Rodent Somatic Cell Hybrids Research Articles

CDNA cloning, expression and mapping of human laminin B2 gene to chromosome 1q31

A laminin B2 chain cDNA clone was isolated from a human lung cDNA library by screening with antibody against mouse laminin. The authenticity of the human cDNA clone was established by comparison of the nucleotide and deduced amino acid sequences of the cDNA insert with those of the previously reported mouse laminin cDNA clones. The human clone (LC7) contained an insert of 0.75 kb (kilobase pair) that corresponded to the last 232 amino acid residues in the carboxyl terminus of the B2 chain. Northern blot analyses with the LC7 probe detected two mRNA transcripts of 8.2 and 5.6 kb in both normal human skin fibroblasts and three human tumor cell lines. The cDNA probe was also used in Southern blot analysis of DNA from human rodent somatic cell hybrids to localize the gene to human chromosome 1. In situ hybridization of the cDNA with metaphase chromosome spreads confirmed the assignment and further mapped the human laminin B2 chain gene to the long arm of chromosome 1 in the band q31.

CDNA cloning and assignment to chromosome 21 of IFI-78K gene, the human equivalent of murine Mx gene.

Recently we have purified to homogeneity and characterized an interferon-induced human protein (p78 protein) which is the equivalent of the interferon-induced murine Mx protein responsible for a specific antiviral state against influenza virus infection. A cDNA library was constructed using mRNAs from interferon-induced human diploid fibroblasts. cDNA clones coding for the human p78 protein were identified and used to determine the chromosomal location of the corresponding gene (termed IFI-78K gene) by hybridization to DNA from a panel of human x rodent somatic cell hybrids. The newly identified gene is located on chromosome 21. This has been confirmed by the observation of a gene dosage effect using chromosome 21 trisomic cells (fibroblasts derived from Down's syndrome patients). Among all interferon-inducible genes mapped so far, the IFI-78K gene is the only one located on chromosome 21, together with the gene for the receptor of type I interferon. Our results also provide further evidence for homology between human chromosome 21 and mouse chromosome 16, since the gene encoding the mouse Mx protein (the presumed mouse homolog protein of human p78 protein) has been assigned to chromosome 16.

Chromosomal localization of the met proto-oncogene in the mouse and cat genome

The met proto-oncogene was mapped in the mouse and cat genomes with the use of mouse × hamster and cat × rodent somatic cell hybrid DNA panels. Based on these analyses we assigned the met gene to mouse chromosome 6 and to cat chromosome A2. We also assigned the cat raf-1 proto-oncogene to the A2 chromosome; met and raf-1 are the first cloned DNAs mapped to this linkage group. Using an interspecies backcross we further localized met on mouse chromosome 6 to a position proximal to the β chain of the T-cell receptor. This places met near the obese locus in a region of mouse chromosome 6 that appears to be homologous with the long arm of human chromosome 7. The close linkage of met to the gene responsible for cystic fibrosis in humans suggests that further genetic analysis of mouse chromosome 6 may be useful in developing a mouse model for the disease.

Highly polymorphic DNA sequences in the distal region of the long arm of human chromosome 18.

Highly polymorphic repeated DNA sequences were detected at the end of the long arm of human chromosome 18 by a recombinant DNA probe containing 56 bp of human DNA (pERT25). This was shown by hybridization of pERT25 to DNA from a panel of human X rodent somatic cell hybrids, by dot blot hybridization to flow-sorted human chromosomes, and by in situ hybridization to metaphase chromosomes. The high degree of polymorphism detected by this 18q DNA fragment makes it potentially useful for various applications, including investigations into the genetics of trisomy 18 (Edwards syndrome), linkage studies, and paternity testing.

Assignment of the human tyrosine aminotransferase gene to chromosome 16

The liver enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5) catalyzes the rate-limiting step in the catabolic pathway of tyrosine. Deficiency in TAT enzyme activity underlies the autosomally inherited disorder tyrosinemia II (Richner-Hanhart syndrome). Using a human TAT cDNA clone as hybridization probe, we have determined the chromosomal location of the TAT structural gene by Southern blot analysis of DNAs from a series of human X rodent somatic cell hybrids. The results assign the TAT gene to human chromosome 16.

Genetic mapping of endogenous RD-114 retroviral sequences of domestic cats.

The RD-114 family of endogenous retroviral sequences in domestic cats has been shown to consist of approximately 20 copies of genetically divergent virogenes per haploid genome. The chromosomal localization for four endogenous sequences (RDV1-4) was accomplished by correlating the occurrence of specific feline chromosomes with diagnostic viral DNA fragments in a panel of cat X rodent somatic cell hybrids. Analysis of the hybrid panel revealed that endogenous RD-114 sequences are dispersed on multiple cat chromosomes, that certain proviral segments are polymorphic with respect to the presence or absence of virus, and that a restriction fragment characteristic of inducible RD-114 resides on a single feline chromosome (B3), probably at a single locus.

Chromosomal assignments of human genes for serine proteases trypsin, chymotrypsin B, and elastase.

The genes for the serine proteases trypsin, chymotrypsin B, and elastase were chromosomally assigned in man using cDNA probes that have been isolated from a rat pancreatic cDNA library. DNA from human X rodent somatic cell hybrids was cleaved with BamHI or EcoRI and analyzed by Southern filter hybridization methods for the segregation of the genes for trypsin-1 (TRY1), chymotrypsin B (CTRB), and elastase-1 (ELA1). TRY1 was assigned to human chromosome 7q22----qter, CTRB to chromosome 16, and ELA1 to chromosome 12. Although the three genes are members of the same gene family, they are dispersed over different chromosomes.

