Plasmacytoma-associated neuronal glycoprotein, Pang, maps to mouse chromosome 6 and human chromosome 3.

A new member of the immunoglobulin/fibronectin superfamily of adhesion molecules, Pang (plasmacytoma-associated neuronal glycoprotein), was recently isolated from a plasmacytoma. In previous studies, Pang was found to be normally expressed in the brain and ectopically activated by intracisternal A-type particle long terminal repeats in plasmacytomas. In this study, Pang was initially mapped to mouse Chr 6 by somatic cell hybrid analysis and further positioned on the chromosome between Wnt7a and Pcp1. Southern blot analysis of human-rodent somatic cell hybrids together with predictions from the mouse map location indicate that human PANG is located at 3p26.

Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21-->q22 and 2p16-->p15 respectively.

Calcineurin (also called protein phosphatase-2B) is a calmodulin-regulated protein phosphatase which plays an important role in signal transduction. The enzyme is a heterodimer of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a small (i.e. 19 kDa) Ca(2+)-binding regulatory subunit (calcineurin B). The highly conserved calcineurin B is encoded by a single gene in all tissues except testes, whereas there are three isoforms of calcineurin A (alpha, beta and gamma) encoded by genes on three different chromosomes. This enzyme can play a critical role in transcriptional regulation and growth control in T lymphocytes by a mechanism believed to involve dephosphorylation of the nuclear factor NF-AT which is essential for transcription of the interleukin-2 gene. To better evaluate the potential role of the calcineurin genes in human genetic disorders, we have studied their chromosome locations. Calcineurin B (PPP3R1) is located on human chromosome 2p16-->p15 and calcineurin A beta (PPP3CB, previous gene symbol CALNB) is present on 10q21-->q22. We confirm the localization of calcineurin A alpha (PPP3CA, previous gene symbol CALNA) to chromosome 4 without regional localization.

Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene.

G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.

Chromosomal organization and transcriptional regulation of human GEM and localization of the human and mouse GEM loci encoding an inducible Ras-like protein.

The mitogen-induced gene, GEM, encodes a GTP-binding protein that belongs to a new family within the Ras superfamily. The regulated expression pattern of Gem suggests a role for this protein in cellular responses to growth stimulation. To facilitate the assessment of the possible role of GEM in heritable and spontaneous disease processes, the genomic organization of human GEM and the chromosomal localization of human and murine GEM have been determined. GEM has been localized to the long arm of human chromosome 8 (8q13-q21) between the D8S85 and CA2 loci by genetic linkage analysis using an MspI restriction fragment length polymorphism within GEM. No consistent somatic chromosomal alterations or heritable diseases are associated with this region. Mouse Gem maps to the proximal region of chromosome 4 between Mos and Cga. To gain insight into the transcriptional regulation of GEM, we have established the transcriptional initiation site of GEM in human T cells and defined a 5' upstream region sufficient for mitogen-responsive, inducible transcription.

Restriction polymorphisms of the ceruloplasmin gene on chromosome 3.

Using a probe isolated from a human liver cDNA library, polymorphisms were observed in the human ceruloplasmin gene with the enzymes PstI and MspI. The PstI polymorphism was frequent (allele frequencies, 0.46 and 0.54) whereas the polymorphisms found with MspI were rare.

Genomic organization and chromosomal localization of the DUSP2 gene, encoding a MAP kinase phosphatase, to human 2p11.2-q11.

The mitogen-induced gene, DUSP2, encodes a nuclear protein, PAC1, that acts as a dual-specific protein phosphatase with stringent substrate specificity for MAP kinase. MAP kinase phosphorylation and consequent enzymatic activation is a central and often obligatory component in signal transduction initiated by growth factor stimulation or resulting from various types of oncogenic transformation. DUSP2 downregulates intracellular signal transduction through the dephosphorylation/inactivation of MAP kinases. To facilitate assessment of the possible role of DUSP2 in growth processes, the genomic structure and chromosomal location of the gene have been determined. DUSP2 has been localized to the pericentromeric region of human chromosome 2 (2p11.2-q11) by analysis of somatic cell hybrids, in situ chromosome hybridization, and genetic linkage analysis using a single-strand conformational polymorphism (SSCP) that has been identified in the 3' UTR of the gene. No consistent translocations or deletions at this chromosomal site have been reported in hematopoietic neoplasias or other tumors.

CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19.

Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic alpha-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses.

Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1).

Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences (Tarbp2-rs1, Tarbp2-rs2). The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells.

Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping.

The nuclear receptor superfamily of transcription factors, which includes the retinoic acid receptors and v-erb A, play important roles in the molecular control of hematopoiesis. To identify nuclear receptors expressed in hematopoietic cells, we screened a human bone marrow cDNA library using a degenerate oligonucleotide and isolated a 1.85-kb full-length cDNA encoding a new human member of this superfamily, the peroxisome proliferator activated receptor gamma (hPPAR gamma). Two different hPPAR gamma transcripts were expressed in hematopoietic cells: a 1.85-kb transcript, which corresponds to the full-length mRNA (PPAR gamma 1), and a 0.65-kb transcript (PPAR gamma 2), which cannot encode all of the nuclear receptor functional domains. Normal neutrophils and peripheral blood lymphocytes, as well as circulating leukemic cells from patients with AML, ALL, and CML, express only PPAR gamma 2 on Northern blot analysis. In contrast, only the PPAR gamma 1 transcript was detected in a variety of human leukemia cell lines and in cultured normal primary bone marrow stromal cells. Both transcripts were detected in various fetal and adult nonhematopoietic tissues. We mapped the location of the hPPAR gamma gene to human chromosome 3p25 by somatic cell hybridization and linkage analysis. PPARs have been shown to be activated by peroxisome proliferating agents, long-chain fatty acids and arachidonic acid. Human PPAR gamma, although homologous to the PPAR gamma s of other species, has unique sequence and amino acid differences. Identification of hPPAR gamma will allow further understanding of its role in human cellular leukotriene, prostaglandin, and peroxide degradative or synthetic pathways, as well as its role in lipid metabolism and regulation of adipocyte differentiation.

Fine mapping of the Darier's disease locus on chromosome 12q.

Darier's disease (DD) is an autosomal dominant genodermatosis characterized by epidermal acantholysis and dyskeratosis. We have performed genetic linkage studies in 10 families with DD (34 affected) by analyzing 14 polymorphic microsatellite markers. Our results confirm recent reports mapping the DD gene to chromosome 12q23-q24.1. Haplotype analysis of recombinant chromosomes in our families, along with previously reported data, narrow the location of the DD gene to a 5 cM interval flanked by the loci D12S354 and D12S84/D12S105. This localization allowed exclusion of two known genes, PLA2A and PAH, as candidate loci for DD. Three other gene loci (PPP1C, PMCH, PMCA1), mapping in 12q21-q24, remain potential candidates.

cDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria.

The pig and human dihydropyrimidine dehydrogenase (DPD) cDNAs were cloned and sequenced. The pig enzyme, expressed in Escherichia coli, catalyzed the reduction of uracil, thymine, and 5-fluorouracil with kinetics approximating those published for the enzyme purified from mammalian liver. DPD could be expressed in significant quantities only when uracil was added to the bacterial growth medium. The pig and human enzymes contained 1025 amino acids and calculated M(r) = 111,416 and 111,398, respectively. Conserved domains corresponding to a possible NADPH binding site and FAD binding site were found in the NH2-terminal half of the proteins and two motifs of putative [4Fe-4S] binding sites were found near to the carboxyl terminus of the enzyme. The latter corresponds to the labile COOH-terminal fragment previously shown to contain the iron sulfur centers. A sequence encompassing a peptide corresponding to the uracil binding site was found between the NADPH/FAD-containing NH2-terminal portion of the protein and the iron-sulfur binding sites near to the COOH terminus. Thus, the DPD appears to be derived from at least three distinct domains. The DPYD gene was localized to the centromeric region of human chromosome 1 between 1p22 and q21.

Fine mapping of the locus for nevoid basal cell carcinoma syndrome on chromosome 9q.

The nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized primarily by multiple basal cell carcinomas, odontogenic keratocysts, and pits of the palms and soles. Tumor deletion studies and linkage analysis in Caucasians have revealed that the gene is on chromosome 9q. To further refine the location of the nevoid basal cell carcinoma syndrome locus, we tested linkage to this region in three families. Evaluation of recombinants suggested that the nevoid basal cell carcinoma syndrome locus lies in the interval defined distally by D9S127. Our data, together with existing published data defining D9S12 as a proximal flanking marker, refine the location of nevoid basal cell carcinoma syndrome to an 8.3-cM interval. Two of the families studied were African-American and show a notable variation in phenotypic expression in which affected individuals developed few skin cancers. However, despite clinical heterogeneity, our data are consistent with the hypothesis that the same locus is involved in these African-American families.

