Gestational length in the koala, Phascolarctos cinereus.

We report a possible case of extended gestation in the koala, Phascolarctos cinereus. Birth of a pouch young was first observed 127 days after the removal of the male from a multi-female colony at Taronga Zoo. No other males were present at that time or had access to the facility. Head measurements and other growth data collected at the time of detection and over the period of pouch life indicates the time from removal of the male and the date of birth to be between 50 and 77 days. DNA fingerprinting using microsatellite loci unambiguously assigned paternity of the pouch young to this male. These observations suggest either an extended period of gestation of at least 50 days, or activation of a dormant blastocyst from the previous breeding season, as the female entered the period of seasonal oestrus.

Genetic diversity and gene flow among southeastern Queensland koalas (Phascolarctos cinereus).

Habitat fragmentation and destruction associated with the rapid urban and rural development of southeast Queensland presents an immediate threat to the survival of koala populations within this region. A sensitive method combining heteroduplex analysis (HDA) with temperature gradient gel electrophoresis (TGGE) was optimized to detect within-species variation in a mitochondrial DNA (mtDNA) control-region fragment, approximately 670 bp in length, from the koala. Eight different haplotypes were characterized in koalas, of which four were novel. Analysis of mtDNA diversity in 96 koalas from five populations in southeast Queensland revealed that the number of haplotypes in a single population ranged from one to five, with an average within-population haplotype diversity of 0.379 +/- 0.016, and nucleotide diversity of 0.22 +/- 0.001%. Nucleotide divergence between populations averaged 0.09 +/- 0.001% and ranged from 0.00 to 0.14%. Significant genetic heterogeneity was observed among most populations, suggesting that koala populations may be spatially structured along matrilines, although this may not be universal. The limited distribution of the central phylogenetic haplotype suggested the possibility of historical population bottlenecks north of the Gold Coast, while the presence of two highly divergent haplotypes at the Moreton site may indicate the occurrence of one or more undocumented translocation events into this area.

Phylogeographical population structure of tiger quolls Dasyurus maculatus (Dasyuridae: Marsupialia), an endangered carnivorous marsupial.

Tiger quolls, Dasyurus maculatus, are the largest carnivorous marsupials still extant on the mainland of Australia, and occupy an important ecological niche as top predators and scavengers. Two allopatric subspecies are recognized, D.m. gracilis in north Queensland, and D.m. maculatus in the southeast of the mainland and Tasmania. D.m. gracilis is considered endangered while D.m. maculatus is listed as vulnerable to extinction; both subspecies are still in decline. Phylogeographical subdivision was examined to determine evolutionarily significant units (ESUs) and management units (MUs) among populations of tiger quolls to assist in the conservation of these taxa. Ninety-three tiger quolls from nine representative populations were sampled from throughout the species range. Six nuclear microsatellite loci and the mitochondrial DNA (mtDNA) control region (471 bp) were used to examine ESUs and MUs in this species. We demonstrated that Tasmanian tiger quolls are reciprocally monophyletic to those from the mainland using mtDNA analysis, but D.m. gracilis was not monophyletic with respect to mainland D.m. maculatus. Analysis of microsatellite loci also revealed significant differences between the Tasmanian and mainland tiger quolls, and between D.m. gracilis and mainland D.m. maculatus. These results indicate that Tasmanian and mainland tiger quolls form two distinct evolutionary units but that D.m. gracilis and mainland D.m. maculatus are different MUs within the same ESU. The two marker types used in this study revealed different male and female dispersal patterns and indicate that the most appropriate units for short-term management are local populations. A revised classification and management plan are needed for tiger quolls, particularly in relation to conservation of the Tasmanian and Queensland populations.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817).

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species' range. Eighteen different haplotypes were defined in the approximately 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 +/- 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.

Genetic variation in captive koalas (Phascolarctos cinereus): parentage determination and individual identification.

Highly repeatable randomly amplified polymorphic DNA (RAPD) markers were developed for parentage studies in the koala (Phascolarctos cinereus). Of the 25 RAPD primers screened, 5 (20.0%) produced 32 repeatable polymorphic RAPD bands (average/primer = 6.4 +/- 4.2). A high level of polymorphism was observed for each group of koalas (Featherdale, 71.9%; Lone Pine, 84.4%). All 25 koalas could be uniquely identified using either RAPD or microsatellite markers. Of the 32 RAPD markers generated in koalas, 25 were informative for parentage analyses. These RAPD markers successfully determined both parents to three offspring and a male parent to a fourth offspring. Paternity analysis (where the female parent is known) succeeded in assigning the correct male parent to seven offspring. Our RAPD-PCR method generates informative genetic markers that are useful for parentage determination and individual identification of captive koalas. This would provide genetic analysis to zoos and wildlife parks as a low-cost alternative to the more expensive microsatellite markers.

