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1.
The nature of thymidine kinase in the human-mouse hybrid cell 总被引:7,自引:0,他引:7
In order to characterize the thymidine kinase in human-mouse somatic cell hybrids derived from mouse parental cells lacking thymidine kinase, we have examined the electrophoretic migration on starch gel and the heat sensitivity of this enzyme in human, mouse, and hybrid cells. The enzyme of hybrid cells migrates similarly to that of human fetal liver and human diploid fibroblasts and faster than that of either L or A9 mouse cells. It is less sensitive to heat than that of the mouse cells. Therefore, the human group E chromosome provides human thymidine kinase for the hybrid cell. The electrophoresis of thymidine kinase makes possible the search for variants.Aided by U.S.P.H.S. Grant No. HD 00486. 相似文献
2.
Regional localization of human gene loci on chromosome 9: studies of somatic cell hybrids containing human translocations.
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T Mohandas R S Sparkes M C Sparkes J D Shulkin K E Toomey S J Funderburk 《American journal of human genetics》1979,31(5):586-600
Somatic cell hybrids were derived from the fusion of (1) Chinese hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) and human cells carrying an X/9 translocation and (2) Chinese hamster cells deficient in thymidine kinase (TK) and human cells carrying a 17/9 translocation. Several independent primary hybrid clones from these two series of cell hybrids were analyzed cytogenitically for human chromosome content and electrophoretically for the expression of human markers known to be on human chromosome 9. The results allow the assignment of the loci for the enzymes galactose-1-phosphate uridyltransferase (GALT), soluble aconitase (ACONs), and adenylate kinase-3 (AK3) to the short arm of chromosome 9 (p11 to pter) and the locus for the enzyme adenylate kinase-1 (AK1) to the distal end of the long arm of human chromosome 9 (hand q34). Earlier family studies have shown that the locus for AK1 is closely linked to the ABO blood group locus and to the locus of the nail-patella (Np) syndrome. Thus the regional localization of AK1 locus permits the localization of the AK1-Np-ABO linkage group. 相似文献
3.
Interspecific hybridization between human and mouse somatic cells: Enzyme and linkage studies 总被引:8,自引:0,他引:8
Human-mouse somatic cell hybrids have been isolated and examined for enzyme and chromosome constitution. The enzymes assayed were lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH), NADP-dependent malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucomutase (PGM), and several esterases. Coexpression of mouse and human genomes and formation of heteropolymeric enzymes were observed in seven different hybrid populations for the enzymes LDH, IDH, MDH, and G6PD. Evidence predicated on the absence of chromosomal rearrangements is provided for the lack of genetic linkage in the human genome for these four enzymes, as well as for thymidine kinase.Supported in part by NIH Grants GM 09966 and 1-F1-GM-39,399 from the Institute of General Medical Sciences and by NIH Training Grant HD-32. 相似文献
4.
D. L. Devore-Carter E. Pietrzak S. Kakati 《In vitro cellular & developmental biology. Plant》1986,22(10):615-620
Summary A method is described to generate microcells from human lymphobalsts for use in microcell-mediated chromosome transfer (MMCT).
Micronuclei were induced in cells from a human lymphoblastic cell line by prolonged colcemid treatment, and were separated
from these lymphoblasts by: (a) attaching the cells to Concanvalin A coated plastic slides designed for enucleation, and (b)
centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 μm in diameter were fused with thymidine
kinase negative mouse fibroblast (LMTK−). HAT medium (hypoxanthine, aminopterin and thymidine) was used to select microcell hybrids expressing thymidine kinase activity.
Positive clones were isolated and Q-banded for chromosome analysis. Unlike previous methods this procedure permits microcells
to be easily generated from lymphoid cells. This methodology of enucleation of microcells may be extended to a variety of
other donor cell types which can be micronucleated but which do not adhere tightly to enucleation slides and do not exhibit
extrusion subdivision. This feature makes our methodology particularly useful for constructing a library of hybrid clones
containing one or a few human chromosomes. 相似文献
5.
