnoi)

The karyotype of the lungfish, Neoceratodus forsteri, is described as 2n = 54, comprising 3 pairs of very large metacentrics, 1 pair of large submetacentrics, 13 pairs of smaller acrocentrics, and 10 pairs of microchromosomes. In addition to centromeric constitutive heterochromatin, C-bands were located on the arms of almost all of the macrochromosomes. The nuclear DNA content of N. forsteri was confirmed as being high (98.6-111.9 pg/nucleus).

Mapping the distribution of the telomeric sequence (T2AG3)n in the 2n = 14 ancestral marsupial complement and in the macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization.

In this study we test the theory that the presence of the conserved vertebrate telomeric sequence (T2AG3)n at the centromeres of Australian marsupial 2n = 14 complements is evidence that these karyotypes are recently derived, which is contrary to the generally held view that the 2n = 14 karyotype is ancestral for Australasian and American marsupials. Here we compare the distribution of the (T2AG3)n sequence and constitutive heterochromatin in the presumed ancestral 2n = 14 complement and in complements with known rearrangements. We found that where there were moderate to large amounts of constitutive heterochromatin, the distribution of the (T2AG3)n sequence reflected its presence as a native component of satellite DNA rather than its involvement in past rearrangements. The presence of centromeric heterochromatin in all Australian 2n = 14 complements therefore suggests that centromeric sites of the (T2AG3)n sequence do not represent evidence for recent rearrangements.

Mapping the distribution of the telomeric sequence (T2AG3)n in the Macropodoidea (Marsupialia) by fluorescence in situ hybridization. II. The ancestral 2n = 22 macropodid karyotype

In marsupial karyotypes with little heterochromatin, the telomeric sequence (T₂AG₃)_n, is involved in chromosome rearrangements. Here we compare the distribution of the (T₂AG₃)_n sequence in chromosomes recently derived by fusions and other rearrangements (7–0.5 MYBP) with its distribution in chromosomes derived earlier (24–9 MYBP). We have previously shown that the (T₂AG₃)_n sequence is consistently retained during chromosome rearrangements that are recent (7–0.5 MYBP). We suggest that in less recent rearrangements (24–9 MYBP) the pattern observed is initial retention followed by loss or amplification. We also suggest that the presence of interstitial (T₂AG₃)_n sequence is related to the evolutionary status of single chromosomes rather than entire karyotypes.

Phylogenetics, population structure and genetic diversity of the endangered southern brown bandicoot (Isoodon obesulus) in south-eastern Australia

The southern brown bandicoot (Isoodon obesulus) has undergone significant range contractions since European settlement, and it is now considered “Endangered” throughout south-eastern mainland Australia. This species currently has a highly fragmented distribution inhabiting a mosaic of habitats. This project uses mitochondrial DNA (mtDNA) and microsatellite data to determine levels of genetic diversity, population structure and evolutionary history, which can aid wildlife managers in setting priorities and determining management strategies. Analyses of genetic diversity revealed low levels of mtDNA variability (mean h=50.42%, π=0.76%) and divergence (mean dA=0.29%) across all regions investigated, and was among the lowest recorded for marsupials. These data indicate a relatively small female effective population size, which is most likely a consequence of a large-scale population contraction and subsequent expansion occurring in pre-history (mismatch distribution analysis, SSDP-value=0.12). Individuals from the Sydney region experienced significant reductions in microsatellite diversity (A=3.8, HE=0.565), with the Garigal National Park (NP) population exhibiting “genetic reduction signatures” indicating a recent population bottleneck. Population differentiation analysis revealed significant genetic division amongst I. obesulus individuals from Sydney, East Gippsland and Mt Gambier regions (θ=0.176–0.271), but could not separate the two Sydney populations (Ku-ring-gai NP and Garigal NP). Based on these data and habitat type, translocations could readily be made between the two Sydney populations, but not between the others. Phylogenetic comparisons between I. obesulus and I. auratus show little support for current Isoodon taxonomy, consistent with the findings of Pope et al. 2001. We therefore recommend the recognition of only three I. obesulus sub-species and suggest that these comprise a single morphologically diverse species that once was widespread across Australia.