Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Identification of the Human βA2 Crystallin Gene (CRYBA2): Localization of the Gene on Human Chromosome 2 and of the Homologous Gene on Mouse Chromosome 1

By using primers synthesized on the basis of the bovine βA2 crystallin gene sequence, we amplified exons 5 and 6 of the human gene (CRYBA2). CRYBA2 was assigned to human chromosome 2 by concordance analysis in human × rodent somatic cell hybrids using the amplified PCR products as probe. Regional localization to 2q34-q36 was established by hybridizing the CRYBA2 probe to microcell and radiation hybrids containing defined fragments of chromosome 2 as the only human contribution. The CRYBA2 probe was also used to localize, by interspecific backcross mapping, the mouse gene (Cryba2) to the central portion of chromosome 1 in a region of known human chromosome 2 homology. Finally, we demonstrate that in both species the βA2 crystallin gene is linked but separable from the γA crystallin gene. The βA2 crystallin gene is a candidate gene for human and mouse hereditary cataract.     

Mapping of the Gene Encoding the Regulatory Subunit RIIα of cAMP-Dependent Protein Kinase (Locus PRKAR2A) to Human Chromosome Region 3p21.3–p21.2

We have determined the chromosomal localization of the gene for the regulatory subunit RIIα of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome 3 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. Furthermore, PCR analysis of a chromosome 3 mapping panel revealed the presence of a human RIIα-specific amplification product only in cell lines containing the region 3p21.3–p21.2. The localization of PRKAR2A was confirmed by PCR mapping using the Stanford G3 Radiation Hybrid Panel as template. The results from this analysis demonstrated that PRKAR2A is most closely linked to D3S3334 (lod score 12.5) and flanked by D3S1322E and D3S1581.     

A Novel Human Gene Encoding a G-Protein-Coupled Receptor (GPR15) Is Located on Chromosome 3

We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have namedGPR15.A comparison of the amino acid sequence of the receptor encoded byGPR15with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1.GPR15was mapped to human chromosome 3q11.2–q13.1.     

Assignment of the Human Fast Skeletal Troponin T Gene (TNNT3) to Chromosome 11p15.5: Evidence for the Presence of 11pter in a Monochromosome 9 Somatic Cell Hybrid in NIGMS Mapping Panel 2

Human fast skeletal troponin T (TnT_f), the tropomyosin binding component of the multisubunit troponin complex, plays an important role in the Ca²⁺regulation of striated muscle contraction. Specific primers designed from the 3′ end of human TnT_fcDNA were used to amplify an intronic region by polymerase chain reaction (PCR). This TnT_f-specific PCR product was detected from two somatic cell hybrids containing human chromosomes 9 and 11, respectively, in NIGMS mapping panel 2. However, further studies with other somatic hybrid cell lines (Bios Laboratory) localized the TnT_fgene (HGMW-approved symbol TNNT3) only to chromosome 11. This observation was further confirmed by fluorescencein situhybridization with a 12-kb TnT_fgenomic probe generated by extended PCR, showing the sublocalization of the gene to band p15.5 on chromosome 11. This locus is of specific interest, as Beckwith–Wiedemann syndrome and various childhood and adult tumor-related abnormalities have been mapped to this region. The study also indicates the presence of an 11pter region in the NIGMS cell hybrid GM10611, which has previously been reported to contain only human chromosome 9.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Chromosomal localization of the human interleukin 1α (IL-1α) gene

The human interleukin 1α gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12–21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1α gene maps to the same general region on the long arm of chromosome 2 as the IL-1β gene, which has been previously assigned.     

The Gene Encoding Protein Kinase SEK1 Maps to Mouse Chromosome 11 and Human Chromosome 17

We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human–rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouseSerk1gene was mapped to chromosome 11, closely linked toD11Mit4,using genomic DNAs from a (C57BL/6J ×Mus spretus)F₁×M. spretusbackcross.     

CD19 maps to a region of conservation between human chromosome 16 and mouse chromosome 7

CD19 is a B lymphocyte cell surface protein expressed from the earliest stages of B lymphocyte development unitl their terminal differentiation into plasma cells. In this report the human CD19 gene (hCD19) was localized to band p11.2 on the proximal short arm of chromosome 16 by in situ hybridization to metaphase chromosomes, using hCD19 cDNA as probe. hCD19 gene localization was confirmed by polymerase chain reaction based analysis with hCD19-specific primers, using a panel of human/hamster somatic cell hybrid DNA as templates. The mouse CD19 gene (MCd19) was mapped to bands F3-F4 of chromosome 7 by in situ hybridization to metaphase chromosomes, using a mCD19 cDNA probe. Segregation analysis of nucleotide sequence polymorphisms in inter-specific backcross progeny revealed linkage of mCd19 with hemoglobin (Hbb), Int-2, and H19, other loci previously mapped to the same region of mouse chromosome 7, confirming the localization of mCd19 to this region. The order of these loci was determined to be centromere — Hbb — mCd19 — H19 — Int-2 —telomere. The genetic distance between the loci examined, calculated from the recombination frequencies, suggested that mCd19 was located centrally between Hbb and H19. This region of mouse chromosome 7 is homologous to the region of human chromosome 16 to which the hCD19 gene maps. Multiple genes with a lymphocyte-related function also map to this conserved region including genes encoding the IL-4 receptor, CD11a, CD11b, CD11c, CD43 (leukosialin), and protein kinase C polypeptide.     

