Monochromosomal Somatic Cell Hybrid Research Articles

Cloning and Mapping of Human Rab7 and Rab9 cDNA Sequences and Identification of a Rab9 Pseudogene

Rab GTPases reside in specific intracellular compartments and are key regulators of vesicular transport. To facilitate studies of the mechanism of lysosomal integral membrane protein (LAMP-1) transport, cDNAs for human Rab7 and Rab9 were isolated, and their nucleotide sequences were determined. During isolation and characterization of these cDNAs a Rab9 pseudogene was identified. The sequences are highly homologous to other mammalian Rab proteins and also share homology with proteins of the Rab GTPase family. Rab7 and the Rab9 pseudogene were mapped to chromosomes 3 and 5, respectively, by amplification of their sequences from human monochromosomal somatic cell hybrids. In addition, preliminary studies using antisense expression indicate that down-regulation of either Rab7 or Rab9 proteins induces severe cell vacuolation that resembles the phenotype seen in fibroblasts from patients with Chediak–Higashi syndrome.

Localization and Physical Mapping of Genes Encoding the A+U-Rich Element RNA-Binding Protein AUF1 to Human Chromosomes 4 and X

Messenger RNAs encoding many oncoproteins and cytokines are relatively unstable. Their instability, which ensures appropriate levels and timing of expression, is controlled in part by proteins that bind to A+U-rich instability elements (AREs) present in the 3′-untranslated regions of the mRNAs. cDNAs encoding the AUF1 family of ARE-binding proteins were cloned from human and murine cDNA libraries. In the present study monochromosomal somatic cell hybrids were used to localize two AUF1 loci to human chromosomes 4 and X.In situhybridization analyses using P1 clones as probes identified the 4q21.1–q21.2 and Xq12 regions as the locations of the AUF1 genes.

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.

Chromosomal Assignment of 79 cDNAs from a Range of Human Tissues

Single-pass DNA sequencing of cDNAs selected at random from a human mixed tissue cDNA library have generated a series of more than 2000 expressed sequence tags. One hundred twenty-eight unique cDNA fragments with little or no known protein or nucleic acid homologies have been selected for further analysis. Oligonucleotide primer pairs have been designed from the cDNAs and used in PCR amplification in combination with genomic DNA from a panel of monochromosomal somatic cell hybrids. This has allowed us to assign 70 of these transcribed genes to a single chromosome, and a further 9 have been located on two or three chromosomes. Additionally, 3 cDNAs contain short tandem repeats that may allow them to be further localized by linkage analysis.

Localization and Physical Mapping of the Prostate-Specific Membrane Antigen (PSM) Gene to Human Chromosome 11

The prostate-specific membrane antigen (PSM) was identified by the monoclonal antibody 7E11-C5.3, which was raised against the human prostatic carcinoma cell line LNCaP (3). The PSM antigen is expressed by normal, neoplastic, and metastatic prostatic tissues. The 2.65-kb cDNA encoding the 100-kDa PSM glycoprotein was cloned from LNCaP cells (4). Studies have shown that the expression of PSM is tissue-specific (5). In the present study monochromosomal somatic cell hybrids were used to localize the PSM gene to human chromosome 11. Using this information, initial mapping studies identified two potential PSM gene loci at 11p11.1–p13 and 11q14. Further high-stringency analysis using cosmid probes identified the 11q14 region as the location of the PSM gene.

Chromosomal localization and expressed sequence tag generation of clones from a normalized human adult thymus cDNA library.

Expressed sequence tags (ESTs) from 298 clones have been generated from a randomly primed, normalized human adult thymus cDNA library. We describe the chromosomal localization of 136 of these ESTs by PCR-based mapping to a human monochromosomal somatic cell hybrid panel. Data base similarities to known genes are also described. A subset (n = 18) of these randomly primed ESTs extended the sequence of ESTs from other tissues currently in dbEST. Of the nonrepetitive human adult thymus ESTs generated in this study, 237 (79.5%) have no similarity to current data base entries. This would suggest that our collection contains approximately 100 new coding regions from thymus tissue, a large proportion of which likely will represent the middle regions of genes. The mapped ESTs should prove useful as new gene-based markers for mapping and candidate gene hunting, particularly when anchored to a well-developed physical map of the human genome.

