Chromosomal localization of three human D5 dopamine receptor genes

It is currently thought that genetic predisposition to imbalances in dopaminergic transmission may underlie several neurological disorders, including schizophrenia, manic depression, Tourette syndrome, Parkinson disease, Huntington disease, and alcohol abuse. Originally two receptors, D1 and D2, were thought to account for all of the pharmacological actions of dopamine. However, through homology screening three additional genes, D3, D4, and D5, and two pseudogenes closely related to D5 have been characterized. To begin our genomic and evolutionary analyses of the human D5 dopamine receptor gene and its two pseudogenes, we have mapped each of them to their respective chromosomes. By combining in situ hybridization results with sequence analysis of PCR products from microdissected chromosomes, somatic cell hybrids, and radiation hybrids, we have assigned DRD5 (the locus containing the functional human D5 receptor gene) to chromosome 4p16.1, DRD5P1 (the locus containing D5 pseudogene 1) to chromosome 2p11.1-p11.2, and DRD5P2 (the locus of D5 pseudogene 2) to chromosome 1q21.1.     

Regional localization of the interferon-beta 2/B-cell stimulatory factor 2/hepatocyte stimulating factor gene to human chromosome 7p15-p21

The human interferon-beta 2 gene (IFNB2) is identical to the genes encoding the B-cell stimulatory factor (BSF-2), the hybridoma growth factor (HGF), and the hepatocyte stimulating factor (HSF). This protein mediates major alterations in the secretion of a wide spectrum of plasma proteins by the liver in response to tissue injury (the acute-phase response). We have used a cDNA probe specific to the human IFNB2 gene in DNA hybridization experiments and report the regional localization of this gene to human chromosome 7p15-p21. Southern blot analyses of DNA extracted from a panel of mouse X human somatic cell hybrids localized this gene to human chromosome 7p. In situ hybridization of the IFNB2 cDNA probe to prebanded human metaphase chromosome spreads allowed the further localization of this gene to 7p15-p21.     

Regional assignment of the human acid sphingomyelinase gene (SMPD1) by PCR analysis of somatic cell hybrids and in situ hybridization to 11p15.1----p15.4

Human acid sphingomyelinase (SMPD1) is the lysosomal phosphodiesterase that cleaves sphingomyelin to ceramide and phosphocholine. The deficient activity of SMPD1 is the enzymatic defect in Types A and B Niemann-Pick disease. Previously, the gene encoding human SMPD1 was assigned to chromosome 17 by the differential thermostability of human and hamster SMPD1 in somatic cell hybrids. The recent isolation of the human SMPD1 cDNA (L. E. Quintern, E. H. Schuchman, O. Levran, M. Suchi, K. Ferlinz, H. Reinke, K. Sandhoff, and R. J. Desnick, 1989, EMBO J. 8: 2469-2473) permitted the mapping of this gene by molecular techniques. Oligonucleotide primers were synthesized to PCR amplify the human, but not murine, SMPD1 sequences in man-mouse somatic cell hybrids. In a panel of 15 hybrid cell lines, amplification of the human SMPD1 sequence was 100% concordant with the presence of human chromosome 11. For each of the other human chromosomes there were at least 6 discordant hybrid lines. Further analysis of somatic cell hybrids containing only chromosome 11 or chromosome 11 rearrangements localized the human SMPD1 gene to the region 11p15.1----p15.4. To provide an independent regional gene assignment, in situ hybridization was performed using the radiolabeled human SMPD1 cDNA. In the 58 metaphase cells examined, 34% of the 122 hybridization sites scored were located in the distal end of chromosome 11 with the major peak of hybridization at band 11p15. The absence of any other in situ hybridization site indicated the absence of pseudogenes or homologous sequences elsewhere in the genome. In contrast to the previous provisional localization to chromosome 17, these results assign a single locus for human SMPD1 to 11p15.1----p15.4.     

Mapping of human chromosome 5 microsatellite DNA polymorphisms.

Thirteen moderately to highly informative microsatellite DNA polymorphisms based on (dC-dA)n.(dG-dT)n repeats were mapped to segments of human chromosome 5 using both linkage analysis and a panel of somatic cell hybrids which contained rearranged chromosomes. The markers were distributed throughout most of the length of the chromosome from the regions p15.3-p15.1 to q33.3-qter. Maps of the sites of meiotic recombination within the reference families proved particularly useful for the purpose of integrating new polymorphisms into the existing linkage map.     

Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome

delta-Aminolevulinate synthase (ALAS) catalyzes the first committed step of heme biosynthesis. Previous studies suggested that there were erythroid and nonerythroid ALAS isozymes. We have isolated cDNAs encoding the ubiquitously expressed housekeeping ALAS isozyme and a related, but distinct, erythroid-specific isozyme. Using these different cDNAs, the human ALAS housekeeping gene (ALAS1) and the human erythroid-specific (ALAS2) gene have been localized to chromosomes 3p21 and X, respectively, by somatic cell hybrid and in situ hybridization techniques. The ALAS1 gene was concordant with chromosome 3 in all 26 human fibroblast/murine(RAG) somatic cell hybrid clones analyzed and was discordant with all other chromosomes in at least 6 of 26 clones. The regional localization of ALAS1 to 3p21 was accomplished by in situ hybridization using the 125I-labeled human ALAS1 cDNA. Of the 43 grains observed over chromosome 3, 63% were localized to the region 3p21. The gene encoding ALAS2 was assigned by examination of a DNA panel of 30 somatic cell hybrid lines hybridized with the ALAS2 cDNA. The ALAS2 gene segregated with the human X chromosome in all 30 hybrid cell lines analyzed and was discordant with all other chromosomes in at least 8 of the 30 hybrids. These results confirm the existence of two independent, but related, genes encoding human ALAS. Furthermore, the mapping of the ALAS2 gene to the X chromosome and the observed reduction in ALAS activity in X-linked sideroblastic anemia suggest that this disorder may be due to a mutation in the erythroid-specific gene.     

Construction of a NotI linking library and isolation of new markers close to the Huntington''s disease gene.

Linking clones contain sequences flanking recognition sites for enzymes cutting rarely in mammalian DNA. They can be used to obtain and correlate both physical and genetic mapping information over subregions of mammalian chromosomes. We have constructed and used a NotI linking clone library representing unmethylated NotI sites from HHW693 DNA, a hamster hybrid cell line containing 4p15-4pter and a fragment of 5p as its only human chromosome contribution. Human clones were identified by hybridisation with a cloned human repeat sequence, and localised further to subregions of human chromosome 4p15-4pter using a panel of additional hybrids. Clones from the region distal to the DNA probes (D4S10, D4S43, D4S95) linked to the Huntington's disease mutation, were further analysed. Four markers close to the HD gene: D4S111, D4S113, D4S114 and clone 417 are described here. In addition to serving as markers in physical and genetic mapping experiments, these linking clones provide probes next to cleavable NotI sites, and can therefore be used to screen NotI based chromosome jumping libraries. They also provide indications for potential gene sequences, identifiable as evolutionarily conserved sequences.     

A human D1 dopamine receptor gene is located on chromosome 5 at q35.1 and identifies an EcoRI RFLP.

Dopaminergic neurons have been shown to affect voluntary movement, hormone secretion, and emotional tone. Mediating these activities are two receptor subtypes, D1 and D2, which are biochemically and pharmacologically distinct. The D1 subtype, the most abundant form of dopamine receptor in the central nervous system, stimulates adenylate cyclase, modulates D2 receptor activity, regulates neuron growth and differentiation, and mediates several behavioral responses. Recently we reported the cloning of a human D1 dopamine receptor gene (DRD1). High-stringency hybridization of the DRD1 clone to human genomic blots suggests that DRD1 is single copy. When used to probe a Southern blot made with DNAs from a rodent-human somatic cell hybrid panel, DRD1 hybridized to a 6.5-kb EcoRI restriction fragment which was assigned to chromosome 5. Fluorescent in situ hybridization of this gene to human metaphase chromosomes refined the location of DRD1 to 5q35.1. A search for RFLPs associated with DRD1 identified a two-allele EcoRI RFLP, allowing confirmation of DRD1's localization by linkage analysis in Centre d'Etude du Polymorphisme Humain families.     

Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid

We have developed an X-irradiation:cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.     

Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280-9284) was hybridized to TaqI digests of a panel of 25 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer's disease.     

Chromosomal distribution of three members of the human natriuretic peptide receptor/guanylyl cyclase gene family

Chromosomal localization of the genes encoding three homologous human proteins, the ANPRA, ANPRB, and ANPRC cell surface receptors, was determined by polymerase chain reaction (PCR) analysis of genomic DNA from somatic cell hybrids. The ANPRA gene was assigned to 1q12----qter by intron-specific PCR. The ANPRB gene was assigned to 9p11----p22 using species-specific length variation in PCR fragments. The ANPRC gene was assigned to chromosome 5 using human-specific PCR primers identified by screening a human primer panel on parental DNA samples (shotgun primer screening). Chromosomal assignments based on PCR analysis were confirmed and the genes further sublocalized by in situ hybridization of cloned cDNA probes to human metaphase chromosomes. The ANPRA gene was sublocalized to 1q21----q22, the ANPRB gene to 9p12----p21, and the ANPRC gene to 5p13----p14.     

Localization of the human coproporphyrinogen oxidase gene to chromosome band 3q12

The human gene encoding coproporphyrinogen oxidase is the defective gene in hereditary coproporphyria. This gene was mapped to chromosome band 3q12 using fluorescent in situ hybridization. The chromosomal localization was confirmed by cosegregation of the human gene with chromosome 3 in a panel of human/rodent somatic hybrids.     

Radiation hybrid map of 13 loci on the long arm of chromosome 5.

