C-->U editing of apolipoprotein B mRNA in marsupials: identification and characterisation of APOBEC-1 from the American opossum Monodelphus domestica.

The C->U editing of RNA is widely found in plant and animal species. In mammals it is a discrete process confined to the editing of apolipoprotein B (apoB) mRNA in eutherians and the editing of the mitochondrial tRNA for glycine in marsupials. Here we have identified and characterised apoB mRNA editing in the American opossum Monodelphus domestica. The apoB mRNA editing site is highly conserved in the opossum and undergoes complete editing in the small intestine, but not in the liver or other tissues. Opossum APOBEC-1 cDNA was cloned, sequenced and expressed. The encoded protein is similar to APOBEC-1 of eutherians. Motifs previously identified as involved in zinc binding, RNA binding and catalysis, nuclear localisation and a C-terminal leucine-rich domain are all conserved. Opossum APOBEC-1 contains a seven amino acid C-terminal extension also found in humans and rabbits, but not present in rodents. The opossum APOBEC-1 gene has the same intron/exon organisation in the coding sequence as the eutherian gene. Northern blot and RT-PCR analyses and an editing assay indicate that no APOBEC-1 was expressed in the liver. Thus the far upstream promoter responsible for hepatic expression in rodents does not operate in the opossum. An APOBEC-1-like enzyme such as might be involved in C->U RNA editing of tRNA in marsupial mitochondria was not demonstrated. The activity of opossum APOBEC-1 in the presence of both chicken and rodent auxiliary editing proteins was comparable to that of other mammals. These studies extend the origins of APOBEC-1 back 170 000 000 years to marsupials and help bridge the gap in the origins of this RNA editing process between birds and eutherian mammals.     

A eutherian X-linked gene, PDHA1, is autosomal in marsupials:A model for the evolution of a second, testis-specific variant in eutherian mammals

We report the cloning and mapping of a gene (PDHA)for the pyruvate dehydrogenase E1α subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.     

In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals

Background

Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals.

Results

The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations.

Conclusion

The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.     

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

M6P/IGF2R imprinting evolution in mammals

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.     

Phylogenetic differences of mammalian basal metabolic rate are not explained by mitochondrial basal proton leak

Metabolic rates of mammals presumably increased during the evolution of endothermy, but molecular and cellular mechanisms underlying basal metabolic rate (BMR) are still not understood. It has been established that mitochondrial basal proton leak contributes significantly to BMR. Comparative studies among a diversity of eutherian mammals showed that BMR correlates with body mass and proton leak. Here, we studied BMR and mitochondrial basal proton leak in liver of various marsupial species. Surprisingly, we found that the mitochondrial proton leak was greater in marsupials than in eutherians, although marsupials have lower BMRs. To verify our finding, we kept similar-sized individuals of a marsupial opossum (Monodelphis domestica) and a eutherian rodent (Mesocricetus auratus) species under identical conditions, and directly compared BMR and basal proton leak. We confirmed an approximately 40 per cent lower mass specific BMR in the opossum although its proton leak was significantly higher (approx. 60%). We demonstrate that the increase in BMR during eutherian evolution is not based on a general increase in the mitochondrial proton leak, although there is a similar allometric relationship of proton leak and BMR within mammalian groups. The difference in proton leak between endothermic groups may assist in elucidating distinct metabolic and habitat requirements that have evolved during mammalian divergence.     

Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.     

Identification of natural killer cell receptor genes in the genome of the marsupial Tasmanian devil (Sarcophilus harrisii)

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genes KLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.     

VpreB1/VpreB2/lambda 5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus

At the precursor B cell stage during bone marrow B cell development, Ig muH chain associates with surrogate L (SL) chain, which is encoded by the three genes VpreB1, VpreB2, and lambda 5, to form the pre-B cell receptor (pre-BCR). Surface expression of the pre-BCR is believed to signal both proliferation and allelic exclusion of the IgH locus. Mice which lack either VpreB1/VpreB2 or lambda 5 show a lack of precursor B cell expansion but normal IgH allelic exclusion. This would suggest that one of either lambda 5 or VpreB can make a pre-BCR-like complex which is still able to signal allelic exclusion but not proliferation. To investigate this, we established mice lacking all components of the SL chain. These mice showed severely impaired B cell development which was similar to that previously found in mice lacking either lambda 5 or VpreB1/VpreB2. Surprisingly, the IgH locus was still allelically excluded and thus the SL chain appears not to be involved in allelic exclusion.     

Divergent T-cell receptor delta chains from marsupials

Complementary DNAs (cDNAs) encoding T-cell receptor delta (TRD) chains from the northern brown bandicoot, Isoodon macrourus, were identified while sequencing expressed sequence tags (ESTs) from a thymus cDNA library. Surprisingly, the I. macrourus TRD sequences were not orthologous to previously published TRD sequences from another Australian marsupial, the tammar wallaby, Macropus eugenii. Identification of TRD genes in the recently completed whole genome sequence of the South American opossum, Monodelphis domestica, revealed the presence of two highly divergent TRD loci. To determine whether the presence of multiple TRD loci accounts for the lack of orthology between the I. macrourus and M. eugenii cDNAs, additional TRD sequences were obtained from both species of marsupials. The results of this analysis revealed that, unlike eutherian mammals, all three species of marsupials have multiple, highly divergent TRD loci. One group of marsupial TRD sequences was closely related to TR sequences from eutherian mammals. A second group of TRD sequences formed a unique marsupial-specific clade, separate from TR sequences from eutherians. An interesting expression pattern of TRD variable (TRDV) and constant (TRDC) segments was evident in cDNAs from I. macrourus and M. eugenii. TRDV and TRDC sequences that were closely related to TRD genes from eutherian mammals were only found in association with each other in cDNAs from both marsupial species. A similar pattern was seen between TRDV and TRDC sequences that were most closely related to other marsupial TRD genes.     

Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley-Liss, Inc.     

Structure and pre-B lymphocyte restricted expression of the VpreB in humans and conservation of its structure in other mammalian species.

DNA from several mammals, including humans, was found to contain one or more restriction enzyme digested DNA fragments which hybridized to the mouse VpreB gene under stringencies demonstrating at least 70% nucleotide sequence homologies, indicating that the VpreB locus may be widespread and highly conserved among mammals. A human VpreB genomic clone was isolated and sequenced. Two exons and the intervening intron are spaced almost identically as in the mouse VpreB1 gene, and show 76% sequence homology to the mouse gene. As in the mouse VpreB1 gene, the 5' end of the human VpreB gene contains characteristic features of Ig domains, while the 3' end is Ig non-related. This 3' Ig non-related structure of the VpreB gene(s) may, therefore, have existed before the speciation of humans and mice over 65 million years ago. Sequences encoding the entire putative second framework region and a stretch in the third framework region are identical in human and mouse VpreB. the human VpreB gene appears to be selectively expressed in human pre-B cell lines as an 0.85 kb poly(A)+ RNA. Its expression promises to be a useful marker for the detection of normal and malignant human pre-B lymphocytes.     

First-generation linkage map of the gray, short-tailed opossum, Monodelphis domestica, reveals genome-wide reduction in female recombination rates

The gray, short-tailed opossum, Monodelphis domestica, is the most extensively used, laboratory-bred marsupial resource for basic biologic and biomedical research worldwide. To enhance the research utility of this species, we are building a linkage map, using both anonymous markers and functional gene loci, that will enable the localization of quantitative trait loci (QTL) and provide comparative information regarding the evolution of mammalian and other vertebrate genomes. The current map is composed of 83 loci distributed among eight autosomal linkage groups and the X chromosome. The autosomal linkage groups appear to encompass a very large portion of the genome, yet span a sex-average distance of only 633.0 cM, making this the most compact linkage map known among vertebrates. Most surprising, the male map is much larger than the female map (884.6 cM vs. 443.1 cM), a pattern contrary to that in eutherian mammals and other vertebrates. The finding of genome-wide reduction in female recombination in M. domestica, coupled with recombination data from two other, distantly related marsupial species, suggests that reduced female recombination might be a widespread metatherian attribute. We discuss possible explanations for reduced female recombination in marsupials as a consequence of the metatherian characteristic of determinate paternal X chromosome inactivation.     

Identification of a Novel PNMA-MS1 Gene in Marsupials Suggests the LTR Retrotransposon-Derived PNMA Genes Evolved Differently in Marsupials and Eutherians

Two major gene families derived from Ty3/Gypsy long terminal repeat (LTR) retrotransposons were recently identified in mammals. The sushi-ichi retrotransposon homologue (SIRH) family comprises 12 genes: 11 in eutherians including Peg10 and Peg11/Rtl1 that have essential roles in the eutherian placenta and 1 that is marsupial specific. Fifteen and 12 genes were reported in the second gene family, para-neoplastic antigen MA (PNMA), in humans and mice, respectively, although their biological functions and evolutionary history remain largely unknown. Here, we identified two novel candidate PNMA genes, PNMA-MS1 and -MS2 in marsupials. Like all eutherian-specific PNMA genes, they exhibit the highest homology to a Gypsy12_DR (DR, Danio rerio) Gag protein. PNMA-MS1 is conserved in both Australian and South American marsupial species, the tammar wallaby and grey short-tailed opossum. However, no PNMA-MS1 orthologue was found in eutherians, monotremes or non-mammalian vertebrates. PNMA-MS1 was expressed in the ovary, mammary gland and brain during development and growth in the tammar, suggesting that PNMA-MS1 may have acquired a marsupial-specific function. However, PNMA-MS2 seems to be a pseudogene. The absence of marsupial orthologues of eutherian PNMA genes suggests that the retrotransposition events of the Gypsy12_DR-related retrotransposons that gave rise to the PNMA family occurred after the divergence of marsupials and eutherians.     

Analysis of VpreB expression during B lineage differentiation in lambda5-deficient mice

The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.     

Localization of four human chromosome 21 genes--SOD1, ETS2, IFNAR, and CBR--to two different chromosomes in the marsupial species Macropus eugenii.

We have mapped the chromosomal location of four genes previously assigned to human chromosome 21--Cu/Zn superoxide dismutase (SOD1), the protooncogene ETS2, the interferon alpha/beta receptor gene (IFNAR), and the carbonyl reductase gene (CBR)--in the tammar, Macropus eugenii. The genes are localized on two separate autosomes: SOD1 and CBR map to chromosome 7 and ETS2 and IFNAR map to chromosome 3 or 4. These results provide the first example of asynteny between SOD1/CBR and ETS2/IFNAR in a mammalian species. The results suggest that either this synteny group has been disrupted in the marsupial lineage, or, alternatively, the genes located on human chromosome 21 may have been joined after the marsupials diverged from the eutherian mammals some 130-150 million years ago.