Assignment of the gene for beta-spectrin (SPTB) to chromosome 14q23----q24.2 by in situ hybridization

Type I hereditary spherocytosis results from a molecular defect in the beta-polypeptide of the erythrocyte cytoskeletal protein spectrin. Using a cDNA probe, we had previously assigned the gene for human erythrocyte beta-spectrin (SPTB) to chromosome 14 based upon analysis of its segregation in panels of human x rodent somatic cell hybrids (Winkelmann et al., 1988). Here we report the regional localization of this gene by in situ hybridization to 14q23----q24.2.     

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.     

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.     

Assignment of the gene coding for the alpha 2-macroglobulin receptor to mouse chromosome 15 and to human chromosome 12q13-q14 by isotopic and nonisotopic in situ hybridization.

The assignment of the gene encoding the alpha 2-macroglobulin receptor (A2MR), which was first described as the low-density lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal human metaphases to the region 12q13-q14. The same human cDNA, which has 95% sequence identity with the mouse A2mr, was hybridized to metaphases containing the Robertsonian translocation Rb(6;15)1Ald. The mouse A2mr gene was assigned to chromosome 15 in the region B2-D1. This locus and other loci on mouse chromosome 15 have been shown to be homologous with loci on human chromosome 12q.     

Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.     

Molecular cloning of alpha 5(IV) collagen and assignment of the gene to the region of the X chromosome containing the Alport syndrome locus.

Type IV collagen is a major structural component of basement membranes. Four constituent polypeptides have been described and characterized to different degrees. Whereas the primary structure of the alpha 1(IV) and alpha 2(IV) chains has been completely established, only short protein sequences have been reported for the recently recognized alpha 3(IV) and alpha 4(IV) subunits. We have isolated overlapping human cDNA clones whose derived amino acid sequence is highly homologous to the alpha 1(IV) and alpha 2(IV) chains. However, these clones code for neither alpha 3(IV) nor alpha 4(IV), and thus this new polypeptide has been designated the alpha 5 chain of type IV collagen. To determine whether the gene encoding the alpha 5(IV) chain is syntenic with the contiguously arranged alpha 1(IV) and alpha 2(IV) genes at 13q34, the alpha 5(IV) cloned DNA was hybridized to genomic DNA from somatic cell hybrids and to metaphase chromosomes. The results demonstrated that the alpha 5(IV) collagen gene is located on the long arm of the X chromosome. Since 14 collagen genes have previously been assigned to nine autosomes, these data represent the first mapping of a collagen gene to the X chromosome. Most important, the alpha 5(IV) gene has been sublocalized to bands Xq22----q23, which are in the same region known to contain the locus for the X-linked form of Alport syndrome. It is therefore possible that this severe dominantly inherited nephritis, manifested by splitting of the glomerular basement membrane, could be caused by mutations in the alpha 5(IV) collagen gene.     

Cloning of the human monocarboxylate transporter MCT3 gene: localization to chromosome 22q12.3-q13.2.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.     

Localization of the beta A4-crystallin gene (CRYBA4) on human chromosome 22 in the region q11.2-->q13.1.

The chromosomal localization of the human gene (CRYBA4) coding for the eye lens protein beta A4-crystallin has been carried out using a nearly full-length cDNA clone encoding bovine beta A4-crystallin. A panel of 21 human-mouse or human-hamster hybrid cell lines derived from different parental combinations was characterized with respect to the human chromosomal content and the presence of well established human chromosome-specific markers. These panels were screened for the presence of CRYBA4 using the bovine cDNA clone as a probe. A 100 percent concordance was observed between the presence or absence of the CRYBA4 and human chromosome 22 indicating that the gene resides on this chromosome. By using cell hybrids containing translocated chromosome 22 segments, the localization could be refined to the region 22q11.2-->q13.1.     

X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex

The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.     

Chromosomal localization of the human gene for brain Ca2+/calmodulin-dependent protein kinase type IV

Cloned cDNAs have been identified as corresponding to a new brain Ca2+/calmodulin-dependent protein kinase. On the basis of structural and immunological features, we refer to this new kinase as CaM Kinase IV. Two cDNA clones were used to identify CaM Kinase IV: The downstream clone, lambda ICM-1, contains the sequence encoding the calmodulin-binding site and the second clone, lambda ICM-2, encodes a partial amino acid sequence similar to the catalytic domain of several known protein kinases. Within the calmodulin-binding site a stretch of 8 amino acids (and 9 of 10) is identical to the corresponding site in the subunits of CaM Kinase II. Southern blot analysis shows the CaM Kinase IV gene is single copy in the mouse and human genomes. Synteny analysis of Southern blot data of DNA from hamster--human hybrid cells shows that the gene is present in human chromosome 5. Hybridization of cDNA probes to metaphase spreads of human chromosomes indicates that the gene is most likely located within the region of bands q21 to q23 of chromosome 5.     

