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Biomedical subjects

F H Ruddle

Publications and source records attributed to F H Ruddle.

At least 271 records · Page 15Linked to original sources
PubMed1979

Partial purification and characterization of DNA from the human X chromosome.

Human X chromosome DNA was partially purified from a mouse-human hybrid cell line containing a single human chromosome, the X. Enrichment of such DNA was accomplished by two sequential reassociations of radiolabeled hybrid cell DNA with large excesses of mouse DNA. Unreassociated hybrid cell DNA was used as a probe for human X chromosome sequences. The human-specific fraction of probe DNA CONTAINED THREE COMPONENTS. Two of these reassociated to human DNAs at rates proportional to the number of X chromosomes present. These two components were thus localized to the X chromosome. One of these X-specific components, representing about 80% of human-specific probe DNA, consisted of single copy or very low order reiterated DNA. The second X-specific component, representing about 10% of human-specific probe DNA, was about 20-30 times more reiterated. The remaining 10% of human-specific probe DNA, although derived from the X chromosome, reassociated to human DNAs at a rate independent of the number of X chromosomes present. This component was thus homologous to autosomal as well as X chromosome DNA. The probe DNA accounts for approximately half of the human X chromosome, suggesting that the remainder may have homology with mouse DNA.

Animals↗
PubMed1979

Chromosomal assignment of the mouse kappa light chain genes.

Mouse-hamster somatic cell hybrids containing a variable number of mouse chromosomes have been used in experiments to determine which mouse chromosome carries the immunoglobulin kappa light chain genes. It has been shown by nucleic acid hybridization that the kappa constant gene and the genes for at least one variable region subgroup are on mouse chromosome 6. This somatic cell genetic mapping procedure appears to be general and can be applied to any expressed or silent gene for which an appropriate nucleic acid probe exists.

Animals↗
PubMed1979

Molecular analysis of chromosome-mediated gene transfer.

Metaphase chromosomes isolated from a cell line carrying the thymidine kinase (TK) gene of herpes simplex virus type I were used to transform the TK-deficient cell line LMTK- to the TK+ phenotype. Four independent transformants were isolated, all of which expressed virus-specific TK. Each of the four transformant cell lines initially became TK- at a rate of 12% per day. All four transformants possessed multiple copies of the TK gene and in one of the four a rearrangement occurred adjacent to the TK sequences. Stable TK+ derivatives of each line, isolated after prolonged cultivation, retained fewer copies of the TK gene than did their unstable parents. The transferred chromosomal fragment was larger than 17 kilobases in each line.

Animals↗
PubMed1979

Biological detection of specific mRNA molecules by microinjection.

We have used direct microinjection of messenger RNA into individual mouse and human cells to assay for specific translation products. We have been able to detect the synthesis of human fibroblast interferon, thymidine, kinase, hypoxanthine phosphoribosyltransferase, adenine phosphoribosyltransferase, and propionyl-CoA carboxylase in response to injected mRNA. Using the interferon system as a model, we have quantitated interferon synthesis and followed partial purification of interferon mRNA sequences on sucrose density gradients. The methods we have utilized should be applicable to other systems in which sensitive assays exist for gene products and should provide a screening procedure for isolating specific mRNA sequences.

Adenine Phosphoribosyltransferase↗
PubMed1979

X-linkage of a human genetic locus that corrects the DNA synthesis lesion in tsC1AGOH mouse cells.

GM 126 diploid fibroblasts were fused with a heat-sensitive mouse cell mutant defective in DNA synthesis, and primary hybrids were selected at permissive and nonpermissive temperatures in HAT medium. Primary hybrids, primary hybrid clones back-selected in 8-azaguanine at the permissive temperature, and subclones of heat-resistant primary hybrids isolated under nonselective conditions or after 8-azaguanine treatment were tested for heat sensitivity, the expression of 26 human enzymes assigned to 19 different human chromosomes, and the presence of human chromosomes. Only the human X chromosome and X-linked marker enzymes exhibited a clear pattern of concordant segregation with the heat-resistant phenotype. On the basis of these observations, we have defined the human genetic locus that corrects the heat-sensitive lesion in tsC1AGOH as hrC1AGOH and have assigned this locus to the X chromosome. This observation provides the first instance where two selectable markers (heat resistance and 8-azaguanine sensitivity) are found on a single human chromosome and suggests that these markers may prove to be a valuable push-pull selective system of use in determining the linear arrangement of genes on human chromosomes by somatic cell genetics.

Animals↗
PubMed1978

Assignment of the gene for dipeptidase 2 to Mus musculus chromosome 18 by somatic cell hybridization.