Metaphase distribution of the mouse chromosomes 总被引:1,自引:1,他引:0
The reports in the literature agree that non-random distribution patterns do occur for the acrocentric human chromosomes in metaphase cell preparations, and it has been suggested that it is a property of acrocentric chromosomes that promotes these non-random patterns. Under this hypothesis, the telocentric chromosomes of the mouse should not show deviation from a random distribution within a cell. This possibility is examined using our data for several types of mouse cells and there is no indication of any significant clustering. However, certain translocations do appear to lead to significant non-random patterns. Alternate hypotheses are presented as possible explanations for this occurrence.This project was supported by: California State Department of Mental Hygiene; Mental Retardation Program, NPI, UCLA; MCH-927, Interdisciplinary Training in Mental Retardation; HD-04612, Mental Retardation Research Center, UCLA; HD-00345, Research Training in Mental Retardation; HD-05615, Developmental Biology in Mental Retardation, and Cancer Research Funds of the University of California. 相似文献
6.
R L Nussbaum R M Walmsley J G Lesko S D Airhart D H Ledbetter 《American journal of human genetics》1985,37(6):1192-1205
Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31----qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites. 相似文献
7.
Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method 总被引:5,自引:0,他引:5
The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415). 相似文献
8.
Summary Cultures of embryonic mouse and rat spinal cord-dorsal root ganglion combinations, and of newborn hamster and rat cerebellum were treated with 5×10-6M I-125 thyroxine and I-125 triiodothyronine. Autoradiographic studies on glutaraldehyde-osmium tetroxide fixed tissue indicated that these hormones were located predominantly in neurons, but could be found in glia cells as well. This pattern was observed in cultures treated for 2 hours to 4 days. Some of the effects of thyroid hormones on the developing nervous system were therefore linked to a direct action of these hormones on nerve cells.Furthermore, the label was seen not only over the cytoplasm, as might be expected from past biochemical studies, but was also present over the nucleus, particularly the nucleolus. This suggested a possible role for these hormones in the process of translation.This work was supported by NIH Grant NB06735, The Nancy Louise Trynor Memorial Grant for Research on Multiple Sclerosis 433-D-10, and United States Public Health Service Grant No. NB-07849-02.Post-Doctoral Fellow, Department of Pathology, Yale University.Kennedy Scholar of the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, New York. 相似文献
9.
Molecular genetic analysis of recessive mutations at a heterozygous autosomal locus in human cells 总被引:5,自引:0,他引:5
We have investigated the genotypic changes that lead to expression of a recessive allele at a heterozygous autosomal locus in a human cell line. Mutant clones lacking thymidine kinase activity were derived from a B-cell lymphoblastoid line initially heterozygous at the tk locus, and restriction mapping was performed to detect intragenic structural alterations in the tk gene. In addition, informative molecular markers located elsewhere on chromosome 17 were analysed in order to detect large-scale (multilocus) events. We report that among 325 spontaneous and induced mutants, allele loss was more common than intragenic rearrangements or point mutations; in many cases, loss of heterozygosity appears to have extended well beyond the locus under selection. Cytogenetic analysis of a subset of these mutants showed that expression of the recessive TK-deficient phenotype and the associated loss of heterozygosity for chromosome 17 markers was not typically associated with detectable chromosomal changes. 相似文献
10.
Human lysosomal genes: Arylsulfatase A and β-Galactosidase 总被引:12,自引:0,他引:12
G. A. P. Bruns B. J. Mintz A. C. Leary V. M. Regina P. S. Gerald 《Biochemical genetics》1979,17(11-12):1031-1059
The segregation of human lysosomal arylsulfatase A (ARS-A) has been evaluated in 50 primary hybrid clones derived from four separate fusions involving WBCs from two unrelated individuals and three hamster cell lines. ARS-A was expressed in the hybrids as a dimeric molecule of very similar or identical subunits. The expression of this enzyme was concordant with that of mitochondrial aconitase (ACON-M), an isozyme assigned to chromosome 22, in all 50 clones and with chromosome 22 segregation in all but one of the 29 karyotyped hybrids. No other human chromosome cosegregated with 22 in these clones, suggesting that this enzyme is specified in hybrid cells by a locus (or loci) on a single chromosome. beta-Galactosidase (B-GAL) expression was analyzed with two different electrophoresis systems and with a number of cell extract preparation methods in 39 of the primary hybrid clones. The B-GAL isozyme expressed in these hybrid cells was concordant with the expression of glutathione peroxidase-1 (GPX-1), an isozyme assigned to chromosome 3, in all 39 clones and with the segregation of this chromosome in 97% of the 29 karyotyped hybrids. These observations substantiate the prior tentative assignments of an ARS-A locus to chromosome 22 and a B-GAL locus to chromosome 3 (Bruns et al., 1978a, b). The implications of the chromosome assignments of loci for 12 human lysosomal enzymes for the cellular assembly of these organelles are discussed. 相似文献
11.
