Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 byin situ hybridization with biotinylated DNA probe

We have made a regional assignment of the type II collagen gene (COL2A1) on human chromosome 12 by means of anin situ hybridization technique with a biotinylated DNA probe. The precise localization of the signal was mapped to the band 12q13.1-q13.2. This result was in agreement with the previous mapping by isotopicin situ hybridization technique (12q13.1-q13.2), but not with the result of Southern hybridization analysis using somatic cell hybrids (12q14.3).     

Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 by in situ hybridization with biotinylated DNA probe

We have made a regional assignment of the type II collagen gene (COL2A1) on human chromosome 12 by means of an in situ hybridization technique with a biotinylated DNA probe. The precise localization of the signal was mapped to the band 12q13.1-q13.2. This result was in agreement with the previous mapping by isotopic in situ hybridization technique (12q13.1-q13.2), but not with the result of Southern hybridization analysis using somatic cell hybrids (12q14.3).     

An extended panel of hamster-human hybrids for chromosome 2q

A hamster-human hybrid containing only the q arm of chromosome 2 has been used to construct a panel of hybrids bearing reduced regions of chromosome 2 using the technique of irradiation fusion gene transfer. The human chromosome 2 carried theEcogpt gene and all hybrids were selected using this marker. The integratedEcogpt gene was localized to the region 2q33-34, resulting in the selective retention of this region in the hybrids. These data were combined with another previously constructed panel of hybrids containing regions of 2q, which were enriched for the region 2q36-37. The combined hybrid panel is useful for the mapping of new markers to defined regions of chromosome 2 and for the cloning of genes located on 2q by a positional strategy.     

Confirmation that the Type I collagen gene on chromosome 17 is COL1A1 (α1(I)), using a human genomic probe

A cloned 15 kb genomic fragment from the human α1 (I) collagen gene (COL1A1) has been used as a probe on restriction digests of DNA from human-mouse somatic cell hybrids. Positive results on hybrids containing chromosome 17 as their only karyotypically visible human material confirm the assignment of this gene to chromosome 17. Hybrids which contain fragments of chromosome 17 are used to confirm the localization to 17q21-qter.     

Phenotypic correction of ataxia-telangiectasia cellular defect by exogenously introduced human or mouse subchromosomal fragments

A human-mouse hybrid containing a human 11q22–23 fragment including theATM locus was used to examine its capability to correct the cellular defect of ataxia-telangiectasia (A-T). Examination of 21 A-T-derived hybrids indicated that the acquired radioresistance was observed in the clones where the 11q22–23 fragment was transferred intact, but not in those where donorderived 11q segment was lost. In one exceptional clone, theATM locus was deleted from the transferred fragment, while it was still partially radioresistant. This partially radioresistant clone was found to include the mouse-derived fragment containing theAtm gene, the mouse homologue of humanATM gene. Similar association of partial radioresistance with the presence of mouseAtm gene was observed in three additional hybrids. The results indicate that the cellular A-T defect can be corrected by the mouse subchromosomal fragment containing theAtm gene as well as by the human 11q22–23 fragment containing theATM gene, but apparently to a lesser extent in the former.     

A hamster-human subchromosomal hybrid cell panel for chromosome 2

We have constructed hamster-human hybrid cell lines containing fragments of human chromosome 2 as their only source of human DNA. Microcell-mediated chromosome transfer was used to transfer human chromosome 2 from a monochromosomal mouse-human hybrid line to a radiation-sensitive hamster mutant (XR-V15B) defective in double-strand break rejoining. The human chromosome 2 carried theEcogpt gene and hybrids were selected using this marker. The transferred human chromosome was frequently broken, and the resulting microcell hybrids contained different sized segments of the q arm of chromosome 2. Two microcell hybrids were irradiated and fused to XR-V15B to generate additional hybrids bearing reduced amounts of human DNA. All hybrids were analyzed by PCR using primers specific for 27 human genes located on chromosome 2. From these data we have localized the integratedgpt gene on the human chromosome 2 to the region q36–37 and present a gene order for chromosome 2 markers.     

Subchromosomal localization of a gene (XRCC5) involved in double strand break repair to the region 2q34-36

We have previously shown that human chromosome 2 can complement both the radiation sensitivity and the defect in double strand break rejoining characteristic of ionizing radiation (IR) group 5 mutants. A number of human-hamster hybrids containing segments of human chromosome 2 were obtained by microcell transfer into two group 5 mutants. In most, but not all, of these hybrids, the repair defect was complemented by the human chromosomal DNA. Two complementing microcell hybrids were irradiated and fused to XR-V15B, an IR group 5 mutant, to generate further hybrids bearing smaller regions of chromosome 2. All hybrids were examined for complementation of the repair defect. The region of chromosome 2 present was determined using PCR with primers specific for various human genes located on chromosome 2. A complementing hybrid bearing only a small region of chromosome 2 was finally generated. From this analysis we deduced that theXRCC5 gene was tightly linked to the marker,TNP1, which is located in the region 2q35.     

