Assignment of the human ARNO3 gene (PSCD3) to chromosome 7p21 by radiation hybrid mapping

The chromosomal localization of the human ARNO3 gene ( PSCD3 ) was determined using monochromosomal somatic cell hybrid and radiation hybrid mapping panel. PCR analysis of a hybrid DNA panel localized the ARNO3 gene to human chromosome 7. The analysis of the Genebridge4 radiation hybrid panel using PCR amplification located the ARNO3 gene between NIB1822 (9.5 cR) and D7S481 (5.5 cR) with a lod score of >3.0. This region is located in the human chromosome band 7p21.     

Isolation and mapping of three new polymorphic markers to chromosomes 3, 20 and 21

Screening of single human chromosome plasmid libraries using a digoxygenin labelled (AAAT)₁₅ oligonucleotide probe led to the identification of several positive clones. DNA sequence analysis of these was carried out and showed the presence of a number of simple DNA repeats. Oligonucleotide primers were designed from the sequences flanking these repeats and tested in PCR amplification reactions of human genomic DNA. Three of the markers tested were shown to be polymorphic with heterozygosities ranging from 40% to 69%. The markers were assigned to chromosomes using a panel of monochromosomal somatic cell hybrids combined with linkage analysis using DNA from the CEPH panel of families. The markers designated (AAAT)₁₁, (AAAT)₁₂ and (CA)₁₉ were thus assigned to chromosomes 3, 21 and 20 respectively.     

Primed in situ (PRINS) Labelling with Alu and Satellite Primers for Rapid Characterization of Human Chromosomes in Hybrid Cell Lines

The primed in situ (PRINS) labelling method was developed as an alternative to classical cytogenetics and fluorescence in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed by PRINS using Alu-specific primers to generate the painting of all human material associated with R-like banding. The representativity of individual human chromosomes was established using primers specific for discriminent α-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Using this methodology, a somatic hybrid clone was shown to be monochromosomal for the der(11) from a t(11;22) patient.     

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.     

A novel human gene whose product shares significant homology with the bovine brain-specific protein p25 on chromosome 5p15.3

Here, we report on the sequence features and chromosomal location of a novel human gene which shares significant homology with the bovine brain-specific protein p25. Based on polymerase chain reaction analysis with a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped on to p15.3 region of chromosome 5. Received: August 27, 1998 / Accepted October 21, 1998     

Regional mapping of RBP4 to 10q23→q24 and RBP1 to 3q21→q22 in man

The human gene coding for RBP4 has been assigned to 10q2324 using a panel of somatic cell hybrids and in situ hybridization experiments. The mapping of the human RBP1, previously assigned by our group to chromosome 3 using a panel of somatic cell hybrids, was restricted to the region 3q2122 using in situ experiments and Southern blots of genomic DNA from a hybrid retaining a portion of chromosome 3.R.F. is recipient of a Research Grant from A.I.R.C.     

New chromosomal mapping assignments for argininosuccinate synthetase pseudogene 1, interferon-β3 gene,and the diazepam binding inhibitor gene

Argininosuccinate synthetase pseudogene 1 (ASSP1), interferon-₃ (IFNB3) gene, and diazepam binding inhibitor (DBI) gene have previously been mapped to human chromosome 2. Their nucleotide sequences, recorded in the GENBANK data base, were used to generate DNA primers to amplify specific sequences using the polymerase chain reaction (PCR). These primers failed to amplify DNA sequences when used to analyze microcell hybrid clones containing human chromosome 2. In order to map these genes, a panel of somatic cell hybrids was analyzed by PCR with these primer sets. The results of these experiments place ASSP1 sequences on human chromosome 6, IFNB3 on human chromosome 8, and DBI on human chromosome 6.     

The assignment of the genes coding for human complement components C6 and C7 to chromosome 5

A panel of 19 somatic cell hybrids was tested for the presence of human sequences coding for complement components C6 and C7 by restriction enzyme digestion and Southern blots probed with human C6 and C7 cDNA probes. C7 was also detected by amplifying part of the human gene in hybrid DNA using the polymerase chain reaction. Detection of human C6 and C7 was completely correlated with the presence of chromosome 5.     