An expressed β-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed β-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human X Chinese hamster somatic cell hybrids cleaved with Hind III or EcoR I. Probes containing the 3′ untranslated regions of the expressed gene M40 and of pseudogene 21β were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21 → 6pter, the 21β pseudogene (TUBBP1) to chromosome 8 region 8q21 → 8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

Molecular Cloning, Chromosomal Localization, and Cell Cycle-Dependent Subcellular Distribution of the A-Kinase Anchoring Protein, AKAP95

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994)J. Biol. Chem.269, 7658–7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414–692 of human AKAP95 was expressed inEscherichia coliand shown to bind RIIα. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIα binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIα was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIα overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIα was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIα may be cell cycle-dependent.     

Identification and regional localization of a human IMPdehydrogenase-like locus (IMPDHL1) at 16p13.13

Sequence-tagged sites (STSs) are versatile chromosomalmarkers for a variety of genome mapping efforts. In this report, we describe a randomly generated STS (323F4) from human chromosome 16 genomic DNA that has 90.0% sequence identity to the type I human inosine-5′-monophosphate dehydrogenase (IMPDH1) gene and 72% identity to the type II human inosine-5′-monophosphate dehydrogenase (IMPDH2) gene. Additional sequencing by primer walking has provided a total of 1380 by of the human chromosome 16 sequence. The IMPDH-like sequence 323F4 was regionally localized by PCR analysis of a panel of somatic cell hybrids containing different portions of human chromosome 16 to 16p13.3-13.12, between the breakpoints found in hybrids CY196/CY197 and CY198. This regional mapping assignment was further refined to subband 16p13.13 by high-resolution fluorescence in situ hybridization using cosmid 323F4 as a probe. We conclude that a third, previously undescribed IMPDH locus, termed IMPDHL1, exists at human chromosome 16p13.13.     

A eutherian X-linked gene, PDHA1, is autosomal in marsupials:A model for the evolution of a second, testis-specific variant in eutherian mammals

We report the cloning and mapping of a gene (PDHA)for the pyruvate dehydrogenase E1α subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.     

Conserved Chromosomal Location and Genomic Structure of Human and Mouse Fatty-Acid Amide Hydrolase Genes and Evaluation ofclasperas a Candidate Neurological Mutation

Fatty-acid amide hydrolase (FAAH) is a membrane-bound enzyme that degrades neuromodulatory fatty acid amides, such as oleamide and anandamide, and is expressed in the mammalian central nervous system. To evaluateFAAHgenes as candidates for neurogenetic diseases in humans and mice, we have mapped the loci in both species and have determined their intron–exon structures. The humanFAAHgene was mapped to region 1p34–p35, closely linked to D1S197 and D1S443, by using PCR analysis of somatic cell hybrid (SCH) and radiation hybrid mapping panels. Analysis of an SCH mapping panel and a mouse interspecific backcross panel has localized theFaahgene to the conserved syntenic region on mouse chromosome 4, close to the neurological mutationclasper. Faahgene rearrangements were excluded by Southern blot analysis of clasper DNA. No sequence abnormality was detected in PCR products containing the 15 exons and splice junctions of the mouseFaahgene. FAAH protein levels were normal in clasper mouse tissues as determined by enzyme activity assays and Western blotting.     

Regional localization of the gene encoding pregnancy specific beta-1-glycoprotein 1 (PSBG1) to human chromosome 19q13.1

Pregnancy specific beta-1-glycoprotein (PSBG), a major product of the human placenta, is encoded by multiple genes. Southern blot hybridization of human x rodent somatic cell hybrid DNAs with a cDNA specific for one member of the PSBG gene family allowed us to map this gene to human chromosome 19. Further analysis using hybrids with subchromosomal segments of 19q revealed that the gene maps to the distal segment of region 19q13.1.     

Chromosomal Locations of Three Human Nuclear Genes (RPSM12, TUFM, and AFG3L1) Specifying Putative Components of the Mitochondrial Gene Expression Apparatus

We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination ofin situhybridization and interspecies hybrid mapping. The genesRPMS12(mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center),TUFM(mitochondrial elongation factor EF-Tu), andAFG3L1(similar to the yeast genesAfg3andRca1involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing.RPMS12maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. TheTUFMgene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17.AFG3L1is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy.     

Genetic Linkage Mapping of the Dehydroepiandrosterone Sulfotransferase (STD) Gene on the Chromosome 19q13.3 Region

In the human liver and adrenal, there is a single hydroxysteroid sulfotransferase, which catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundantly circulating steroid in humans, and also catalyzes the sulfation of a series of other 3β-hydroxysteroids as well as cholesterol. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in several peripheral tissues, indicating that hydroxysteroid sulfotransferase plays a pivotal role in controlling the hormonal action of sex steroids by regulating their bioavailability. We recently elucidated the structure of the gene encoding hydroxysteroid sulfotransferase (STD), also designated dehydroepiandrosterone sulfotransferase, which spans 17 kb and contains six exons. The STD gene was preliminarily assigned to chromosome 19 by polymerase chain reaction (PCR) amplification of DNA from a panel of human/rodent somatic cell hybrids. To locate the STD gene, the novel biallelic polymorphism found in intron 2 was genotyped in eight CEPH reference families by direct sequencing of PCR products. Two-point linkage analysis was first performed between the latter polymorphism and chromosome 19 markers from Généthon and NIH/CEPH. The closest linkage was observed with D19S412 (Z_max= 9.23; θ_max0.038) and HRC (Z_max= 5.95; θ_max0.036), located on the 19q13.3 region. A framework map including six Généthon markers flanking the polymorphic STD gene was created by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the region was constructed, yielding to the following order: qter–D19S414–D19S224–D19S420–D19S217–(APOC2–D19S412)–(STD–HRC)– KLK–D19S22–D19S180–PRKCG–D19S418–tel.