Assignment of human dihydrofolate reductase gene to band q23 of chromosome 5 and of related pseudogene ΨHD1 to chromosome 3

The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a number of human-Chinese hamster ovary cell hybrids containing terminal and interstitial deletions in human chromosome 5. A correlation of genomic blotting data with the chromosome 5 constitution of the individual hybrids has allowed the assignment of the human DHFRgene to 5q23. The present work also establishes the location of the related intronless pseudogene HD1 in chromosome 3.     

Regional localization of the gene coding for sphingolipid activator protein SAP-1 on human chromosome 10

Sphingolipid activator protein SAP-1 is required for the enzymatic hydrolysis of GMI ganglioside and sulfatide. The gene coding for SAP-1 was previously mapped to human chromosome 10 using monospecific antibodies prepared against SAP-1 in synteny analysis of somatic cell hybrids. In this study, we used a cDNA probe for SAP-1 and in situ hybridization to regionally localize theSAP1 gene to the long arm of chromosone 10, region q21–22. Additional mapping data using cell hybrids containing partial chromosome 10 and skin fibroblasts with trisomy 10p are consistent with the in situ hybridization mapping results.     

Metastasis suppressor gene(s) for rat prostate cancer on the long arm of human chromosome 7

Allelotype analyses of human prostate cancer indicate that allelic losses on human chromosome arms 7q, 8p, 10q, 13q, 16q, 17q, and 18q are observed frequently. For the study of the possible biological significance of the frequently observed deletions on chromosome arm 7q in human prostate cancer, human chromosome 7 was introduced into highly metastatic rat prostate cancer cells by use of a microcell‐mediated chromosome transfer technique. The introduction of human chromosome 7 resulted in the suppression of metastatic ability of the microcell hybrids, whereas no suppression of tumorigenicity was observed. To identify the portion of chromosome 7 containing the metastasis‐suppressive function gene, the derivative chromosome 7 that was generated with the initial transfer was retransferred into rat prostate cancer cells. Human chromosome 7‐containing rat prostate cancer cells could be used as the donor cells, because rodent cells produced a sufficient number of microcells with colchicine treatment. Cytogenetic and molecular analyses of these clones demonstrated that loss of segments on 7q was related to the reexpression of the metastatic phenotype. These results show that human 7q contains a metastasis suppressor gene or genes for rat prostate cancer. The findings also suggest that this gene may play an important role in the progression of human prostate cancer. Genes Chromosomes Cancer 24:1–8, 1999. © 1999 Wiley‐Liss, Inc.     

Irradiation-reduced human chromosome 21 hybrids

Rodent-human somatic cell hybrids have been constructed which contain fragments of human chromosome 21 as their only human material. This was done by irradiating rodent-human somatic cell hybrids containing a complete chromosome 21 to fragment the genome and then rescuing human GAR synthetase and various amounts of flanking chromosome 21 DNA by fusing with GAR synthetase-deficient hamster cells and selecting for growth in purine-free medium. Four irradiation-reduction hybrids were produced by this method and contain the distal, proximal, and central portions of the long arm of human chromosome 21, all centered about GAR synthetase. These irradiation-reduction hybrids were used as a panel to regionally map single-copy and individual copies of repetitive sequences. Using these hybrids along with another independently constructed hybrid, the GAR synthetase gene was mapped distal to SOD-1 and proximal to CP21G1(D21S60). Of special interest is the regional mapping of the gene for the amyloid -protein distal to pPW236B(D21S11) and proximal to SOD-1.     

Two cell-surface markers for human chromosome 1 in interspecies hybrids

Monoclonal antibodies AbAJ9 and AbT87 define two distinct human cell-surface antigen systems (1, 2). Both antibodies react with a wide variety of cultured human cell types but not with rodent cell lines. AbAJ9 identifies a glycoprotein of 140,000 mol wt and AbT87 identifies a glycoprotein of 60,000 mol wt. The genetic control of antigen expression was studied in rodent-human somatic cell hybrids containing different subsets of the entire human chromosome complement, using an immune rosetting assay for serological analysis and hybrid selection. The presence of human chromosome 1 was found to be both necessary and sufficient for expression of antigens AJ9 and T87 in hybrid cells. Two independent gene loci on chromosome 1, designated MSK-1 for AJ9 and MSK-2 for T87, control the expression of these antigens. We have assigned MSK-1 to region 1 cen-p22 and MSK-2 to region 1 q32-qter, using hybrids containing only fragments of human chromosome 1.     