Rapid characterization of human chromosomes in hybrid cell lines by primed in situ (PRINS) labeling

The primed in situ (PRINS) labeling technique allows a rapid and specific labeling of human chromosomes in situ. This method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. We have developed a PRINS protocole for the cytogenetic analysis of somatic hybrid cell lines. Painting of human chromosomes is performed using Alu specific primers. Individual human chromosomes are identified using chromosome-specific alpha-satellite primers. The method was successfully tested to 3 different human-hamster hybrid cell lines. This approach provides an interesting alternative to classical cytogenetic and in situ hybridization techniques for the characterization of the human content of hybrid cell lines.     

Polymerase chain reactions with alphoid-repeat primers in combination with Alu or LINEs primers, generate chromosome-specific DNA fragments

Y alphoid primers in combination with Alu and LINEs primers generated new DNA fragments in polymerase chain reactions (PCR) on DNA from a Y-only somatic cell hybrid but not from X-only, 3-only, or 21-only hybrids. X alphoid primers used in a similar manner generated new DNA fragments from the X-only hybrid, and 1 of the primers (X₂) also generated new DNA fragments on 3-only and 21-only hybrids when used in conjunction with Alu or LINEs primers. In all but one case, consensus alphoid primers generated new chromosome-specific fragments in PCR reactions with the Alu or LINEs primers. A search for cryptic Alu- or alphoid-alone PCR products as the source for one Alu -alphoid band (chosen at random) was negative. Partial sequencing of products demonstrated that alphoid and Alu sequences were indeed contiguous in some newly synthesized DNA fragments. While Alu or LINEs primers generate smears of DNA fragments on total human DNA, the alphoid-nonalphoid repeat combinations generated electrophoretically distinguishable bands of DNA when the template was total DNA. While these were distinguishable with different chromosome-specific alphoid primers, the DNA fragments were not of the same sizes as those generated with the chromosome-only hybrids.     

Production of Chinese hamster monoclonal antibody to a human cell-surface antigen using a hamster-human somatic cell hybrid as antigen

Hybridomas producing monoclonal antibody (MAb) of predefined specificity were isolated from a Chinese hamster that had received injections of a Chinese hamster-human somatic cell hybrid. A standard method for the production of murine hybridomas was used to produce Chinese spleen lymphocyte X murine plasmacytoma hybridomas, and they were screened for complement-mediated cytotoxicity against a panel of Chinese hamster-human somatic cell hybrids and agglutination of human erythrocytes. Two hybridomas were established in tissue culture following limiting dilution cloning, and their reactivity with a panel of Chinese hamster-human somatic cell hybrids indicates that they are specific for the previously identified human cell-surface antigen a1. The Chinese hamster appears to respond preferentially to human antigens of Chinese hamster-human somatic cell hybrids, and it will serve as a useful tool for the production of MAb specific for human cell-surface antigens expressed in the many well-characterized Chinese hamster-human somatic cell hybrids available.     

Construction and characterization of radiation hybrids for chromosome 9, and their use in mapping cosmid probes on the chromosome

Radiation hybrids were produced from a monochromosomal microcell hybrid (PK87-9) which contains only human chromosome 9 with an inserted marker on 9p. Doses of radiation ranging from 1000 to 8000 rads were used to produce a series of hybrids with different size fragments of human chromosome 9. The inserted dominant selectable marker was used to select for hybrids that preferentially maintain fragments of 9p. A panel of 53 radiation hybrids were characterized for 17 chromosome 9 markers. In addition, 17 hybrids were analyzed by fluorescent in situ hybridization (FISH). Hybrids were produced with breaks on both 9p and 9q, many of which appear to contain a single fragment of human chromosome 9. These hybrid cell lines were used to regionally localize 31 cosmids isolated from a chromosome 9 cosmid library. Six cosmids were mapped to intervals on 9p, six cosmids mapped to the centromeric region of the chromosome, and 19 mapped to 9q.     

