The evolution of marsupial and monotreme chromosomes.

Marsupial and monotreme mammals fill an important gap in vertebrate phylogeny between reptile-mammal divergence 310 million years ago (mya) and the eutherian (placental) mammal radiation 105 mya. They possess many unique features including their distinctive chromosomes, which in marsupials are typically very large and well conserved between species. In contrast, monotreme genomes are divided into several large chromosomes and many smaller chromosomes, with a complicated sex chromosome system that forms a translocation chain in male meiosis. The application of molecular cytogenetic techniques has greatly advanced our understanding of the evolution of marsupial chromosomes and allowed the reconstruction of the ancestral marsupial karyotype. Chromosome painting and gene mapping have played a vital role in piecing together the puzzle of monotreme karyotypes, particularly their complicated sex chromosome system. Here, we discuss the significant insight into karyotype evolution afforded by the combination of recently sequenced marsupial and monotreme genomes with cytogenetic analysis, which has provided a greater understanding of the events that have shaped not only marsupial and monotreme genomes, but the genomes of all mammals.

A review of complementary mechanisms which protect the developing marsupial pouch young.

Marsupials are born without a functioning adaptive immune system, into a non-sterile environment where they continue to develop. This review examines the extent of exposure of pouch young to microorganisms and describes the protective mechanisms that are complementary to adaptive immunity in the developing young. Complementary protective mechanisms include the role of the innate immune system and maternal protection strategies, such as immune compounds in milk, prenatal transfer of immunoglobulins, antimicrobial compounds secreted in the pouch, and chemical or mechanical cleaning of the pouch and pouch young.

Physical mapping of innate immune genes, mucins and lysozymes, and other non-mucin proteins in the tammar wallaby (Macropus eugenii).

Sequencing of the tammar wallaby (Macropus eugenii) genome has the potential to be an extremely valuable resource for investigating evolutionary and developmental aspects of the mammalian immune system. However, the tammar wallaby genome has only been sequenced to a 2-fold depth and consists of small contigs, leaving many sequence gaps, many putative orthologs unpredicted and the location of genes within the genome unknown. In the case of low sequenced genomes, physical maps of genes on chromosomes can help identify specific genes if they map to conserved regions. Genes corresponding to adaptive immunity have been mapped in the tammar wallaby; however, genes corresponding to the innate immune system have not been investigated. We predict 2 types of genes important to the innate immune system, mucins and lysozymes, in the tammar wallaby and compare the predicted peptide sequences and locations of the genes with the South American opossum (Monodelphis domestica) and human. We use fluorescence in situ hybridization to physically map the genes to tammar wallaby chromosomes, demonstrating the importance of identifying and mapping genes when genomes have low sequence coverage. As mucins and lysozymes play protective roles in young animals, we also propose that their immunological role in developing marsupials warrants further investigation.

Physical mapping of immune genes in the tammar wallaby (Macropus eugenii).

The tammar wallaby (Macropus eugenii) is a model marsupial that has recently had its genome sequenced to a depth of 2-fold coverage. Although this is a great resource for comparative genomic studies, information on gene location is essential if this resource is to be used to its full potential. In this study, tammar wallaby bacterial artificial chromosomes (BACs) containing key immune genes were isolated from the tammar wallaby BAC library. BACs containing T cell receptor (TCR) and immunoglobulin (Ig) genes were physically mapped using fluorescence in situ hybridisation (FISH) to tammar wallaby chromosomes. Congruence between the locations of these immune genes in the tammar wallaby genome, with those predicted from chromosome painting data, highlights the conservation of genomic context of these important immune genes in marsupials. The isolation and mapping of these key immune genes in the tammar wallaby will aid in the assembly of the recently sequenced light coverage genome and assignment of sequence to chromosomes.

Class I genes have split from the MHC in the tammar wallaby.

Genes within the Major Histocompatibility Complex (MHC) are critical to the immune response and immunoregulation. Comparative studies have revealed that the MHC has undergone many changes throughout evolution yet in tetrapods the three different classes of MHC genes have maintained linkage, suggesting that there may be some functional advantage obtained by maintaining this clustering of MHC genes. Here we present data showing that class II and III genes, the antigen processing gene TAP2, and MHC framework genes are found together in the tammar wallaby on chromosome 2. Surprisingly class I loci were not found on chromosome 2 but were mapped to ten different locations spread across six chromosomes. This distribution of class I loci in the wallaby on nearly all autosomes is not a characteristic of all marsupials and may be a relatively recent phenomenon. It highlights the need for the inclusion of more than one marsupial species in comparative studies and raises questions regarding the functional significance of the clustering of MHC genes.

Linkage mapping and physical localization of the major histocompatibility complex region of the marsupial Monodelphis domestica.