Complementary physical and genetic techniques map the vinculin (VCL) gene on chromosome 10q.

Vinculin is a cytoskeletal protein component of adherens type cell junctions. The gene had been mapped to 10q11.2-qter. We have used a combination of physical and genetic mapping techniques to refine this localization. Hybridization of the vinculin cDNA probe, HV1, to a human-rodent somatic hybrid panel initially suggested a position of either 10q11.2 or 10q22.1-10q23. Genetic recombination mapping in three-generation families with multiple endocrine neoplasia type 2 (MEN2) indicated a position distal to D10S22 (10q21.1) in 10q22.1-10q23. This was confirmed by hybridization of the vinculin cDNA to flow-sorted translocation derivative chromosomes containing the q21-qter portion of chromosome 10. We conclude that the vinculin locus maps in 10q22.1-q23, distal to D10S22.     

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.     

Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase

A full-length cDNA encoding a novel protein was isolated and sequenced from a human hepatocellular cDNA library. This cDNA consists of 7037 base pairs and has a predicted open reading frame encoding 1924 amino acids. It possesses an RNA-helicase motif containing a DEXH-box in its amino-terminus and an RNase motif in the carboxy-terminus. From a striking homology to Caenorhabditis elegans K12H4.8, it might be a human homolog of the K12H4.8. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 14q31 near the marker D14S605.     

Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

Identification and localization of MEP1A-like sequences (MEP1AL1-4) in the human genome.

The human MEP1A gene encodes the meprin alpha subunit that consists of a protease domain conserved in the astacin family of metalloendopeptidases and several C-terminal interaction domains present in other proteins. Using the alpha subunit cDNA, we identified two clones from a human P1-derived artificial chromosome (PAC) library. Fluorescence in situ hybridization (FISH) mapped both PACs (1e12, 65a14) to chromosome 6p21, confirming the MEP1A location. FISH also mapped PAC 65a14 to chromosome 13cen, and to chromosome 9 in three different regions, 9p12-13, 9q21, and 9q22. Southern blot analysis showed that sequences of PAC 65a14 and MEP1A were similar in the 3' end but different in the 5' end, revealing for the first time that the human genome may encode multiple interaction domains highly similar to those of the meprin alpha subunit. The symbols of MEP1AL1, MEP1AL2, MEP1AL3, and MEP1AL4 have been designated for MEP1A-like sequences on 9p12-13, 9q21, 9q22, and 13cen, respectively.     

Identification of the candidate ALS2 gene at chromosome 2q33 as a human aldehyde oxidase gene

Denver, Tokyo, and Salt Lake City investigators recently published different complimentary deoxyribonucleic acid (cDNA) sequences for human liver xanthine dehydrogenase/xanthine oxidase (XD/XO). The gene encoding the Denver cDNA was subsequently linked to juvenile familial amyotrophic lateral sclerosis (JFALS) at chromosome 2q33 and has been proposed as the ALS2 locus. The present investigation was undertaken to elucidate the differences between the three cDNA sequences, and we provide evidence that the Denver cDNA encodes aldehyde oxidase (AO): first, the Denver cDNA sequence diverged significantly from the Tokyo and Salt Lake City cDNA sequences which were very similar; second, the deduced protein sequence from the Denver cDNA was very similar to the amino acid sequence of purified rabbit liver AO protein; third, the deduced Denver protein sequence was 76% identical to the encoded 101 amino acid long peptides from partial cDNAs for rabbit and rat AO and 81.7% identical to 300 amino acids from an incomplete cDNA encoding bovine AO; fourth, the Denver gene was expressed in liver, kidney, lung, pancreas, prostate, testes, and ovary while the Tokyo XD gene was expressed predominantly in liver and small intestine; fifth, the Denver gene was previously mapped to chromosome 2q33 which is syntenic to the mouse AO locus on chromosome 1. Our results have revealed dramatic similarities in protein and DNA sequence in the human molybdenum hydroxylases, have uncovered unanticipated complexity in the human molybdenum hydroxylase genes, and advance the potential for AO derived oxygen radicals in JFALS and other human diseases.