The human myoglobin gene: a third dispersed globin locus in the human genome.

Human myoglobin is specified by a single gene. Unique sequence DNA probes were isolated from the cloned gene and used to test for the presence of the human myoglobin gene in a series of human rodent somatic cell hybrids containing various complements of human chromosomes. The myoglobin gene cosegregated with human chromosome 22. Somatic cell hybrids containing translocation chromosomes carrying part of chromosome 22 were used to locate the myoglobin gene to the region 22q11----22q13. The myoglobin gene is therefore not linked to the alpha-globin gene cluster on chromosome 16 or the beta-globin cluster on chromosome 11, and represents a third dispersed globin locus in the human genome.

Regional assignment of the human gene for platelet-type phosphofructokinase (PFKP) to chromosome 10p: novel use of polyspecific rodent antisera to localize human enzyme genes.

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), liver-type (L), and platelet or fibroblast-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign the human PFKP locus to a specific human chromosome, in this study, we have examined ten human X rodent somatic cell hybrids for the expression of human P subunits using a mouse anti-human P subunit-specific antiserum in an active-enzyme-immunoprecipitation technique. In nine of ten hybrids studied, the expression of the PFKP locus segregated concordantly with chromosome 10 and none other, indicating that PFKP is located on chromosome 10; the discordancy rates for all the other chromosomes were 0.2 or greater. In the one discordant hybrid, only the long arm of chromosome 10 was retained and PFKP was not expressed. Human fibroblasts from a patient with duplication of the short arm of chromosome 10 consistently exhibited PFK activity values 180% of normal. These data indicate that human PFKP is located on the short arm of chromosome 10, and that a gene dosage effect is demonstrable in fibroblasts with a duplication of 10p. The use of rodent antihuman antibody combined with immunoprecipitation aided by staphylococci-bearing protein A may find general application in mapping human enzyme genes, when human and rodent gene-products are not distinguishable by other means.

Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells.

The identification of transforming genes in human tumor cells has been made possible by DNA mediated gene transfer techniques. To date, it has been possible to show that most of these transforming genes are activated cellular analogues of the ras oncogene family. To better understand the relationship between these oncogenes and other human genes, we have determined their chromosomal localization by analyzing human rodent somatic cell hybrids with molecularly cloned human proto-oncogene probes. It was possible to assign N-ras to chromosome 1 and regionally localize c-K-ras-1 and c-K-ras-2 to human chromosomes 6pter-q13 and 12q, respectively. These results along with previous studies demonstrate the highly dispersed nature of ras genes in the human genome. Previous reports indicated that the c-myb gene also resides on chromosome 6. It has been possible to sublocalize c-myb to the long arm of chromosome 6 (q15-q21). The non-random aberrations in chromosomes 1, 6 and 12 that occur in certain human tumors suggest possible etiologic involvement of ras and/or myb oncogenes in such tumors.

A human quantitative polymorphism related to Xg blood groups.

A monoclonal antibody, 12E7, raised against lymphocytes from a patient with a T-cell acute lymphocytic leukaemia reacts strongly with cortical thymocytes and, to a lesser extent, with other human cells. The antigen defined by 12E7 is not expressed on mouse or hamster cells; this species specificity allowed us to investigate the genetics of the expression of the 12E7 antigen using human--rodent somatic-cell hybrids. Hybrid cells which contain the human X chromosome as the only human genetic contribution react with 12E7. The X-linkage of the gene, or genes, controlling 12E7 antigen expression and the fact that 12E7 antigen is also expressed on red blood cells led us to investigate the possibility of a relationship with the X-linked Xg blood group system. Although the 12E7 and Xga antigens seem to be different antigenic determinants specified by different loci, we report here a sex-limited effect involving the Xg phenotype on the expression of 12E7 antigen.

The beta-globin gene is on the short arm of human chromosome 11.

Investigations on the regulation of human globin gene expression are assisted by a knowledge of the chromosomal location of the globin genes. Previous studies have mapped the alpha-globin locus to human chromosome (HC) 16, and have shown that the human globin gene complex gamma-delta-beta co-segregates with lactate dehydrogenase A (LDH-A) and the presence of HC 11 in somatic cell hybrids. Radioactively labelled globin complementary DNA (cDNA) probes were used in molecular hybridisation experiments to determine the chromosomal locations of the alpha- and beta-globin genes. When human x rodent somatic cell hybrids are used which contain well-defined parts of human chromosomes, direct mapping of genes of chromosomal regions or single bands is possible. We have regionally localised the human beta-globin gene using two sets of hybrid cell lines: (1) Chinese hamster x human hybrid cells containing the HC 11 long arm or both the short and long arms and (2) mouse x human hybrids containing only the HC 11 short arm. The techniques of liquid molecular hybridisation and Southern blotting with 32P-labelled human beta-globin cDNA (from plasmid JW102) have been used to localise the beta-globin gene sequence to region 11p11 leads to 11p15. Similar results were reported recently by Jeffreys et al.

The expression of creatine kinase isozymes in human cultured cells.

The BB isozyme of creatine kinase is consistently present in cultured human fibroblasts and shows great variation in activity in long-term lymphoid lines. One mouse line tested, PG 19, had strong activity, but all other rodent lines tested did not express CK BB. Human and mouse CK BB can be expressed independently of each other in human--rodent somatic cell hybrids. There is some evidence that the structural locus for CK BB may be on chromosome 14, but the involvement of other chromosomes, expecially no.17, cannot be excluded. The MM and MB isozymes of creatine kinase were not seen in any human cultured cells.