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A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine‐gated strychnine‐sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent‐human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2‐p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.

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Using a positional cloning approach, we have isolated an expressed gene from a flow-sorted Y chromosome cosmid library. The isolation of this gene was based on the identification of the Y-231 cosmid that contains CpG rich sequences (HTF islands) in its human insert. The Y-231 cosmid was capable of detecting a 1.3 kb transcript in poly (A)+ RNA samples from human testis. Several cDNA clones were isolated from a human testis cDNA library constructed in lambda gt10. In addition, DNA-mediated gene transfer and restriction enzyme mapping experiments demonstrated that two functional transcriptional units are present within the Y-231 cosmid. DNA sequencing analysis showed that the largest cDNA clone contains 1075 bp of unique sequence and a poly (A) track at the 3' end of the corresponding mRNA. An open reading frame of 762 bp that encodes a predicted protein of 253 amino acids with a calculated molecular weight of 28.9 kD was identified. The Y-231 structural gene encompasses approximately 2.7 kb of genomic sequence and contains six exons that are interrupted by five introns. The Y-231 gene shares very high (97%) identity at the DNA level to a previously described Y-specific gene, testis specific protein Y-encoded (TSPY) gene, suggesting the possibility that these two genes are related, if not identical. However, the TSPY gene has been postulated to be intronless. Further PCR and RT-PCR analyses of these two genes and their transcripts have provided evidence supporting the hypothesis that they are the same gene and are members of a Y-specific repeated gene family containing intronic sequences. The Y-231 (TSPY) gene is conserved in the male genome and expressed in the testis of the chimpanzee, suggesting that it may play an important role in the physiology of this organ in man and the great ape.