Glen A. Evans

We report the isolation and sequencing of cDNAs encoding two human glutamate decarboxylases (GADs; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), GAD65 and GAD67. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues, whereas human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues. Both cDNAs direct the synthesis of enzymatically active GADs in bacterial expression systems. Each cDNA hybridizes to a single species of brain mRNA and to a specific set of restriction fragments in human genomic DNA. In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31. We conclude that GAD65 and GAD67 each derive from a single separate gene. The cDNAs we describe should allow the bacterial production of test antigens for the diagnosis and prediction of insulin-dependent diabetes mellitus.

The PPP2R1B gene, which encodes the beta isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), was identified as a putative human tumor suppressor gene. Sequencing of the PPP2R1B gene, located on human chromosome 11q22-24, revealed somatic alterations in 15% (5 out of 33) of primary lung tumors, 6% (4 out of 70) of lung tumor-derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated a truncated PP2A-Abeta protein that was unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control.

Translocations involving chromosome 11, band q23, are frequent recurring abnormalities in human acute lymphoblastic and acute myeloid leukemia. We used 19 biotin-labeled probes derived from genes and anonymous cosmids for hybridization to metaphase chromosomes from leukemia cells that contained four translocations involving band 11q23: t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13). The location of the cosmid probes relative to the breakpoint in 11q23 was the same in all translocations. Of the cosmid clones containing known genes, CD3D was proximal and PBGD, THY1, SRPR, and ETS1 were distal to the breakpoint on 11q23. Hybridization of genomic DNA from a yeast clone containing yeast artificial chromosomes (YACs), that carry 320 kilobases (kb) of human DNA including CD3D and CD3G genes, showed that the YACs were split in all four translocations. These results indicate that the breakpoint at 11q23 in each of these translocations occurs within the 320 kb encompassed by these YACs; whether the breakpoint within the YACs is precisely the same in the different translocations is presently unknown.

A genomic clone encoding H-2Ld, a mouse major transplantation antigen, has been identified and the structure of the H-2Ld gene has been partially determined. We isolated 35 genomic clones from a BALB/c (H-2d) genomic library by hybridization to mouse or human probes. One of these clones encodes H-2Ld as determined by two criteria. First, the gene encodes a protein that is identical at the 76 known amino acid positions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that is recognized by anti-H-2Ld monoclonal antibodies. The sequence of the H-2Ld protein predicted by the DNA sequences shows more than 80% homology to known H-2 antigens. H-2L-like sequences are found in mutant H-2Kb molecules, suggesting that gene conversion or reciprocal recombination may play a role in the development of H-2 polymorphism.