The human cytochrome b5 gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively

Using very high stringency hybridization conditions for the Southern blot hybridization analysis of hamster-human cell hybrid DNA, we were able to map the human cytochrome b₅ gene and two of its pseudogenes (psgb₅1 and psgb₅2) unambiguously to chromosomes 18, 14, and 20. These localizations were confirmed and extended to 18q23, 14q31-32.1, and 20p11.2 by using a combination of nonisotopic in situ hybridization of chromosomal spreads and the polymerase chain reaction analysis of DNA samples isolated from somatic cell hybrids retaining deletions or translocations of chromosome 18.     

Isolation of DNA fragments from a human chromosomal subregion by Alu PCR differential hybridization

The recent advent of Alu element-mediated PCR (Alu PCR) allows the rapid isolation of human-specific fragments from mixed DNA sources. This technique greatly facilitates the isolation of DNA fragments from specific regions of the human genome. We report a novel technique utilizing Alu PCR products as differential hybridization probes to isolate human DNA fragments from a chromosomal subregion. We used the Alu PCR products from a pair of somatic cell hybrids in which the human DNA content differs only in the 5q11.2-q13.3 region as differential hybridization probes. One hybrid (GM10114) retains an intact chromosome 5, while the other (HHW1064) contains a chromosome 5 deleted for the q11.2-q13.3 region. Phage from a flow-sorted chromosome 5 library were hybridized with the Alu PCR synthesis product from the chromosome 5 hybrid. Positively hybridizing phage were then screened with the Alu PCR product from the deletion 5 hybrid. Phage that hybridized to the Alu PCR product of the chromosome 5 hybrid but did not hybridize to the Alu PCR product of the deletion 5 hybrid were further characterized. We isolated five phage from 5q11.2-q13.3 using this differential hybridization procedure. Only one of these phage corresponded to a detectable difference between the ethidium bromide-stained Alu PCR products of the two somatic cell hybrids. This technique should be applicable to any somatic cell hybrid-deletion hybrid pair.     

Mapping of the versican proteoglycan gene (CSPG2) to the long arm of human chromosome 5 (5q12-5q14).

Versican is a major chondroitin sulfate proteoglycan of vascularized connective tissues whose eponym reflects its functional versatility in macromolecular affinity and interactions. In this report we have localized the versican gene (CSPG2) to the long arm of human chromosome 5 by utilizing a combination of somatic cell hybrids, Southern blotting, polymerase chain reaction, and chromosomal in situ hybridization. The proteoglycan gene segregated concordantly with hybrid cell lines containing the long arm of chromosome 5, comprising the 5q12-q14 band regions. To refine this locus further, we screened a chromosome 5-specific library and isolated several genomic clones encoding a portion of the 5' end of versican. One of these genomic clones was used as a probe for in situ hybridization of human chromosome metaphases. The results corroborated the data obtained using somatic cell hybrids and further refined the assignment of the versican gene to the narrow band region of 5q12-5q14, with the primary site likely to be 5q13.2. The availability of novel genomic clones and the mapping data presented here will make possible the identification of any defect genetically linked to this proteoglycan gene.     

Isolation of 1001 new markers from human chromosome 11, excluding the region of 11p13-p15.5, and their sublocalization by a new series of radiation-reduced somatic cell hybrids.

The determination of the physical map of human chromosome 11 will require more clones than are currently available. We have isolated an additional 1001 new markers in a bacteriophage vector from a somatic cell hybrid cell line that contains most of chromosome 11, except the middle of the short arm. These markers were localized to five different regions, 11p15-pter, 11p12-cen, 11q11-q14, 11q14-q23, and 11q23-qter, by a panel of previously characterized somatic cell hybrids. The region 11q11-14 harbors genes that have been shown to be important in breast cancer, B-cell lymphomas, centrocytic lymphomas, asthma, and multiple endocrine neoplasia, type 1 (MEN1). To determine the positions of the recombinant clones located there, we developed a new series of radiation-reduced somatic cell hybrids. These hybrids, together with those previously characterized, allowed us to map the 11q11-q14 markers into 11 separate segregation groups.     

Assignment of a serotonin 5HT-2 receptor gene (HTR2) to human chromosome 13q14-q21 and mouse chromosome 14

A gene for serotonin 5HT-2 receptor (HTR2) is assigned to human chromosome 13 by somatic cell hybrids and to region 13q14-q21 by in situ hybridization. It is assigned to mouse chromosome 14 by somatic cell hybrid analysis.     

Regional assignment of seven genes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. II. Results obtained after induction of breaks in chromosome 1 by X-irradiation.

The position of genes coding for PGD, PPH1, UGPP, GuK1, PGM1, Pep-C, and FH on human chromosome 1 was investigated by analysis of karyotype and enzyme phenotypes in man-Chinese hamster somatic cell hybrids carrying aberrations involving chromosome 1. Suitable hybrid cell lines were obtained by X-irradiation of hybrid cells carrying an intact chromosome 1 and by fusion of human cells from a clonal population carrying a translocation involving chromosome 1 with Chinese hamster cells. The latter human cell population had been isolated following X-irradiation of primary Lesch-Nyhan fibroblasts. In addition, products of de novo chromosome breakage in the investigated hybrid lines were utilized. By integrating the results of these analyses with earlier findings in our laboratory, the following positions of genes are deduced: PGD and PPH1 in 1p36 leads to 1p34; PGM1 in 1p32; UGPP in 1q21 leads to 1q23; GuK1 in 1q31 leads to 1q42; Pep-C in 1q42; and FH in 1qter leads to 1q42.     

