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Biomedical subjects

F H Ruddle

Publications and source records attributed to F H Ruddle.

At least 181 records · Page 10Linked to original sources
PubMed1984

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene.

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.

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PubMed1984

Receptors for human alpha and beta interferon but not for gamma interferon are specified by human chromosome 21.

We examined the proposed role of human chromosome 21 in determining the cellular sensitivity to human alpha, beta, and gamma interferons (HuIFN-alpha, -beta, and -gamma) and the expression of the receptors for the HuIFNs with the use of mouse-human hybrid cells containing human chromosome 21. Hybrid cells (WA17) containing three copies of human chromosome 21 showed specific displaceable binding of 125I-labeled HuIFN-alpha 2 (125I-HuIFN-alpha 2), which was not observed with mouse parent (A9) cells. Crosslinking of 125I-HuIFN-alpha 2 bound to WA17 cells with disuccinimidyl suberate yielded a complex of Mr approximately equal to 150,000 similar to the 125I-HuIFN-alpha 2-receptor complex obtained with human cells as described earlier. Such a complex was not obtained with mouse parent (A9) cells or with hybrid cells containing certain other human chromosomes but not chromosome 21. Mice inoculated with mouse-human hybrid cells containing human chromosome 21 produce antibodies that block the antiviral action of HuIFN-alpha and -beta on human cells. Such antibodies could immunoprecipitate the 125I-HuIFN-alpha 2-receptor complex obtained from human cells but not free 125I-HuIFN-alpha 2, indicating that these antibodies were directed against the receptor. WA17 hybrid cells were highly sensitive to the antiviral action of HuIFN-alpha 2, -alpha (Le) and -beta but were completely insensitive to HuIFN-gamma. Furthermore, 125I-HuIFN-gamma showed specific binding to human WISH cells but not to WA17 hybrid cells or A9 mouse cells. The results indicate that the receptors for HuIFN-alpha and -beta but not for HuIFN-gamma are specified by human chromosome 21. Hybrid cells containing one, two, or three copies of human chromosome 21 were found to be increasingly sensitive to HuIFN-alpha 2, indicating that a chromosome 21-specified component (possibly the HuIFN-alpha receptor) may be a limiting factor in the cellular sensitivity to HuIFN-alpha.

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PubMed1984

Human c-fos oncogene mapped within chromosomal region 14q21----q31.

The human cellular homolog (c-fos) of the transforming gene of Finkel-Biskis-Jinkins (FBJ) murine osteosarcoma virus was mapped to a single human chromosome. DNA from a series of 31 mouse-human somatic cell hybrid lines was probed with v- and c-fos molecular clones by Southern blotting. Human c-fos segregated with the distal region of the long arm of human chromosome 14. In situ hybridization of 125I-labeled human c-fos probe to normal human metaphase chromosomes independently confirmed these results and localized the c-fos oncogene to region 14q21----q31.

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PubMed1984

Assignment of the genes for the alpha and beta subunits of thyrotropin to different mouse chromosomes.

A series of mouse-hamster somatic cell hybrids, containing reduced numbers of mouse chromosomes and a complete set of hamster chromosomes, was used to determine the chromosomal locations of the genes for the alpha and beta subunits of mouse thyrotropin. Cloned cDNA probes for each subunit, in conjunction with Southern blot analysis of DNA treated with the restriction enzyme BamHI, allowed for assignment of the alpha-subunit gene to mouse chromosome 4 and of the beta-subunit gene to chromosome 3. Mouse alpha-subunit gene sequences always segregated with chromosome 4 (concordant in 14 hybrids) and the enzyme markers phosphoglucomutase 2 and 6-phosphogluconate dehydrogenase. Mouse beta-subunit gene sequences always segregated with chromosome 3 (concordant in 15 hybrids). Thus, the genes for at least one of the glycoprotein hormones, thyrotropin, are on different chromosomes.

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PubMed1984

Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes.

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 large number of human-mouse cell hybrids containing overlapping subsets of human chromosomes. A correlation of genomic blotting data with the chromosome constitution of the individual cell hybrids has allowed the assignment of the human DHFR gene to chromosome 5. This chromosome assignment has been confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin, a marker associated with chromosome 5, in two human-mouse cell hybrids selected for resistance to the toxin. Six EcoRI fragments of human DNA containing DHFR pseudogenes or other DHFR-related sequences have been assigned to chromosomes other than chromosome 5.

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PubMed1984

Partial purification and characterization of the mRNA complementing a temperature-sensitive S-phase cell cycle mutation.

E36ts24 is a temperature-sensitive (ts) cell cycle mutant derived from the Chinese hamster lung cell line E36. At the restrictive temperature for growth (40.3 degrees C), the mutant cells are arrested at early S phase. We have microinjected poly(A)+ RNA isolated from the wild-type E36 cells into the cytoplasm of E36ts24 cells arrested at early S phase after 24 hr of incubation at 40.3 degrees C. The ts mutation was transiently complemented in a significant fraction of the microinjected cells as evidenced by the incorporation of [3H]thymidine assayed by autoradiography. Microinjection of mRNA fractionated by methylmercuric hydroxide/agarose gel showed that the mRNA capable of transiently complementing the mutation in E36ts24 contains about 940 nucleotides. Hence, it can code at most for a protein containing about 230 amino acids. We estimate that the partial purification by fractionation of the mRNA active in the transient complementation is on the order of 100- to 200-fold.