We used genetic linkage mapping and fluorescence in situ hybridization (FISH) to conduct the first analysis of genic organization and chromosome localization of the major histocompatibility complex (MHC) of a marsupial, the gray, short-tailed opossum Monodelphis domestica. Family based linkage analyses of two M. domestica MHC Class I genes (UA1, UG) and three MHC Class II genes (DAB, DMA, and DMB) revealed that these genes were tightly linked and positioned in the central region of linkage group 3 (LG3). This cluster of MHC genes was physically mapped to the centromeric region of chromosome 2q by FISH using a BAC clone containing the UA1 gene. An interesting finding from the linkage analyses is that sex-specific recombination rates were virtually identical within the MHC region. This stands in stark contrast to the genome-wide situation, wherein males exhibit approximately twice as much recombination as females, and could have evolutionary implications for maintaining equality between males and females in the ability to generate haplotype diversity in this region. These analyses also showed that three non-MHC genes that flank the MHC region on human chromosome 6, myelin oligodendrocyte glycoprotein (MOG), bone morphogenetic protein 6 (BMP6), and prolactin (PRL), are split among two separate linkage groups (chromosomes) in M. domestica. Comparative analysis with eight other vertebrate species suggests strong conservation of the BMP6-PRL synteny among birds and mammals, although the BMP6-PRL-MHC-ME1 synteny is not conserved.

Analysis of the genomic region containing the tammar wallaby (Macropus eugenii) orthologues of MHC class III genes.

Major histocompatibility complex (MHC) molecules are central to development and regulation of the immune system in all jawed vertebrates. MHC class III cytokine genes from the tumor necrosis factor core family, including tumor necrosis factor (TNF) and lymphotoxin alpha and beta (LTA, LTB), are well studied in human and mouse. Orthologues have been identified in several other eutherian species and the cDNA sequences have been reported for a model marsupial, the tammar wallaby. Comparative genomics can help to determine gene function, to understand the evolution of a gene or gene family, and to identify potential regulatory regions. We therefore cloned the genomic region containing the tammar LTB, TNF, and LTA orthologues by "genome walking", using primers designed from known tammar sequences and regions conserved in other species. We isolated two tammar BAC clones containing all three genes. These tammar genes show similar intergenic distances and the same transcriptional orientation as in human and mouse. Gene structures and sequences are also very conserved. By comparing the tammar, human and mouse genomic sequences we were able to identify candidate regulatory regions for these genes in mammals. Full length sequencing of BACs containing the three genes has been partially completed, and reveals the presence of a number of other tammar MHC III orthologues in this region.

Characterisation of and immunity to the aerobic bacteria found in the pouch of the brushtail possum Trichosurus vulpecula.

The bacterial composition of the brustail possum (Trichosurus vulpecula) pouch was characterized throughout the reproductive cycle using brushtails from an Australian captive breeding colony (45 swabs) and a wild population in New Zealand (26 swabs). Gram-positive coccal species predominate throughout the reproductive cycle. Enteric Gram-negative rods, particularly Escherichia coli, were most prevalent when a pouch young was present and was most likely the result of faecal contamination from the pouch young. As culturing is only able to detect a proportion of bacteria present in a particular environment, molecular 16S rDNA sequencing was carried out on DNA extracted from a pouch wash of a female carrying a pouch young to gain a more accurate assessment of the pouch microflora. This approach identified approximately five times the number of bacterial species when compared to culture results. The majority detected were Gram negative rods or most closely related to Gram-negative rods species. Brushtails are immunologically immature at birth yet survive in a pouch colonised with potentially pathogenic bacteria. A haemagglutination assay was used to determine whether antibodies to a frequently isolated bacterium (Klebsiella pneumoniae) were transferred via milk from mother to pouch young. IgG antibodies were detected in maternal serum, milk and pouch young serum. In young over 70 days, antibody titres were significantly higher than those found in maternal serum, suggesting that the young is capable of producing adult type antibodies to pouch bacteria at this time.

Developmental expression of the androgen receptor during virilization of the urogenital system of a marsupial.

In the marsupial tammar wallaby, virilization begins approximately 3 wk after the onset of testosterone synthesis. In the eutherian mammal, in contrast, the onset of virilization of the male urogenital tract occurs shortly after the onset of androgen synthesis. Androgen action requires the presence of the androgen receptor to mediate a response in target tissues. We therefore investigated the developmental expression of the androgen receptor (AR) in both sexes of the tammar wallaby. AR gene transcript was detected in fetal gonad and brain as early as Day 19 of the 26.5-day gestation, 7 days earlier than the first rise in testicular testosterone (Days 0-5 postpartum [p.p.]). Immunoreactive AR was identified in the male urogenital sinus (UGS) 2 days before birth and in the female UGS and mammary glands by the day of birth. AR was present in the UGS, vagina, and prostate until Day 152 p.p., the oldest age examined. AR was identified in the gubernaculum testis at Day 2 p.p. and became more abundant by Day 32. In the phallus of both sexes, AR was identified by Day 4 p.p. and until Day 157, the oldest age examined. AR was not detected in the scrotum at any age from the day of birth to Day 157. Maturation of the phallus, wolffian duct, and epididymis was marked by appearance of epithelial immunostaining. AR was localized in the epithelium of the UGS in females by Day 50 p.p. but was not found in the epithelium of the male UGS up to Day 152 p.p., the oldest examined. AR were found in the mesenchyme of the UGS of male and female tammars 3-4 wk before virilization is first evident in the male at Day 25 p.p. We conclude that the presence of AR is not the initiating signal for virilization of the UGS in this marsupial male.