Evolution of MHC class I loci in marsupials: characterization of sequences from koala (Phascolarctos cinereus)

We demonstrate that koala (Phascolarctos cinereus) MHC class I constitutes a variable multigene family. A total of nine partial exon 2 and 3 major histocompatibility complex (MHC) class I sequences are presented, including six sequences from at least three loci from one koala. Variation was detected by examination of sequences from a number of individuals and family groups. The koala is the second marsupial species characterized to date, and comparisons reveal approximately 80% similarity with sequences from the red necked wallaby (Macropus rufogriseus). The latter sequences represent at least two, and probably three, different loci. Phylogenetic analysis demonstrates that all koala sequences are more related to one another than they are to any of the wallaby loci. This indicates that the koala sequences are probably not orthologous to the wallaby genes, and thus represent a new class I gene family. In addition, marsupial gene families cluster away from human gene families, supporting a different origin of MHC genes for marsupials and eutherians.

Low genetic variability of the koala Phascolarctos cinereus in south-eastern Australia following a severe population bottleneck.

Genotyping of koalas at CA-repeat microsatellite loci has revealed significant differences in the levels of allelic diversity (A) and expected heterozygosity (H(E)) between populations from north-eastern and south-eastern Australia. In the 10 populations studied, allelic diversity ranged from 8.0 in the Nowendoc population to 1.7 in the Kangaroo Is. population, and values of H(E) ranged from 0.831 in the Nowendoc population to 0.331 in the Kangaroo Is. population. Data from pooled populations revealed koalas from the north-eastern region had significantly higher levels of allelic diversity (A = 11.5 +/- 1.4) than those from south-eastern Australia (A = 5.3 +/- 1.0). Furthermore significantly higher heterozygosity levels were found in the north-eastern (H(E) = 0.851) vs. the south-eastern (H(E) = 0.436) regions of Australia. Following a near-extinction bottleneck in the 1920s, mainland Victorian and Kangaroo Is. koalas have been involved in an extensive program of relocations. The source populations of the relocated animals were islands in Westernport Bay, which were founded by very few individuals in the late 1800s and early 1900s. The significantly lower levels of variation between south-eastern Australian populations suggests that human intervention has had a severe effect on levels of genetic diversity in this region, and this may have long-term genetic consequences.

Paternity exclusion in koalas using hypervariable microsatellites.

Koala microsatellite loci containing the dinucleotide motif (CA)n were isolated from a size-fractionated (250-500 bp) koala genomic library and sequenced. Six locus-specific primer pairs were designed and synthesized for DNA amplification using the polymerase chain reaction (PCR). Microsatellite genotyping of 12 individuals generated unique "fingerprints" for each koala. All six microsatellite loci were polymorphic, with a mean of 6.5 +/- 0.6 alleles/locus. This level of allelic diversity is capable of generating > 4 x 10(9) DNA profiles, making it the most powerful technology for fingerprinting koalas currently available. Observed heterozygosities (H(o)) in the eight unrelated individuals surveyed ranged from 0.25 to 0.75, with a mean of 0.54 +/- 0.06. Mendelian inheritance of the observed polymorphism was confirmed by family studies. We demonstrate that microsatellite loci are ideal genetic markers for paternity exclusion and pedigree analysis of koalas, which have shown little genetic variation using most other methods.

N-glycosylation of HIV-gp120 may constrain recognition by T lymphocytes.

The HIV envelope protein gp120 is heavily glycosylated, having 55% of its molecular mass contributed by N-linked carbohydrates. We investigated the role of N-glycosylation in presentation of HIV-gp120 to T cells. T cell clones obtained from humans immunized with a recombinant nonglycosylated form of HIV-gp120 (env 2-3) were studied for their ability to recognize both env 2-3 and glycosylated gp120. We found that 20% of CD4+ T cell clones specific for env 2-3 fail to respond to glycosylated gp120 of the same HIV isolate. Using synthetic peptides, we mapped one of the epitopes recognized by such clones to the sequence 292-300 (NESVAINCT), which contains two asparagines that are glycosylated in the native gp120. These findings suggest that N-linked carbohydrates within an epitope can function as hindering structures that limit Ag recognition by T lymphocytes.