Mapping of a vaccinia host range sequence by insertion into the viral thymidine kinase gene. 总被引:6,自引:4,他引:2
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A vaccinia virus mutant deleted of ca. 18 kilobase pairs at the left-hand end of the genome is unable to multiply on many human cell lines. To determine whether all or some of the deleted sequences were responsible for the host range property, the corresponding region from wild-type DNA was cloned in three pieces into a vaccinia transplacement vector containing the thymidine kinase gene on the HindIII J fragment. The next step was to transfer these pieces to the genome of the host range deletion mutant by in vivo homologous recombination around the thymidine kinase locus. Transfer of one 5.2-kilobase-pair EcoRI fragment was found to restore a wild-type phenotype on the host range mutant, thus demonstrating that only a small portion of the 18-kilobase-pair deletion contains the host range function(s). This result also illustrates that the method initially devised for inserting foreign genes into vaccinia virus DNA is useful for studies of the vaccinia genome. 相似文献
12.
13.
Dwight E. Wilson Mark A. McKinlay John Staczek Carol Whitkop Betty Harrison Susan Povey 《Biochemical genetics》1980,18(9-10):981-1001
The herpes simplex type 1 biochemically transformed human cell line, HB-1, was fused with thymidine kinase deficient rodent cells, and 18 hybrids were isolated using the HAT-ouabain selection system. The selected enzyme, viral thymidine kinase, was present in all 18 hybrids. In 16 of 18 hybrids the viral gene for thymidine kinase cosegregated with the human gene for adenylate kinase-1 (AK-1). Thirty-six bromodeoxyuridine (BrdUrd) resistant sublines were isolated from the 16 human AK-1 positive hybrids. Each BrdUrd-resistant subline was examined for the presence of the viral TK gene by back-selection in HAT medium, and for human AK-1. In all 36 BrdUrd-resistant sublines the viral TK gene cosegregated with the human AK-1 gene. These results indicate that the transforming viral DNA fragment was associated with a specific human chromosomal region in HB-1 cells. 相似文献
14.
The Locus for Human Adenine Phosphoribosyltransferase on Chromosome No. 16 总被引:2,自引:0,他引:2
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Evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT) to human chromosome No. 16 is presented. Hybrids of APRT-deficient mouse cells and of human fibroblasts having normal APRT were isolated by fusing the parental cells with Sendai virus, blocking de novo purine nucleotide synthesis with azaserine and selecting for hybrids that could use exogenous adenine. The hybrid clones that were studied had only APRT activity that was indistinguishable from human APRT with regard to electrophoretic migration and reaction with antibodies against the partially purified human enzyme. No. 16 was the only human chromosome consistently present in all of the clones, and in one clone, it was the only human chromosome detected. Selection against hybrid cells with 2,6-diaminopurine (DAP) yielded DAP-resistant survivors that lacked chromosome No. 16. One hybrid that originally had an intact No. 16 yielded adenine-utilizing subclones that lacked No. 16 but had a new submetacentric chromosome. The distribution of centromere-associated heterochromatin and the fluorescence pattern indicated that this chromosome consisted of a mouse telocentric chromosome and the long arm of No. 16. Cells having the submetacentric chromosome had human APRT. Both the enzyme and the chromosome were absent in DAP-resistant derivatives. These results suggest that the structure of APRT is defined by a locus on the long arm of human chromosome No. 16. 相似文献
15.
Taylor Harold A. Riley Susan E. Parks Stephen E. Stevenson Roger E. 《In vitro cellular & developmental biology. Plant》1978,14(5):476-478
Summary The establishment of cultured cell lines from skin biopsies stored at −196°C for periods up to 1 year has been investigated.
Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome
constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples.
This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable
cells when it is impossible or impractical to initiate a cell culture immediately.