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.     

Physical localization of chromosome 20 markers using somatic cell hybrid cell lines and fluorescence in situ hybridization.

A panel of somatic cell hybrid cell lines containing different parts of human chromosome 20 and fluorescence in situ hybridization have been used to physically localize markers to human chromosome 20. Through these complementary approaches and genetic linkage analysis, D20S16, which is closely linked to the maturity onset diabetes of the young (MODY) locus, was mapped to band 20q12 --> q13.1. The gene for growth hormone-releasing factor (GHRF) was physically mapped and reassigned to 20q11, suggesting that GHRF plays no direct role in MODY. In addition, the genes for the chromosome 20-linked glycogen phosphorylase (GYPB) and the bone morphogenetic protein (BMP2A) have been assigned to chromosome 20p, and the interleukin-6-dependent DNA-binding protein (TCF5) has been assigned to 20q12 --> q13 by hybridization to genomic DNA from the panel of somatic cell hybrid cell lines. These approaches are useful for rapid localization of candidate genes for MODY and other DNA markers mapped to chromosome 20.     

Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

Regional assignment of 41 human DNA fragments on chromosome 7 by means of a somatic cell hybrid panel

Summary To detect new restriction fragment length polymorphisms that would cover human chromosome 7 with a network of genetic landmarks, a chromosome 7-specific phage gene library was screened for human single-copy fragments. With use of a somatic cell hybrid panel containing defined regions of human chromosome 7, 41 cloned human single-copy sequences were assigned to five regions of this chromosome. Of special importance are the cell hybrid clones GM1059Rag5 and 7851Rag10-1, derived from human cells with interstitial deletions spanning the bands 7q22-q32, within which the cystic fibrosis gene is located. Twelve new probes are described in 7q22-q32, five of which detect a total of six RFLPs.     

Two human genes encoding zinc finger proteins,ZNF12 (KOX 3) and ZNF 26 (KOX 20), map to chromosomes 7p22-p21 and 12q24.33, respectively

Summary Two members of the human zinc finger Krüppel family, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization. The presence of individual human zinc finger genes in mouse-human hybrid DNAs was correlated with the presence of specific human chromosomes or regions of chromosomes in the corresponding cell hybrids. Analysis of such mouse-human hybrid DNAs allowed the assignment of the ZNF 12 (KOX 3) gene to chromosome region 7p. The ZNF 26 (KOX 20) gene segregated with chromosome region 12q13-qter. The zinc finger genes ZNF 12 (KOX 3) and ZNF 26 (KOX 20) were localized by in situ chromosomal hybridization to human chromosome regions 7p22-21 and 12q24.33, respectively. These genes and the previously mapped ZNF 24 (KOX 17) and ZNF 29 (KOX 26) genes, are found near fragile sites.     

The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase expressed in immature lymphocytes of the thymus and bone marrow, as well as certain leukemic cells. Chromosomal assignment of the gene coding for human TdT was accomplished by in situ hybridization of a 3H-labeled cDNA probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs. The human TdT gene was mapped to the region q23----q24 of chromosome 10. Breaks at this site have been reported in different translocations in human leukemias. The mouse TdT gene was assigned to chromosome 19 by Southern blot analysis of mouse X Chinese hamster somatic cell hybrids. This result adds a fourth locus to the conserved syntenic group on mouse chromosome 19 and human chromosome 10.     

Analysis of chromosomal aberrations involving chromosome 1q31-->q53 in a DMBA-induced rat fibrosarcoma cell line: amplification and overexpression of Jak2

In a study of DMBA-induced rat fibrosarcomas we repeatedly found deletions and/or amplifications in the long arm of rat chromosome 1 (RNO1). Comparative genome hybridization showed that there was amplification involving RNO1q31-->q53 in one of the DMBA-induced rat fibrosarcoma tumors (LB31) and a cell culture derived from it. To identify the amplified genes we physically mapped rat genes implicated in cancer and analyzed them for signs of amplification. The genes were selected based on their locations in comparative maps between rat and man. The rat proto-oncogenes Ccnd1, Fgf4, and Fgf3 (HSA11q13.3), were mapped to RNO1q43 by fluorescence in situ hybridization (FISH). The Ems1 gene was mapped by radiation hybrid (RH) mapping to the same rat chromosome region and shown to be situated centromeric to Ccnd1 and Fgf4. In addition, the proto-oncogenes Hras (HSA11p15.5) and Igf1r (HSA15q25-->q26) were mapped to RNO1q43 and RNO1q32 by FISH and Omp (HSA11q13.5) was assigned to RNO1q34. PCR probes for the above genes together with PCR probes for the previously mapped rat genes Bax (RNO1q31) and Jak2 (RNO1q51-->q53) were analyzed for signs of amplification by Southern blot hybridization. Low copy number increases of the Omp and Jak2 genes were detected in the LB31 cell culture. Dual color FISH analysis of tumor cells confirmed that chromosome regions containing Omp and Jak2 were amplified and were situated in long marker chromosomes showing an aberrant banding pattern. The configuration of the signals in the marker chromosomes suggested that they had arisen by a break-fusion-bridge (BFB) mechanism.     