Evidence is presented for the assignment of the gene for dipeptidase 2 to Mus musculus chromosome 18 by synteny testing and karyotypic analysis of Chinese hamster x mouse somatic cell hybrid clones. DIP-2 and chromosome 18 were expressed concordantly in 24/24 clones examined (ten primary clones and 14 secondary clones). Synteny testing indicated that DIP-2 was not expressed concordantly with the expression of any marker enzymes.

Animals↗
PubMed1978

Assignment of the gene for glyoxylase I to mouse chromosome 17 by somatic cell genetics.

Evidence is presented for the assignment of the gene for glyoxylase I to mouse chromosome 17 using mouse X Chinese hamster somatic cell hybrids. GLO I was not expressed concordantly with any known marker enzymes which represented 11 linkage groups. The presence of chromosome 17 and expression of GLO I were concordant in 31/31 clones. GLO I is thus linked to the H-2 histocompatibility locus in the mouse.

Animals↗
PubMed1978

Assignment of the genes for triose phosphate isomerase to chromosome 6 and tripeptidase-1 to chromosome 10 in Mus musculus by somatic cell hybridization.

Evidence is presented for the assignment of the gene for triose phosphate isomerase to Mus musculus chromosome 6 and tripeptidase-1 to chromosome 10 by synteny testing and chromosome assignment in Chinese hamster X mouse somatic cell hybrid clones. Neither TPI nor TRIP-1 were expressed concordantly with any known isozyme markers in 45 hybrid clones (13 primary and 32 secondary). Karyotypic analysis of 21 clones showed that the expression of TPI and chromosome 6 were concordant in all cases as was expressed of TRIP-1 and chromosome 10. Both chromosomes were previously unmarked by isozymes.

Aminopeptidases↗
PubMed1978

Assignment of the gene locus for human alpha-L-fucosidase to chromosome 1 by analysis of somatic cell hybrids.

The alpha-L-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human alpha-L-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human alpha-L-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human alpha-L-fucosidase. It is concluded that the structural gene locus for human alpha-L-fucosidase is located on chromosome 1.

Animals↗
PubMed1978

A herpes simplex virus 1 integration site in the mouse genome defined by somatic cell genetic analysis.

Transfection experiments with HSV 1 in which one uses herpes simplex virus (HSV) thymidine kinase (TK) as a selectable prototrophic marker yield two classes of transformed cells: stable and unstable. In this report, we test the hypothesis that the stability phenotype can be explained by virus genome integration into a recipient cell chromosome. The method of analysis is by means of somatic cell genetics. We have isolated a series of microcell hybrids between a TK- Chinese hamster cell line and a transformed mouse cell line expressing the TK encoded by HSV 1. Several of the hybrid lines contain a single murine chromosome and express only the viral TK. Karyotypic analysis of these hybrids and of TK- derivatives generated by BrdUrd counterselection reveals that the TK+ phenotype is correlated with the presence of the terminal portion of the long arm of a specific murine chromosome. Results of extensive isozyme analyses of the hybrids and their TK- segregants fully corroborate the karyologic data. The results are consistent with the hypothesis that the viral tk gene is covalently integrated into this chromosomal region which itself does not appear to carry the endogenous murine tk locus. Other more complicated models are discussed. Our findings also show that somatic cell genetics can be used to localize viral integration sites in host chromosomes with high resolution.

Animals↗
PubMed1978

Cotransfer of thymidine kinase and galactokinase genes by chromosome-mediated gene transfer.

The Chinese hamster genes for thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) and galactokinase (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) have been cotransferred to mouse cells by chromosome-mediated gene transfer. Hamster metaphase chromosomes were incubated with mouse B82 cells and 22 independent colonies were isolated in a selective medium. All of the 12 colonies analyzed expressed the donor form of thymidine kinase; the hamster form of galactokinase was also expressed in 2 of these colonies, indicating cotransfer with a frequency of about 20%. There was coordinate loss of both transferred genes from each colony when selection was applied for the loss of thymidine kinase alone. Comparison of the regional localization of these two linked genes with the frequency of cotransfer suggests that the transgenome is probably not larger than about 0.25% of the donor genome.

Cells, Cultured↗
PubMed1978

Chromosomal localization of human beta globin gene on human chromosome 11 in somatic cell hybrids.

We have successfully used a DNA.cDNA molecular hybridization assay to directly determine the presence or absence of human beta globin gene sequences in 20 human-mouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human beta globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human beta globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human beta globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT-4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT-4. 157-BNPT-4 contained the human beta globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human gamma globin cDNA; two lines positive for human beta globin gene sequences also contained human gamma globin gene sequences while one line was negative for both beta and gamma gene sequences. On the basis of these results, the human beta and gamma globin genes have been assigned to human chromosome 11.

Chromosomes, Human, 6-12 and X↗