Development of a panel of monochromosomal somatic cell hybrids for rapid gene mapping

We have assembled a panel of monochromosomal somatic cell hybrids for use in gene mapping. DNA from each individual hybrid was used as a probe on normal human metaphases to identify the human chromosome and any fragments by reverse painting. To test the efficiency of the panel PCR amplification of DNA from the monochromosomal somatic cell hybrid panel was used in combination with human specific oligonucleotide primers to assign alpha-catenin (CTNNA1) and p21/WAF1 to chromosomes 5 and 6 respectively. These genes were localized further using hybrids containing specific translocations to 5q11-qter and 6p21 respectively. We also developed primers to enable us to assign 17 ESTs sequenced by the HGMP Resource Centre. The hybrid panel was developed with support of the UK HGMP and the DNA is available to all registered users.

The novel gene g17, located in the human major histocompatibility complex, encodes PBX2, a homeodomain-containing protein

Recent characterization of the class III region of the human major histocompatibility complex (MHC), located in chromosome 6p21.3, has revealed that it is very gene dense and contains at least 47 transcriptional units. One of these is the gene G17, which lies 250 kb telomeric of the class II gene DRA. DNA sequence analysis of 5.5 kb of DNA corresponding to the G17 gene has revealed that it encodes PBX2, a homeo-domain-containing protein with extensive similarity to PBX1 (which is involved in t(1;19) chromosomal translocations in acute pre-B-cell leukemias). Comparison of the genomic DNA sequence with the published PBX2 cDNA sequence, which are 99.7% identical, indicates that the G17 gene is split into 9 exons, with the intron/exon boundaries conforming to the normal pattern (AG/…/GT) for splice sites. Of the 9 differences observed between the PBX2 cDNA sequence and the G17 genomic sequence, only 1 is contained in the coding sequence and alters the derived amino acid sequence. This results in an Ile (PBX2)—Met (G17) substitution at amino acid 393 near the C-terminus. The PBX2 gene was originally localized only to human chromosome 3q22–q23. However, comparison of genomic and cosmid Southern blots clearly indicates that another copy(ies) of the PBX2 (G17) gene exist(s) in the genome. PCR amplification of exons III and IX of the G17 (PBX2) gene, corresponding to the coding and 3′ untranslated regions, respectively, using as template genomic DNA from a panel of monochromosomal somatic human-rodent cell hybrids, gave specific products in hybrids that contain human chromosomes 6, 3, and 1. These results confirm that copies of the PBX2 gene are located on human chromosomes 6 and 3 and indicate that a gene homologous to PBX2 could exist on human chromosome 1. Further PCR analysis of the genes and reverse transcribed mRNA from the hybrid cell lines has revealed that the copies of the PBX2 gene on human chromosomes 6 and 1 are expressed, while the copy on human chromosome 3 may be a processed pseudogene.

A collection of tri- and tetranucleotide repeat markers used to generate high quality, high resolution human genome-wide linkage maps.

We report a collection of tri- and tetranucleotide repeat sequence polymorphic markers used to construct genome-wide human linkage maps. Using a strategy of marker selection to create libraries highly enriched for the presence of specific tandem repeat elements, we have developed over 2000 high heterozygosity, easy-to-use tri- and tetranucleotide short tandem repeat polymorphisms (STRPs). To date, over 1300 of these markers have been genotyped on the CEPH reference families. Additional STRPs were assigned to chromosomes using human monochromosomal somatic cell hybrids. The linkage maps constructed with these markers have been integrated with other CEPH genotypes into a comprehensive high density linkage map. These STRPs have been shown to be robust for genotyping in a variety of laboratories using a variety of methods. The high quality of these STRPs makes them ideal candidates for use in genome-wide linkage searches. The integration of these markers with physical mapping reagents and other genetic markers will create a resource for moving from genome-wide linkage searches to rapid sublocalization of disease loci.

Mapping of CD24 and Homologous Sequences to Multiple Chromosomal Loci

The human cell surface antigen, CD24, is a glycosyl phosphatidylinositol (GPI)-linked glycoprotein that has been implicated in the differentiation and activation of granulocytes and B lymphocytes. Changes in expression of the antigen occur at critical times during B lineage development. CD24 was cloned by its homology to mouse heat-stable antigen. Southern blot analysis suggested the presence of multiple CD24 related sequences in the human genome. We have now mapped CD24 homologous sequences to chromosomes 6q21, 15q21-q22, and Yq11 by screening a panel of somatic cell hybrid DNAs and by in situ hybridization. At least two additional homologues, one located on chromosome 1 at band p36 and one tentatively mapped to chromosome 20, that are distantly related to CD24 were identified. Southern analysis of male and female DNA samples confirmed the presence of CD24 homologous sequences on the human Y chromosome. Sequencing of DNA fragments amplified from monochromosomal somatic cell hybrids showed that the CD24 cDNA was derived from a transcript originating from a gene on chromosome 6. The CD24 gene on the Y chromosome had many base changes compared to the cDNA but had retained an open reading frame, leaving open the question of whether this gene is functional. Both ATGs in the translation initiation region of CD24 homologous sequences on chromosome 15 were converted to GTGs, making it unlikely that this gene, if functional, encodes a protein similar to CD24. CD24, therefore, is a member of a multigene family, but it remains to be determined whether any of the related genes are functional.