The human chromosome content in human x rodent somatic cell hybrids analyzed by a screening technique using Alu PCR

The ubiquitous nature of the Alu sequence throughout the human genome forms the basis of an assay we present here for analyzing the human chromosome content of human x rodent somatic cell hybrids. A human-specific Alu primer was used both to amplify sequences and to 32P label the products in a polymerase chain reaction (PCR) technique. Unlabeled inter-Alu PCR products from two series of human x rodent hybrids were used to prepare dot blots which were probed with labeled inter-Alu products prepared from between 10(3) and 10(4) hybrid cells. In the first series we demonstrate that a labeled inter-Alu probe from the hybrid DL18ts, containing a single chromosome 18, on a dot blot hybridized only with those inter-Alu products containing chromosome 18. Similar specificity for human chromosome 5 was shown when a Southern blot of the PCR products was hybridized with a probe made from the hybrid HHW 213, which contains only chromosome 5p. Using a dot blot from a second series of control hybrids, 15 of which contained single human chromosomes, hybridization of a labeled probe from the hybrid 18X4-1 was shown to react specifically with the controls that expressed chromosome 18. Application of the technique reported here allows simple and rapid characterization of the human chromosome content in human x rodent hybrids.     

The thyroglobulin gene is syntenic with the MYC and MOS protooncogenes and carbonic anhydrase II and maps to chromosome 14 in cattle

Using a panel of bovine x Chinese hamster hybrid somatic cells, sequences homologous to genes spanning human chromosome arm 8q have been syntenically assigned in cattle. Thyroglobulin (TG), carbonic anhydrase II (CA2), and the protooncogenes MYC and MOS were assigned to a newly identified bovine syntenic group, U23. Additionally, in situ hybridization of the thyroglobulin probe to bovine metaphase chromosomes revealed this syntenic group to be on bovine chromosome 14 and the bovine thyroglobulin gene to reside at 14q12----q15.     

Chromosome localization of human ARH genes, a ras-related gene family

The human ARH genes (previously called RHO) share several properties with the ras gene family. Three members of the ARH family, the H6, H9, and H12 genes, have been localized to human chromosomes 2, 5, and 3, respectively. Analysis of DNAs from a rodent-human somatic cell hybrid panel demonstrates linkage of H6 to chromosome region 2p12----2pter and H9 to region 5q33----5qter. In situ chromosome hybridization also showed that the primary site for H9 is in the 5q31----qter region. The H12 gene was some-what difficult to localize using rodent-human hybrids because the probe detects a family of rodent genes as homologous to the human probe as in the human cognate gene. However, chromosome in situ hybridization revealed grains clustered in region 3p14----3p22 with a significant peak in band 3p21. We conclude that H6 is in 2p12----pter, H9 in 5q31----5qter, and H12 in 3p21.     

Regional localization of the human platelet-derived endothelial cell growth factor (ECGF1) gene to chromosome 22q13.

Using in situ hybridization and a panel of human X rodent somatic cell hybrids, which discriminates between four different regions of human chromosome 22, we have localized the gene for human platelet-derived endothelial cell growth factor (ECGF1) to 22q13, placing ECGF1 distal to the PDGFB locus at 22q12.3----q13.1.     

Localization of the interferon-alpha gene cluster to rat chromosome bands 5q31----q33 by fluorescence in situ hybridization.

The chromosomal location of the rat interferon-alpha (IFNA) gene cluster was determined by fluorescence in situ hybridization. The fluorescent signals were localized to 5q31----q33. A previous report, using somatic cell hybrids, had suggested that the IFNA locus maps at a different region on rat chromosome 5.     

Molecular analysis of a t(7;14)(q35;q32) chromosome translocation in a T cell leukemia of a patient with ataxia telangiectasia

Molecular analysis of somatic cell hybrids derived from T cells carrying a t(7;14)(q35;q32) chromosomal translocation from a patient with ataxia telangiectasia and T cell leukemia indicates that the breakpoint on chromosome 14 is proximal to the IgH locus and to the D14S1 locus, while the breakpoint on chromosome 7 involves the T cell receptor beta chain locus immediately 5' to J beta 1.5 on chromosome 7. The separation of V beta and C beta observed in somatic cell hybrids defined the orientation of the T cell receptor beta chain locus on chromosome 7 where the V beta genes are centromeric and the C beta genes are telomeric. A novel chromosomal alteration, undetected cytogenetically, was revealed as being an inversion with duplication of the distal band of chromosome 14q32. The importance of the 14q32 region in the leukemogenic process is discussed.