The SON gene encodes a conserved DNA binding protein mapping to human chromosome 21

We report the identification and characterization of a clone for the DNA binding protein SON , which we have isolated from a human keratinocyte cDNA library. Using this clone we have found that the SON gene is expressed in different cell types and that homologous sequences can be detected in vertebrate and insect genomic DNA. Using the polymerase chain reaction (PCR) to amplify SON sequences from a panel of somatic cell hybrids we have assigned the gene encoding human SON to chromosome 21. By use of hybrids containing regions of chromosome 21 the localization has been refined to 21q 22. 1-q22.2.     

Assignment of retinoblastoma susceptibility gene to mouse chromosome 14

A human cDNA probe was used to screen a panel of mouse-Chinese hamster somatic cell hybrids to determine the chromosomal location of the retinoblastoma susceptibility gene (Rb-1)in mouse. The Rb-1gene mapped to mouse chromosome 14. Thus, the retinoblastoma susceptibility gene is syntenic with esterase 10 (the mouse homolog of human esterase D). The chromosomal assignment of the mouse Rb-1gene was further confirmed by using the same probe to study mouse-rat microcell hybrids. Since the human retinoblastoma susceptibility gene (RB1)along with the gene for esterase D is on chromosome 13q14, these data indicate this linkage group is conserved in man and mouse.     

Generation of a panel of radiation-reduced hybrids containing human 11q22-23 fragments bearing aHPRT selective marker: Identification of hybrids carrying various subregions around the ataxia-telangiectasia locus

A human-mouse monochromosomal hybrid that contains a human t(X;11) translocated chromosome carrying pter→q23 segment of chromosome 11 was used to construct a panel of radiation-reduced hybrids. The hypoxhanthine phosphoribosyltransferase (HPRT) gene located close to the translocation breakpoint was used as a marker to select for the hybrids that preferentially retain the 11q22-23 region. Twenty-three HAT-resistant hybrids were isolated and screened by polymerase chain reaction (PCR) for the retention of 31 loci on 11q22-23 region. Among the 14 hybrids that had breakpoints within the 11q22-23 region, 6 hybrids contained fragments that extend either from centromere or telomere to the 5-Mb region spanned by GRIA4 and FDX, carrying various breakpoints within the region. This subpanel could be a potential resource to analyze the ataxia-telangiectasia disease locus and its neighboring region.     

Cosmids and transcribed sequences from chromosome 11q23

Summary To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to theneo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH-(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.     

Chromosomal mapping of the human interleukin-1 receptor antagonist gene (IL-1RN) and isolation of specific YAC clones

Using a panel of somatic rodent-human cell hybrids, we show that the interleukin-1 receptor antagonist gene (IL-1RN) maps to the long arm of human chromosome 2. Linkage studies permitted the regional localization of this gene to band q14-21. This is the same region in which the IL-1α and IL-1β genes are localized. Three yeast artificial chromosome (YAC) clones containing the IL-1RN gene were isolated, and these will be used for further characterization of this chromosome 2 region.     

Proximity of thyroglobulin and c-myc genes on human chromosome 8

The human thyroglobulin structural gene (TG) was mapped to the long arm of chromosome 8 by blot hydridization of a TG cDNA probe to DNA from 21 human × mouse somatic cell hybrids containing overlapping subsets of human chromosomes. In situ hybridization of the TG probe to metaphase chromosomes from a karyotypically normal human lymphoblastoid cell line, JS, localized the TG gene to within the region 8q23 q24.3. Thus, the TG and c-myc genes map to the same chromosome band in normal human cells. In a human colon carcinoma cell line (COLO 320 DM) which contains amplified c-myc, the TG gene is not amplified and hence it lies outside the amplification domain.     

Suppression of endometrial carcinoma cell tumorigenicity by human chromosome 18

Presumptive tumor suppressor genes may be localized to specific chromosomes by the procedure of microcell fusion, whereby individual chromosomes derived from normal human cells are introduced into tumor cells. Allelic loss on chromosome I 8 is commonly seen in endometrial carcinoma, and the DCC gene on chromosome arm 18q is a potential human tumor suppressor gene. In this study, we investigated the hypothesis that a gene on chromosome 18, possibly DCC, is capable of suppressing the tumorigenicity of endometrial carcinoma cells. Microcells from the mouse A9 cell clone containing one human chromosome I8 tagged with the pSV2-neo plasmid were fused with the highly tumorigenic endometrial carcinoma cell lines HHUA and Ishikawa, and G418-resistant microcell hybrids containing an extra copy of chromosome I8 were isolated. Clones isolated from the HHUA cell line were completely suppressed for tumorigenicity in nude mice, and clones from the lshikawa line were suppressed or inhibited for tumorigenicity. In contrast, growth rates in vitro were not significantly affected in clones from either parental cell line. DCC expression was elevated in most of the suppressed hybrids. These results indicate that a gene on human chromosome I 8 is capable of suppressing the tumorigenicity of endometrial carcinoma cells, and that DCC is a candidate for this endometrial carcinoma tumor suppressor gene. © 1995 Wiley-Liss, Inc.