Identification of a novel tissue-specific processed HPRT gene and comparison with X-linked gene transcription in the Australian marsupial Macropus robustus

The genome of the Australian marsupial Macropus robustus contains a highly conserved processed hypoxanthine phosphoribosyltransferase homologue, HPRT-2. Using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and protein isoelectric focusing (IEF) we have shown this processed gene to be fully functional, but liver specific. In contrast, the unprocessed X-linked parent gene HPRT-1 was expressed in all somatic tissues. Expression of the HPRT-2 gene effectively doubles the total HPRT enzyme activity in liver compared to other tissues. Analysis of the 5′-flanking sequence of HPRT-2 revealed regions with homology to the liver-specific regulatory motifs C/EBP, NF-IL6, LF-A1 and LF-B1, although the functional significance of these regions remains unknown. Consistent with X chromosome inactivation in female mammals, transcript levels of the unprocessed X-linked gene HPRT-1 were similar in males and females in all tissues examined. No HPRT-2 activity was detected in testes, indicating that this gene does not compensate for sex chromosome inactivation during spermatogenesis. Moreover, the demonstration of very high HPRT-1 enzyme levels in testes indicated that such a compensatory mechanism may not be required. Phylogenetic analyses attribute considerable antiquity to the processed gene and PCR with conserved primers spanning exons 4–8 of genomic DNA from several different kangaroo species inferring the existence of a conserved processed HPRT-2 homologue in these marsupial species. However, no such conserved PCR product was obtained with DNA from eutherian species, suggesting that integration of HPRT-2 occurred after the separation of the metatherian and eutherian lineages.