Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol

Davidson, Richard L.; Gerald, Park S.

doi:10.1007/bf01542629

Cited by 513 publications

(172 citation statements)

References 15 publications

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“…In the following experiments hybrids designated A and B were derived I~' t *82 (Emr-2/6-TGr)-1 and (Emr-2/6-TGr)-2 <2 X 10-6 (Emr-2/6-TGr)-1 and (Emr-2/6-TGr)-1 <2 x 10-6 (Emr-2/6-TGr)-2 and (Emr-2/6-TGr)-2 2 X 10-6 EmS/TK-and EmS/TK-6 X 10-6 Mixtures of 106 parental cells of each clone indicated were inoculated into 60-mm culture dishes in nonselective medium. After one day, the monolayer cultures were treated with 5096 (wt/wt) PEG-6000 for 60 sec at room temperature (6) (i) As anticipated from the sum of the parental clones' chromosome numbers (Table 2), hybrids contained 37-89 chromosomes. This was consistent with the hypothesis that each hybrid clone arose from the fusion of one 6&TGr cell (n = 21) with one TK-cell (n = 18).…”

Section: Resultsmentioning

confidence: 99%

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Emetine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, S20.

Boersma¹,

McGill²,

Mollenkamp³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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Emr-2 is an emetine-resistant (Emr) Chinese hamster ovary cell mutant that contains a single electrophoretically altered ribosomal protein, a component of its 40S ribosomal subunit [Boersma, D., McGill, S., Mollenkamp, J. & Roufa, D. J. (1979) J. Biol. Chem. 254, in press]. This report describes a genetic experiment designed to test linkage between the genes that specify the altered ribosomal protein, S20*, and emetine resistance in Emr clones that segregated from cultures of Emr-2 cells hybridized with emetine-sensitive cells. The data described indicate that Emr and S20* phenotypes are due to mutations linked to the same chromosome in the Chinese hamster genome; most likely they are due to the same mutation. The data also confirm earlier speculations by others that the Emr locus in Chinese hamster cells is hemizygous. Several Chinese hamster cell mutants that express genetically altered ribosomal functions have been isolated in our laboratory. Within this collection are nine mutagen-induced clones whose ribosomes are resistant to emetine (Emr), an alkaloid drug inhibitor of protein biosynthesis (1). We used two-dimensional polyacrylamide gel electrophoresis to analyze 72 proteins extracted from Emr mutants' ribosomes. We observed that only one ribosomal protein prepared from mutant clone Emr-2 was altered in a manner detectable by our assay. The altered protein, S20*, a component of the 40S ribosomal subunit, differs from wild-type S20 protein in its electrophoretic migration at pH 5. S20 and S20* migrated with identical mobilities in buffer containing sodium dodecyl sulfate (i.e., appeared to possess the same molecular weight) (1). These observations were consistent with S20* having arisen by a point mutation. The identification of an altered 40S ribosomal subunit protein from an Emr clone agreed with other investigators' assignment of emetine resistance in Chinese hamster cells to the 40S ribosomal subunit (2).Three genetic models could account for the relationship between the altered ribosomal protein S20* and Emr-2's Emr phenotype: (i) Emetine resistance might be independent of the alteration in S20 protein.(ii) The mutation responsible for emetine resistance might affect a gene coding for a protein that modifies S20 protein. S20* thus might be the nonmodified, electrophoretically distinguishable precursor to normal S20. (iii) The structural gene that codes for S20 protein might contain a mutation responsible for both S20* and the mutant's resistance to emetine.The electrophoretic and genetic experiments described in this report were designed to investigate the relationship between S20* protein and the Emr phenotype of clone Emr-2. We fused a derivative of Emr-2 with emetine-sensitive (Ems) Chinese hamster cells and selected several approximately tetraploid hybrid clones. Spontaneous Emr mitotic segregants were isolated from hybrid clones, and proteins from the 40S ribosomal subunits of hybrid and segregant clones were analyzed by two-dimensional polyacrylamide gel electrophoresis. By these experi...

