Tania A. Jones

Localization of the gene encoding a type I protein phosphatase catalytic subunit to human chromosome band 11q13

A cDNA encoding one isoform (PP1 alpha) of the catalytic subunit of human protein phosphatase 1 has been isolated and used to map the human PP1 alpha gene (PPP1A) to chromosome band 11q13 by analysis of somatic cell hybrids and in situ hybridization. Neoplasms that map to 11q13 are discussed in the light of the recent findings that PP1 alpha is a putative tumor suppressor and that it plays a key role in the control of mitosis.

Genomic structure and domain organisation of the human Bak gene.

The Bcl-2 homologue, Bak, is a potent inducer of apoptosis. FISH data presented here located the gene to 6p21.3. Mapping was consistent with its location centromeric of the HSET locus and approximately 400kb from the MHC. The construction of a contig of genomic clones across the locus facilitated the sequencing of a PAC containing the gene. Comparison of the gene structure to functional and physical domains revealed a good agreement between the physical structure and the intron-exon organisation. The position of a single intron was conserved in comparison to other members of the Bcl-2 family, namely Bax, CED-9, Bcl-X and Bcl-2, but all other introns were displaced, consistent with a divergent phylogeny.

Localisation of the gene encoding the catalytic γ subunit of phosphorylase kinase to human chromosome bands 7p12-q21

Skeletal muscle phosphorylase kinase has the structure (alpha beta gamma delta)4 where the alpha and beta subunits are regulatory components, the gamma subunit possesses catalytic activity and the delta subunit is identical to the calcium binding protein calmodulin. A rabbit skeletal muscle cDNA for the gamma subunit has been used to map the human gene (PYKG1) to 7p12-q21, by analysis of somatic cell hybrids and in situ hybridisation. The data suggest that the skeletal muscle gamma subunit gene is located just above the centromere of chromosome 7, with further cross-hybridising sequences at 7q21 and 11p11-14. The liver gamma subunit is distinct and its mRNA does not cross-hybridize with the skeletal muscle gamma subunit cDNA. These results indicate that autosomal human phosphorylase kinase deficiencies affecting both liver and muscle are likely to be caused by a defect in the autosomally determined beta subunit, rather than the gamma subunit.

Localization of the genes encoding the catalytic subunits of protein phosphatase 2A to human chromosome bands 5q23→q31 and 8p12→p11.2, respectively

The gene for the alpha isoform of the catalytic subunit of human protein phosphatase 2A (PPP2CA) was localized to chromosome 5 using somatic cell hybrids, and then more finely mapped to chromosome region 5q23-->q31 by in situ hybridization using a tritiated cDNA probe. The gene for the beta isoform of the catalytic subunit of this enzyme (PPP2CB) was mapped by the polymerase chain reaction to human chromosome 8 using somatic cell hybrids. Fluorescence in situ hybridization was then used to localize the PPP2CB gene to 8p12-->p11.2, using a mixture of three genomic probes that ranged from 3.5 to 8 kb in size. Finally, Southern blot analysis of somatic cell hybrid DNA suggested that a PPP2CB catalytic subunit pseudogene (PPP2CBP) is on chromosome 16.

Localization of an epithelial-specific receptor kinase (EDDR1) to chromosome 6q16

A protein receptor tyrosine kinase (EDDR1) has been isolated from a complementary DNA library of SKOV-3, an epithelial ovarian cancer cell line. The primary structure of the predicted amino acid sequence of the protein shows a novel N-terminal region that has homology to a factor VIII-like domain. The C-terminal catalytic domain has all of the canonical sequence motifs of a receptor tyrosine kinase with homology to the TRK-2H protein (49%), which suggests that it is a type II receptor. It is expressed in epithelial cells of several tissues. To determine the chromosomal localization of the gene, somatic cell hybrids were analyzed by PCR amplification using oligonucleotide primers specific for EDDR1. Segregation was observed to a hybrid containing human chromosome 6. Cosmids for EDDR1 were isolated from a human chromosome 6 cosmid library and were shown by fluorescence in situ hybridization to map to 6q16.

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12–p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 transactivator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent-human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to three different chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12-p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.

A somatic cell hybrid panel for regional mapping of human chromosome 18

We have produced and characterized a panel of seven somatic cell hybrids defining eight potential intervals on human chromosome 18. Using 24 markers that have previously been assigned to this chromosome, we have placed markers into six of these regions. The gene for alpha 2-plasmin inhibitor (PLI) and the anonymous DNA segment locus D18S23 are excluded from this chromosome.

Complex Duplications at 6p22.1, 6pll.2, 5q13, 5p15.1 and 5p13 Revealed by Fluorescent In Situ Hybridisation

A complex pattern of fluorescent in situ hybridisation (FISH) has been detected using PAC clones from the short arm of chromosome 6, proximal to the haemochromatosis gene at 6p22.1. Cross-hybridisation to 6p22.1, 6p11.2, 5q13, 5p15.1 and 5p13 was consistently detected with several PAC clones covering a genomic region greater than 200 kb. These results indicate that large sections of genomic DNA are shared by these 5 disparate chromosomal segments, indicative of large scale duplication events. These results were in part accounted for by the identification of several expressed sequence tags (ESTs).