The human DNA polymerase beta gene structure. Evidence of alternative splicing in gene expression.

DNA polymerase beta (beta-pol) is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. Using two human genomic libraries we have cloned the complete human beta-pol gene and determined the organization of the beta-pol coding sequence within the gene. The human beta-pol gene spans 33 kb and contains 14 exons that range from 50 to 233 bp. The 13 introns vary from 96 bp to 6.5 kb. Information derived from this study was used in defining the location of a deletion/insertion type restriction fragment length polymorphism (RFLP) 5' to exon I of the human beta-pol gene. This RFLP was utilized in linkage analysis of DNAs from CEPH families and the results confirm the previous assignment of the human beta-pol gene to chromosome 8 (p12-p11). Analysis of mRNA from six human cell lines using the polymerase chain reaction showed the expression of two beta-pol transcripts. Sequence analysis revealed that the size difference in these transcripts was due to deletion of the 58 bp sequence encoded by exon II, suggesting that the smaller transcript results from an alternative splicing of the exon II sequence during processing of the beta-pol precursor RNA.

Divergent structure of the human synexin (annexin VII) gene and assignment to chromosome 10.

The human synexin (annexin VII) gene occurs as a single copy at chromosome 10q21.1-21.2 and substantially deviates in size and in the location of splice junctions from the other two well-characterized members of the annexin gene family, lipocortin I (annexin I) and calpactin I (annexin II). The synexin gene contains 14 exons, including an alternatively spliced cassette exon, and spans approximately 34 kb of DNA. Only five of the fourteen splice junctions are conserved compared to other annexins, and the differences are particularly pronounced in the exons that encode the C-terminal third and fourth conserved repeats in the gene product. Although parallels between exons and protein domains were not apparent, we did observe clustering of splice junctions corresponding to either the unique N-terminal domain or the conserved C-terminal tetrad repeat domain, which is common to all annexins. Furthermore, a complete analysis of the 5' flanking region of the annexin VII gene revealed an entirely different set of cis-acting and enhancer elements compared to other annexin genes. We conclude that the annexin VII gene may have arisen by a divergence from the evolutionary pathway taken by both annexins I and II.

The molecular basis of X-linked severe combined immunodeficiency: the role of the interleukin-2 receptor gamma chain as a common gamma chain, gamma c.

X-linked severe combined immunodeficiency is characterized by severe and persistent infections from early life resulting from profound impairment of both cellular and humoral immune function. XSCID is characterized by an absence or diminished number of T cells and histologic evidence of hypoplastic and abnormal differention of the thymic epithelium. The discovery that this disease results from the mutations of the IL-2R gamma chain was surprising since IL-2-deficient mice and human SCID patients had milder phenotypes. This led to the speculation that IL-2R gamma would prove to be a common gamma chain, gamma c, which would play important roles in other cytokine receptors in addition to the IL-2 receptor. There is now compelling evidence to support a role in at least two other cytokine receptors, namely the IL-4 and IL-7 receptors. Thus, with inactivation of gamma c, multiple cytokine systems are simultaneously affected, resulting in the profoundly impaired phenotype of XSCID. It is possible and even likely that gamma c will be found to be a functional component of additional receptors as well. These findings have resulted in a significant improvement in our understanding of the pathophysiologic development of the defects in XSCID and also have important ramifications for prenatal and postnatal diagnosis, carrier female identification, and gene therapy for XSCID.

Localization of the MAGE1 gene encoding a human melanoma antigen to chromosome Xq28.

MAGE1 encodes a tumor specific antigen MZ2-E that elicited a cytotoxic T lymphocytic response (CTL) in the patient from whom it was derived. In this study, cDNA and genomic probes have been used to localize this gene by Southern analysis of a human-rodent somatic cell hybrid panel. The probes detect a small multigene family, and both MAGE1 and several other members of this family are located on the long arm of the human X chromosome. A cosmid with a 12-kb insert including the entire MAGE1 gene was biotinylated and used to further localize the gene to Xq28 by in situ hybridization of metaphase spreads. The function of this antigen in normal cells and tumor cells currently remains unclear.