Radiation hybrid mapping was used in conjunction with a natural deletion mapping panel to predict the order of and distance between 13 loci in the distal portion of the long arm of human chromosome 5. A panel of irradiation hybrids containing fragments of 5q was generated from an HPRT+ Chinese hamster-human cell hybrid containing a derivative chromosome 5 [der(5)t(4;5)(5qter----5p15.1::4p15.1----4pter)] as its only human DNA. One hundred nine radiation hybrids containing human DNA were screened with polymerase chain reaction primer sets representing nine genes encoding growth factors, growth factor receptors, or hormone receptors (IL3, IL4, IL5, CSF1R, FGFA, ADRB2, GRL, GABRA1, and DRD1) as well as four other loci (FER, SPARC, RPS14, and CD14) to generate a radiation hybrid map of the area 5q21-q35. A physical map predicting the order of and distance between the 13 loci was constructed based on segregation of the 13 loci in hybrid clones. The radiation hybrid panel will be useful as a mapping tool for determining the location and order of other genes and polymorphic loci in this region as well as for generating new DNA probes from specific regions.     

Regional localization of DNA probes on the short arm of chromosome 11 using aniridia-Wilms' tumor-associated deletions

Summary We are interested in the precise localization of various DNA probes on the short arm of chromosome 11 for our research on the aniridia-Wilms' tumor association (AWTA), assigned to region 11p13 (Knudson and Strong 1972; Riccardi et al. 1978). For this purpose we have screened lymphocyte DNA and material derived from somatic cell hybrids from individuals with constitutional 11p deletions with a range of available probes: D11S12; calcitonin/CGRP (CALC1/CALC2); insulin (INS); Harvey ras 1 (HRAS 1); beta-globin gene cluster (HBBC); human insulin-like growth factor 2 (IGF-2); parathyroid hormone (PTH); human pepsinogen A (PGA). Using this material, it has been possible to map all probes used, except insulin, outside the region 11p111-p15.1, resulting in an SRO (same regional overlap) of 11p15.1-p15.5 for most probes. We found an SRO for PGA of 11p111-q12 and an SRO for CALC2 of 11p15.1-p15.5 or 11p111-q12. We have localised the insulin gene to band 11p15.1.     

The hemopexin gene maps to the same location as the beta-globin gene cluster on human chromosome 11

Using human hemopexin cDNA clones isolated from lambda gt11 cDNA library as probes, we have carried out Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the hemopexin gene and human chromosome 11. In situ hybridization of 3H-labeled hemopexin cDNA to metaphase chromosomes prepared from human lymphocytes further localized the gene to the region p15.4-p15.5, the same location as the beta-globin gene cluster.     

Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23.

The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism. To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM). The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein. The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand. We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes. High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.     

The gene for human complement component C9 mapped to chromosome 5 by polymerase chain reaction

The gene for human complement component C9 has been mapped to chromosome 5. This was achieved by using a novel application of the polymerase chain reaction to amplify specifically the human C9 gene on a background of rodent DNA in somatic cell hybrids. The assignment to chromosome 5 was confirmed by in situ hybridization to human metaphase chromosomes, giving a regional localization of 5p13.     

Six genes of the human connexin gene family coding for gap junctional proteins are assigned to four different human chromosomes

Connexin genes code for proteins that form cell-to-cell channels known as gap junctions. The genes for the known connexins 26, 32, 43, and 46 have been assigned to human chromosomes, 13, X, 6, and 13, respectively, by analysis of a panel of human-mouse somatic cell hybrids using rat cDNA probes. A pseudogene of connexin 43 that lacks an intron of the cx43 gene has been located on human chromosome 5. Furthermore, the genes of the two new connexins 37 and 40 have both been assigned to human chromosome 1. Thus the human chromosomes 1 and 13 each carry at least two different connexin genes. Their exact location on these chromosomes is not yet known. From our results subchromosomal assignments can be deduced for the human cx32 gene to Xq13-p11, the human cx37 gene as well as the human cx40 gene to 1pter-q12, and the human cx43 gene to 6q14-qter. The generation of the connexin multigene family from a hypothetical ancestral connexin gene is discussed.     

Assignment of human aldolase C gene to chromosome 17, region cen→q21.1

Summary The mapping of the gene coding for human aldolase C has been studied using a specific cDNA probe and genomic blots from a panel of human-hamster somatic cell hybrids. The results show that the aldolase C gene is on chromosome 17. In situ experiments have restricted the mapping to the region 17cenq21.1. Using the same panel of human-hamster somatic cell hybrids, we have confirmed the localization of aldolase A and B on chromosomes 16 and 9, respectively.     

Chromosomal localization of the type-I 15-PGDH gene to 4q34–q35

The gene encoding the human NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase, designated type-I 15-PGDH, was mapped to chromosome 4 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This gene was further localized to bands 4q34–q35 by in situ hybridization on human chromosomes. Received: 7 October 1996