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PubMed1984

Chromosomal location of mouse gene 202 which is induced by interferons and specifies a 56.5 kD protein.

The 202 gene which specifies a 56.5 kD protein can be induced in Ehrlich ascites tumor cells by treatment with mouse beta interferon. This treatment increases the level of the gene 202-specific mRNA at least 12-fold. For determining the chromosomal location of this gene a 1.5 kb fragment of the gene was hybridized to EcoR1 digested DNA samples from a set of mouse-hamster somatic cell hybrids. Each of the cell hybrids used contained a complete array of hamster chromosomes and one or more mouse chromosomes. The 202 gene fragment hybridized to every DNA sample from cell hybrids containing mouse chromosome 1 (8 hybrids in total) and to none of the DNA samples from hybrids lacking this chromosome (7 hybrids in total). These and other data indicate that the 202 gene is located on mouse chromosome 1.

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PubMed1984

Chromosomal assignment of the murine gene encoding the transformation-related protein p53.

p53 is a transformation-related protein that is encoded by the cellular genome and is synthesized at elevated levels in a wide range of different cell line types and in primary tumors of various species. By using several independently established anti-p53 monoclonal antibodies, it was possible to distinguish between p53 of mouse origin and p53 of Chinese hamster origin. By analysis of a series of mouse X Chinese hamster hybrid cell lines containing various mouse chromosomes, we mapped the p53 gene product to mouse chromosome 11.

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PubMed1984

The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed.

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PubMed1984

The PKU locus in man is on chromosome 12.

Classical phenylketonuria (PKU) is a typical example of inborn errors in metabolism and is characterized by a complete lack of the hepatic enzyme phenylalanine hydroxylase, which normally converts phenylalanine to tyrosine. The genetic disorder causes impairment of postnatal brain development, resulting in severe mental retardation in untreated children. The disease is transmitted as an autosomal recessive trait and has a collective prevalence of about one in 10,000 among Caucasians, so that 2% of the population are carriers of the PKU trait. We have recently reported the cloning of human phenylalanine hydroxylase cDNA and that the human chromosomal phenylalanine hydroxylase gene is encoded by a unique DNA sequence. Using the human phenylalanine hydroxylase cDNA clone to analyze a clonal human/mouse hybrid cell panel by Southern hybridization, the phenylalanine hydroxylase gene has been assigned to human chromosome 12. Since the hypothesis that classical PKU is caused by structural mutations in the phenylalanine hydroxylase gene itself rather than through some transregulatory mechanisms has recently been confirmed by gene mapping, the PKU locus in man is determined to be on chromosome 12.

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PubMed1983

Production of interspecies T cell hybrids which retain differentiation specific surface antigens.

Genetic mapping of differentiation specific surface antigens has been hampered by difficulty in preparation of interspecies hybrid cells which continue to express differentiated functions. A method has been developed for production of interspecies T cell hybrids which continue to express T cell specific cell surface molecules. Hybrids were constructed from either the human leukemic cell line MOLT-4 or freshly isolated human peripheral blood T cells and the mouse T lymphoma line BW5147. Optimal fusion efficiency resulted with pre-treatment of the human parental line with phytohemagglutinin followed by hybridization with 40% polyethylene glycol and plating without thymocyte feeder layers. Immortalization of hybrid lines was accomplished through addition of rat T cell growth factor to cultures.

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PubMed1983

Somatic cell genetics and gene families.

The utility of somatic cell genetic analysis for the chromosomal localization of genes in mammals is well established. With the development of recombinant DNA probes and efficient blotting techniques that allow visualization of single-copy cellular genes, somatic cell genetics has been extended from the level of phenotypes expressed by whole cells to the level of the cellular genome itself. This extension has proved invaluable for the analysis of genes not readily expressed in somatic cell hybrids and for the study of multigene families, especially pseudogenes dispersed in different chromosomes throughout the genome.

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PubMed1983

Somatic cell genetics and flow cytometry.

Human genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluorescence on viable cells, the genes themselves can be monitored and manipulated using flow cytometry and sorting. In this paper, we review ways that we have used cell sorting to develop a somatic cell genetic analysis of the human cell surface.

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PubMed1983

The genes coding for the cardiac muscle actin, the skeletal muscle actin and the cytoplasmic beta-actin are located on three different mouse chromosomes.

The actins are a group of highly conserved proteins encoded by a multigene family. We have previously reported that the skeletal muscle actin gene is located on mouse chromosome 3, together with several other unidentified actin DNA sequences. We show here that the gene coding for the cardiac muscle actin, which is closely related to the skeletal muscle actin (1.1% amino acid replacements), is located on mouse chromosome 17. The gene coding for the cytoplasmic beta-actin is located on mouse chromosome 5. Thus, these three actin genes are located on three different chromosomes.

Actins↗
PubMed1983

Assignment of murine cellular Harvey ras gene to chromosome 7.

Mouse-Chinese hamster somatic cell hybrids containing various combinations of mouse chromosomes were analyzed for the presence of the mouse c-Ha-ras (1) sequences after restriction endonuclease digestion and hybridization with a 32P-labeled Ha-ras specific probe according to the procedure of Southern (2). The presence of the mouse c-Ha-ras containing fragment was correlated with the presence of mouse chromosome 7 in the hybrids.

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