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Section: Resultsmentioning

confidence: 99%

“…Cells were fused by cocultivating 106 cells of each parental clone in a 60-mm dish and treating them with 50% (wt/wt) PEG-6000 (6). Hybrid clones were selected in medium containing 100 ,gM hypoxanthine, 5 ,tM amethopterin, and 20 1,M thymidine (HAT) (5).…”

Section: Methodsmentioning

confidence: 99%

Emetine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, S20.

Boersma¹,

McGill²,

Mollenkamp³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…A panel of 16 mouse-human somatic cell hybrids was derived by polyethylene glycol-mediated fusion of mouse B82 cells (GM 0347A) and human male fibroblasts (IMR91), using standard procedures (26)(27)(28)(29). These clones were grown up in multiple dishes for DNA extraction and chromosome studies.…”

Section: Methodsmentioning

confidence: 99%

Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1, -C2, and -E and LDLR) and the genes C3, PEPD, and GPI.

Lusis¹,

Heinzmann²,

Sparkes³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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We report the regional mapping of human chromosome 19 genes for three apolipoproteins and a lipoprotein receptor as well as genes for three other markers. The regional mapping was made possible by the use of a reciprocal whole-arm translocation between the long arm of chromosome 19 and the short arm of chromosome 1. Examination of three separate somatic cell hybrids containing the long arm but not the short arm of chromosome 19 indicated that the genes for apolipoproteins CI, CII, and E (APOCI, APOC2, and APOE, respectively) and glucose-6-phosphate isomerase (GPI) reside on the long arm, whereas genes for the low density lipoprotein receptor (LDLR), complement component 3 (C3), and peptidase D (PEPD) reside on the short arm. When taken together with previous studies, our results suggest the following physical gene map: pter-LDLR-C3-pl3.2-PEPD-centromere-(APOE, APOCI, APOC2, GPI)-qter. In addition, we have isolated a single A phage carrying both APOCI and part ofAPOE. These genes are tandemly oriented and are separated by about 6 kilobases of genomic DNA. Since previous family studies indicate tight linkage of APOE and APOC2, the apolipoprotein genes APOCI, APOC2, and APOE form a tight complex on the long arm of chromosome 19, suggesting the possibility of coordinate regulation.

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“…Transport assays using digitonin-permeabilized cells and recombinant proteins are very instructive and allow detailed complementation studies (9). The nuclear translocation of endogenous proteins can be studied by inducing the formation of interspecies heterokaryons (10). This relatively simple and widely used assay is helpful in detecting nucleocytoplasmic shuttling activity, but further analyses such as kinetic studies of flux rates are not possible.…”

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confidence: 99%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-b antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-a, but by diverse mechanisms. The translocating antibody blocked the association with importin-a, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-a binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives. ' 2008 International Society for Advancement of Cytometry Key terms antibody; native protein; microinjection; nucleocytoplasmic shuttling; import; export; GFP TRANSPORT of proteins and macromolecules between the nucleus and the cytoplasm of eukaryotic cells is an important mechanism for cytoplasmic regulation of nuclear activities. Nucleocytoplasmic exchange occurs through specialized channels called ''nuclear pore complexes'' (NPCs), which perforate the double layer of the nuclear envelope (1). The NPCs function as selectivity filters allowing passive diffusion of molecules up to $40 kDa, whereas components of higher molecular weight are usually restricted from autonomous exchange (2,3). A well-characterized pathway for nuclear transport of proteins relies on the GTPase Ran and the transport factors (also called carriers or karyopherins) importin-a and importin-b or CRM1 for import or export, respectively (4,5). In addition, certain large proteins can enter the nucleus, independent of metabolic energy and transport factors via direct interactions with constituents of the nuclear pore (6), and an increasing number of proteins, e.g. STAT1 and importin-b are known to use both pathways consecutively or i...

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Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol

Cited by 513 publications

References 15 publications

Emetine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, S20.

Emetine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, S20.

Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1, -C2, and -E and LDLR) and the genes C3, PEPD, and GPI.

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

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