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Identification and expression of a novel marsupial cathelicidin from the tammar wallaby (Macropus eugenii).

ABSTRACT: Cathelicidins are important components of the innate immune system and have been identified in skin and epithelia of a range of mammals. In this study molecular techniques, including RACE-PCR, were used to identify the full cDNA sequence of a cathelicidin gene, MaeuCath8, from the Australian marsupial, the tammar wallaby, Macropus eugenii. This cathelicidin was not homologous to other such genes previously isolated from a tammar wallaby mammary gland EST library, however, it did contain 4 conserved cysteine residues which characterise the pre-propeptide and had 80% identity with a previously isolated bandicoot cathelicidin. Reverse transcriptase-PCR established the expression profile of MaeuCath8 in a range of tissues, including spleen, thymus, gastrointestinal tract, skin and liver, of the tammar wallaby from birth to adulthood. Expression of MaeuCath8 was observed in spleen and gastrointestinal tract of newborn animals and was observed in most tissues by 7 days post-partum. The results indicate that pouch young could synthesize their own antimicrobial peptides from an early age suggesting that this ability most likely plays a role in protecting the pouch young from infection prior to the development of immunocompetence.

SUBMITTER: Carman RL

PROVIDER: S-EPMC2887693 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Identification and expression of a novel marsupial cathelicidin from the tammar wallaby (Macropus eugenii).

Carman Rebecca L RL Old Julie M JM Baker Michelle M Baker Michelle M Jacques Nicholas A NA Deane Elizabeth M EM

Veterinary immunology and immunopathology 20081101 3-4

Cathelicidins are important components of the innate immune system and have been identified in skin and epithelia of a range of mammals. In this study molecular techniques, including RACE-PCR, were used to identify the full cDNA sequence of a cathelicidin gene, MaeuCath8, from the Australian marsupial, the tammar wallaby, Macropus eugenii. This cathelicidin was not homologous to other such genes previously isolated from a tammar wallaby mammary gland EST library, however, it did contain 4 conser ...[more]

PMID: 19046773

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Project description:BackgroundThe tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species.ResultsA second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map.ConclusionsConstruction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably extends that of the first-generation map. It will be a valuable resource for ongoing tammar wallaby genetic research and assembling the genome sequence. The sex-pooled map is available online at http://compldb.angis.org.au/.

Project description:The tammar wallaby (Notamacropus eugenii) is one of the most intensively studied of all macropodids and was the first Australasian marsupial to have its genome sequenced. However, comparatively little is known about genetic diversity and differentiation amongst the morphologically distinct allopatric populations of tammar wallabies found in Western (WA) and South Australia (SA). Here we compare autosomal and Y-linked microsatellite genotypes, as well as sequence data (~600 bp) from the mitochondrial DNA (mtDNA) control region (CR) in tammar wallabies from across its distribution. Levels of diversity at autosomal microsatellite loci were typically high in the WA mainland and Kangaroo Island (SA) populations (A = 8.9-10.6; He = 0.77-0.78) but significantly reduced in other endemic island populations (A = 3.8-4.1; He = 0.41-0.48). Autosomal and Y-linked microsatellite loci revealed a pattern of significant differentiation amongst populations, especially between SA and WA. The Kangaroo Island and introduced New Zealand population showed limited differentiation. Multiple divergent mtDNA CR haplotypes were identified within both SA and WA populations. The CR haplotypes of tammar wallabies from SA and WA show reciprocal monophyly and are highly divergent (14.5%), with levels of sequence divergence more typical of different species. Within WA tammar wallabies, island populations each have unique clusters of highly related CR haplotypes and each is most closely related to different WA mainland haplotypes. Y-linked microsatellite haplotypes show a similar pattern of divergence although levels of diversity are lower. In light of these differences, we suggest that two subspecies of tammar wallaby be recognized; Notamacropus eugenii eugenii in SA and N. eugenii derbianus in WA. The extensive neutral genetic diversity and inter-population differentiation identified within tammar wallabies should further increase the species value and usefulness as a model organism.

Project description:BACKGROUND:Kangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby. RESULTS:We describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken. CONCLUSIONS:This is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.

Dataset Information

Identification and expression of a novel marsupial cathelicidin from the tammar wallaby (Macropus eugenii).

Publications

Identification and expression of a novel marsupial cathelicidin from the tammar wallaby (Macropus eugenii).

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