Regional assignment of human amylase (AMY) to p22 → p21 of chromosome 1

A human genomic DNA segment (hal) which hybridizes with rat pancreatic amylase cDNA was used to regionally assign amylase genes in human-mouse somatic cell hybrids. The assignment of amylase genes to chromosome 1 was confirmed and regionally mapped to the short arm region p22.1p21 using a cell hybrid retaining a translocation involving chromosome 1 [46,XX,t(1;2)(p21;q37)]. Restriction length polymorphisms at the amylase loci are reported for gene linkage studies.     

The human dendritic cell marker CD83 maps to chromosome 6p23

The chromosomal localization of the human CD83 gene was determined using somatic cell hybrids, a radiation hybrid mapping panel and FISH analysis on human metaphase chromosomes. PCR-based analysis of a single chromosome hybrid panel identified the presence of the CD83 gene on human chromosome 6 and subsequent analysis of the Genebridge4 radiation panel located the gene between AFMa192wg9 and AFMb322wd1 with a lod score of 9.2. Finally, using FISH analysis the CD83 gene was localized to chromosome 6 band p23.     

Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of humanmouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p ⁺ (7qter>7p22::Xq21>Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-q21.Preliminary report of this research was presented at Ninth International Workshop on Human Gene Mapping, Abstract supplement p. 5 (1987).     

Human gastrin-releasing peptide gene maps to chromosome band 18q21

A complementary DNA clone encoding human pre-pro gastrin-releasing peptide, a 27-amino acid neuropeptide and putative growth factor, was used to determine the chromosomal location of this gene. Southern blot hybridization to genomic DNA isolated from a panel of human-rodent somatic cell hybrids unambiguously maps this gene to human chromosome 18. In situ chromosomal hybridization confirms the hybrid data and further localized the gene to chromosome band 18q21. Karyotypic abnormalities in tumors and inherited disease states which involve chromosome band 18q21 may now be studied for correlated changes in the structure and expression of the human GRP gene.     

Localization of the oncogene c-erbA2 to human chromosome 3

The human c- erb Al gene has been previously mapped to chromosome 17. We have now mapped c- erb A2 to the short arm of chromosome 3, using a human genomic probe in Southern analysis of DNA from a panel of human/mouse somatic cell hybrids. In situ hybridization using the same probe on metaphase chromosomes has enabled fine chromosome mapping of c- erb A2 to the chromosome region 3p21-pter.     

Molecular cytogenetic resources specific for chromosome 12.

We have generated a panel of 20 somatic cell hybrids retaining fragments of human chromosome 12. Each hybrid was characterized cytogenetically by reverse fluorescence in situ hybridization (FISH) and molecularly by 24 sequence tagged sites (STSs) spaced evenly along the chromosome. The panel can be exploited to map subregionally DNA sequences on chromosome 12 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome. Furthermore, a panel of 58 yeast artificial chromosomes (YACs) mapping to chromosome 12 was characterized by FISH experiments on normal human metaphases. A subset of this panel is recognized by the STSs used in the somatic cell hybrid characterization. In this way a correlation between the genetic and the physical maps of this chromosome can be established.     

Processed pseudogene from the von Hippel-Lindau disease gene is located on human chromosome 1.

The von Hippel-Lindau (VHL) disease gene is a tumor suppressor located at 3p25-26. While amplifying intron 1 of this gene, a smaller-than-expected product was found. This fragment was sequenced and was approximately 78% similar in sequence to the VHL gene and completely lacked sequence from the intron. No stop codons were found in the sequenced region. Using this DNA fragment as a probe for Northern blot hybridization analysis, no evidence was found for expression of a unique RNA. Because of the lack of intron 1 sequence and the likely lack of expression, the new sequence is most probably a part of a VHL processed pseudogene. The putative pseudogene was mapped to human chromosome band 1q12 using the polymerase chain reaction with template DNA from human/rodent somatic cell hybrids, a radiation hybrid panel, and a set of primers that were chosen to be maximally divergent from the genuine VHL gene. The human/rodent somatic cell hybrid DNAs were then used on Southern blots to determine which human bands are from the pseudogene and which are from the functional gene. This knowledge is valuable in interpreting Southern blot evidence of VHL gene abnormalities.     

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.