Junctional sequences influence the specificity of gamma/delta T cell receptors.

T lymphocytes bearing the gamma/delta T cell receptor (TCR-gamma/delta) express a limited number of germline variable gene segments, generating receptor sequence diversity primarily through junctional mechanisms. To examine the role of V(D)J junctional sequences in antigen recognition by TCR-gamma/delta, we derived an alloreactive murine TCR-gamma/delta+ T cell line, LKD1, specific for the I-Ad class II major histocompatibility complex (MHC) molecule, and compared its receptor with that expressed by a previously characterized class II MHC alloreactive T cell line, LBK5, specific for I-Ek,b,s Ia molecules. Both LKD1 and LBK5 express receptors encoded by rearranged V gamma 1.2J gamma 2 and V delta 5D delta 2J delta 1 gene elements, differing in sequence only in the V(D)J junctional regions of the gamma and delta genes. These results demonstrate that junctionally encoded sequences corresponding to the putative third complementarity determining region can influence the antigen specificity of TCR-gamma/delta.

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.

Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum.

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.

Cross-linking of Ly 6-linked alloantigens: association between ThB and Ly 5.

The bifunctional cross-linking reagent dithiobis(succinimidyl propionate) (DSP) was used to cross-link 125I surface-labelled glycoproteins from viable thymocytes. The cells were solubilized, and the cross-linked material immunoprecipitated and analysed by SDS-PAGE. When DSP cross-linked thymocyte material was immunoprecipitated with either anti-ThB or anti-Ly 5 monoclonal antibodies, and then cleaved, molecules with masses identical to Ly 5 (Mr 180 kD) and ThB (Mr 16-18 kD) were obtained. However, if the cross-linker was not cleaved, the intact product had a molecular mass of greater than 200 kD. The identity of these co-precipitated, cross-linked moieties was formally proved by limited proteolysis peptide map analysis. The data indicated that the ThB and Ly 5 antigens were associated on the thymocyte cell surface but no such association could be found on peripheral lymphocytes. The ThB-Ly 5 interaction may indicate an association relevant to the differentiation of thymocytes.

Systematic development of distinct T cell receptor-gamma delta T cell subsets during fetal ontogeny.

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.

Structure and specificity of T cell receptor gamma/delta on major histocompatibility complex antigen-specific CD3+, CD4-, CD8- T lymphocytes.

Analyses of TCR-bearing murine and human T cells have defined a unique subpopulation of T cells that express the TCR-gamma/delta proteins. The specificity of TCR-gamma/delta T cells and their role in the immune response have not yet been elucidated. Here we examine alloreactive TCR-gamma/delta T cell lines and clones that recognize MHC-encoded antigens. A BALB/c nu/nu (H-2d)-derived H-2k specific T cell line and derived clones were both cytolytic and released lymphokines after recognition of a non-classical H-2 antigen encoded in the TL region of the MHC. These cells expressed the V gamma 2/C gamma 1 protein in association with a TCR-delta gene product encoded by a Va gene segment rearranged to two D delta and one J delta variable elements. A second MHC-specific B10 nu/nu (H-2b) TCR-gamma/delta T cell line appeared to recognize a classical H-2D-encoded MHC molecule and expressed a distinct V gamma/C gamma 4-encoded protein. These data suggest that many TCR-gamma/delta-expressing T cells may recognize MHC-linked antigens encoded within distinct subregions of the MHC. The role of MHC-specific TCR-gamma/delta cells in immune responses and their immunological significance are discussed.

The 33,000 protein precipitated by Ly-6A.2-specific antibodies is not associated with the Ly-6 polymorphism.

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12,000-14,000, in contrast to initial studies which showed the relative mass to be 33,000. Using polymorphic Ly-6-specific antibodies, the 33,000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2+, Ly-6A.2- strains and a Ly-6A.2- mutant cell line BW(Thy-1-e). This clearly demonstrated that 33,000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2+ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12,000-14,000, although a minor 33,000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipitate the 12,000-14,000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12,000-14,000 molecules were only immunoprecipitated from Ly-6A.2+ cells, whereas 33,000 molecules were precipitated from both Ly-6A.2+ cells and Ly-6A.2- cells. These findings suggest that the 33,000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.

Interrelationships of the "Ly-6 complex" antigens.

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes--Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10-20% or greater than 90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B.2 antigens are distinct from Ly-6A.2 and Ly-6D.2, however. ThB is a 16-18 kd antigen which is not associated on the cell surface with any other "Ly-6" antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.