This project was supported by the South Carolina Department of Mental Retardation and the Self Foundation. 相似文献
16.
Andrew J. Griffith Daniel L. Burgess David C. Kohrman Jennifer Yu Jillian Blaschak Susan H. Blanton Michael Boehnke Jacqueline T. Hecht Joan Overhauser Miriam H. Meisler 《Genomics》1996,34(3):299
The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locusax.To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci. 相似文献
17.
Martin K. Feldman Donald L. Wong 《In vitro cellular & developmental biology. Plant》1977,13(5):275-279
Summary Mammary epithelil cells from 16- to 17-day pregnant BALB/c mice were cultured in various mammalian sera to determine the kind
of serum which stimulates optimal attachment efficiency and thymidine incorporation. Of those sera tested, horse, bovine,
lamb, goat and fetal bovine provided the highest attachment efficiency, whereas rat, mouse and human gave the lowest. Rabbit
serum stimulated the highest thymidine incorporation into TCA-insoluble material with goat and rat providing the lowest. These
results suggest that sera which provide the highest attachment efficiency for primary cultures are not the best stimulants
of DNA synthesis and show that an inverse relationship exists between cell attachment and thymidine incorporation for any
given type of mammalian serum.
This research was supported in part by Grant No. CA 15764 from the National Cancer Institute, National Institutes of Health,
DHEW. 相似文献
18.
The expression of human mitochondrial thymidine kinase (mt TK) was investigated by polyacrylamide electrophoresis in 19 independent human-mouse somatic cell hybrids which allowed all human chromosomes to be analyzed. In 8 hybrid clones the presence of this enzymatic activity could be demonstrated. Human mt TK segregated concordantly with human adenine phosphoribosyltransferase (APRT) and human chromosome 16. Discordant segregation with all other human chromosomes was demonstrated by karyotype and isozyme analyses. These results suggest that human mt TK is coded for by a gene on chromosome 16 of the nucleus. Thus human mt TK is genetically different from human cytosol thymidine kinase which is coded for by a gene on chromosome 17. The appearance of one heteropolymer band after electrophoretic separation of human and murine mt TK supports the notion that both enzymes have dimeric structures. 相似文献
19.
B Shafit-Zagardo E A Devine M Smith F Arredondo-Vega R J Desnick 《American journal of human genetics》1981,33(4):564-575
The structural gene for human acid beta-glucosidase (GBA) has been assigned to chromosome 1 using somatic cell hybridization techniques for gene mapping. The human enzyme was detected in mouse RAG cell-human fibroblast cell hybrids by a sensitive double antibody immunoprecipitation assay using a mouse antihuman GBA antibody. No cross-reactivity between mouse beta-glucosidase and human GBA or neutral beta-glucosidase (GBN) was observed. Fifty-two primary, secondary, and tertiary manmouse hybrid lines, derived from three separate fusion experiments, were analyzed for human GBA and enzyme markers for the human chromosomes. Without exception, the presence of human GBA in these hybrid clones was correlated with the presence of human chromosome 1 or its enzymatic markers, phosphoglucomutase 1 (PGM1), and fumarate hydratase (FH). All other human chromosomes were eliminated by the independent segregation of GBA and their respective enzyme markers and/or chromosomes. Using a RAG X human fibroblast line with a mouse-human rearrangement of human chromosome 1, the locus for GBA was limited to the region 1p11 to 1qter. 相似文献
20.
Regional chromosome mapping of the human skin type I procollagen gene using adenovirus 12-fragmentation of human-mouse somatic cell hybrids 总被引:8,自引:0,他引:8
Prevous work, using human-mouse somatic cell hybrids, has localized the structural gene for human skin type I procollagen (COL 1) to chromosome 17. One of these hybrids contained only the long arm of human chromosome 17, translocated onto a mouse chromosome, as human chromosomal material. This hybrid was treated with adenovirus 12, and various clones were picked which contained different-sized fragments of human chromosome 17 that were still translocated onto a mouse chromosome. Measurements of these fragments, combined with assays for human COL 1 production and galactose kinase (GAK) activity (also localized on the long arm of human chromosome 17), has allowed us to regionally map the structural gene for human COL 1 to an area just distal to the thymidine kinase (TK) and GAK genes within bands q21 and q22 on human chromosome 17. 相似文献