Regional localization of 56 new human chromosome 18-specific yeast artificial chromosomes.

Fifty-six chromosome 18-specific yeast artificial chromosomes (YAC) were isolated from a human monochromosomal somatic cell hybrid DNA library. Fluorescence in situ hybridization revealed that the clones were evenly distributed throughout the chromosome. The clones, with an average insert size of 250 kb, cover about 18% of the euchromatic part of chromosome 18. Of 90 STS markers tested, 17 were represented in this YAC collection. Together with our previously reported set of unique chromosome 18-specific YACs, we now have available 111 regionally mapped, essentially nonchimeric, clones that provide more than 30% coverage and form a framework for the complete physical map.

Homologous loci DXYS156X and DXYS156Y contain a polymorphic pentanucleotide repeat (TAAAA)n and map to human X and Y chromosomes

We report the isolation and characterization of a polymorphic pentanucleotide repeat (TAAAA)n, which was mapped to human chromosomes X and Y (loci DXYS156X and DXYS156Y) by PCR amplification of DNA from a monochromosomal somatic cell hybrid panel (NIGMS panel 2). The (TAAAA)n repeat of loci DXYS156 occurs within a human LINE element at a position where the consensus sequence contains a single TAAAA motif. In 72 unrelated CEPH individuals seven alleles were detected which ranged in size from 125 to 165 bp in 5 bp intervals. The two largest alleles (160 and 165 bp) were observed only in males, which suggests that they were amplified from the Y chromosome DXYS156Y locus. The other 5 alleles were present in two copies in females and in a single copy in males, which suggests that they were amplified from the X chromosome DXYS156X locus. Locus DXYS156X was polymorphic in CEPH families with an observed heterozygosity in females of 46% (27 of 59). Linkage analysis with DNA markers on the X chromosome revealed significant lod scores for a location of DXYS156X close to markers DXS1002 (theta = 0.000; zeta = 8.43), DXYS1X (theta = 0.015; zeta = 17.3), DXS3, and PGK1 in the region of chromosome Xq13. The sequence of DXYS156Y derived from the 165 bp allele has been deposited in Genbank with accession number X71600.

Mapping the midkine family of developmentally regulated signaling molecules.

Midkine (Mdk) and heparin-binding neurotrophic factor (Hbnf)/pleiotrophin (Ptn) comprise the Midkine family of developmentally regulated signaling molecules. We have determined the chromosomal localization of these genes in the mouse by use of single-strand conformation polymorphisms (SSCPs), which facilitated the typing of Mdk and Hbnf alleles in recombinant inbred (RI) strains and interspecific backcrosses. Mapping was performed relative to other cloned genes, as well as simple sequence length polymorphisms (SSLPs) in the interspecific backcrosses. Mdk maps to mouse Chromosome (Chr) 2, linked to the Hoxd gene cluster. Hbnf maps to proximal mouse Chr 6, linked to the Cftr and Cpa genes. Comparative mapping of human MDK and HBNF employing species-specific polymerase chain reaction (PCR) primers and human monochromosomal somatic cell hybrids assigns MDK to human Chr 11 and HBNF to human Chr 7q32-qter.

“PCR-Karyotype” of human chromosomes in somatic cell hybrids

Amplification of human DNA sequences in 16 monochromosomal somatic cell hybrids containing different human chromosomes was performed by the polymerase chain reaction (PCR) using primers directed at human-specific regions of Alu or L1, the two major classes of interspersed repetitive sequences (IRS-PCR). A chromosome-specific pattern of amplification products was observed on agarose gels run with ethidium bromide, producing a “PCR-karyotype.” This simple gel analysis provides a rapid method for identifying and monitoring the human chromosomal content of monochromosomal somatic cell hybrids without conventional cytogenetic analysis. Hybrids containing multiple human chromosomes produce complex gel patterns, but identification of chromosome content can be achieved by hybridization of PCR products against a reference panel of monochromosomal or highly reduced hybrids representing each human chromosome. This dot-blot method also enables identification of human marker chromosomes or translocated pieces in hybrids that are not identifiable by cytogenetic methods. These IRS-PCR methods should greatly reduce the need for more laborious cytogenetic, isozyme, and Southern blot characterizations of human-rodent cell hybrids.