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Biomedical subjects

R Sheinin

Publications and source records attributed to R Sheinin.

At least 19 recordsLinked to original sources
PubMed1991

Effect of transfection manipulations on mouse cell cycle progression.

BalB/C-3T3 mouse fibroblasts and a temperature-sensitive derivative, ts 2e, were transfected by the calcium phosphatedimethyl sulphoxide procedure to examine the effect of this manipulation on cell cycle progression. Cells were synchronized by growth to confluence in the presence of [2-14C]thymidine to generally label cellular DNA, and then subcultured from the G0 state. Plasmid pSV3-neo or pSV2-neo DNA was added to cells at 24 h post-plating, at peak S phase. At designated intervals prior to, during, and after the transfection procedure, cells were labelled with [methyl-3H]thymidine for 1 h to monitor nascent DNA synthesis and thereby assess cell cycle position. In all experiments performed, irrespective of the time of DNA addition, the transfection manipulations resulted in a reproducible, transient interruption of cell cycle progression, of about 5 h, and manifested as a delay in movement across the subsequent G1-S interface. Thereafter, the cycle resumed normally. The results indicated that the temporal sequence of the cell duplication cycle is altered when cells are exposed to exogenous DNA:Ca3 (PO4)2.

3T3 Cells↗
PubMed1990

Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication.

The temperature-sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human-ts A1S9 somatic cell hybrids. We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus. The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons. Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast. Northern blot analysis of serum-starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle. Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.

Amino Acid Sequence↗
PubMed1990

DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication during cell cycle progression.

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.

Animals↗
PubMed1990

Localization of the human A1S9 gene complementing the ts A1S9 mouse L-cell defect in DNA replication and cell cycle progression to Xp11.2----p11.4.

The temperature-sensitive ts A1S9 mouse L-cell mutant is defective in an X-linked gene essential for progression of cells through the S phase of the cell division cycle. A single copy fragment derived from the complementing human A1S9 gene was used as a probe to localize the gene on the X chromosome. Southern blot analysis of human x rodent hybrids and in situ hybridization to human metaphase chromosomes allowed the regional assignment of the human A1S9 gene to Xp11.2----p11.4.

Animals↗
PubMed1989

Molecular cloning of human A1S9 locus: an X-linked gene essential for progression through S phase of the cell cycle.

The temperature-sensitive (ts) A1S9 mouse L-cell mutant is defective in an X-linked gene essential for the progression of cells through the S phase of the cell duplication cycle. We recently reported the complementation of the ts A1S9 cell defect with total human DNA and the isolation of independent temperature-resistant transformants that retained a common set of human specific Alu-containing fragments. Here we describe the molecular cloning of these human DNA sequences from one of the secondary transformants. ST-1-0. A genomic library prepared from ST-1-0 was screened with a total human DNA probe, and two recombinant bacteriophages carrying overlapping segments were isolated. The cloned region was extended in both directions using a human X-chromosome specific library. In total, a human region spanning 42 kb in length, and containing all the Alu-specific DNA sequences found in ST-1-0, was isolated in five overlapping recombinant phages. The A1S9 gene appeared to be larger than the DNA recovered in individual phage isolates, as was assessed by transfection experiments. A single-copy probe derived from the phage DNA was shown to be conserved in independent primary, secondary, and tertiary transformants of ts A1S9 cells and mapped to the X chromosome by molecular hybridization. Northern blot hybridization of this probe with poly(A)+ mRNA derived from ST-1-0 cells identified a transcript of about 3.6 kb.

Bacteriophages↗
PubMed1989

Gene on short arm of human X chromosome complements murine tsA1S9 DNA synthesis mutation.

We have created somatic cell hybrids between the temperature-sensitive mouse cell line tsA1S9 and human cell lines in order to localize the human gene (A1S9T) complementing the cell cycle defect of the murine line. Segregation of the human X chromosome is completely concordant with growth at the nonpermissive temperature. Hybrids retaining the X chromosome are temperature-resistant, whereas those without a human X are temperature-sensitive. Further hybrids made using human cell lines with X-autosome translocations indicate that the A1S9T gene is located on the short arm of the human X chromosome.

Animals↗
PubMed1988

Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer.

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.

Animals↗
PubMed1988

Pearl Jane Manson Sproule, MD (1879-1961) Canada's first woman otolaryngologist: a belated tribute.

Pearl Jane Manson enrolled in the Ontario Medical College for Women in 1904 and graduated with an M.D. degree from the University of Toronto in 1907. She was admitted to the Royal College of Surgeons of England in 1911 and worked at a hospital in England during World War I. On returning to Toronto she held positions both at Toronto General Hospital and Women's College Hospital, where she became Chief of the ENT Department in 1926. She was keenly interested in epidemiology and the prevention of infections in women and children. In 1931 she became a Fellow of the Royal College of Physicians and Surgeons of Canada and also the American College of Surgeons. She was the first woman physician in Canada to become a specialist and gain recognition as a clinical scientist.

Canada↗
PubMed1986

DNA synthesis in BalB/C-3T3 ts 2 cells is restricted by a temperature-sensitive function of late G1 phase.

ts 2 BalB/C-3T3 mouse fibroblasts are cdc mutants, which arrest late in G1, at or near the G1/S traverse, upon full expression of the heat-sensitive lesion. The kinetics of temperature inhibition of DNA synthesis in logarithmically growing cultures reveal three stages of heat inactivation. During the first generation time equivalent, normal semiconservative, semidiscontinuous replication proceeds but is reduced as cells exit and do not reenter S phase. During a second such period, a minimal rate of normal DNA synthesis is maintained. Thereafter, as the cells move into a third aborted cell division cycle, the rate of DNA synthesis increases. However, all semiconservative synthesis is then replaced by DNA repair replication. Temperature inactivation of the ts 2 protein results in shutdown of nuclear DNA synthesis. In contrast, normal replication of mitochondrial DNA proceeds at control rate throughout the first stage of temperature inactivation. Synthesis of this organellar genome is quantitatively reduced as the cells move into the second phase of heat inhibition. Titration of chromatin-bound DNA with ethidium bromide revealed that wild-type cells exhibit a changing DNA topology as the temperature is raised. Temperature-inactivated ts 2 cells behave as though their DNA has been topologically frozen in the configuration of control cells at or near entry into S phase.

Animals↗
PubMed1985

Cell cycle arrest of heat-inactivated ts 2 BALB/c-3T3 mouse fibroblasts, temperature-sensitive for DNA synthesis.

The ts 2 derivative of BALB/c-3T3 mouse fibroblasts is a cell division cycle (cdc) mutant. Upon expression of the heat-sensitive defect, ts 2 cells arrest late in G1 at, or very near the G1/S traverse. This conclusion derives from three kinds of experiment. In the first the cells were brought to different stages of the cell cycle by physiological manipulation, or with specific anti-metabolites. They were then released from the resulting blocks, and their subsequent cell-cycle progression, at the permissive- and non-permissive temperature (npt), was followed. The second experiment was an execution point analysis. In the third, premature chromosome condensation was performed between metaphase HeLa cells and temperature-blocked ts 2 cells. The resulting prematurely-condensed chromosomes were largely of the morphotype of very late G1 cells. The ts 2 cells are prevented from expressing their defect by temporary incubation at 38.5 degrees C in the G0, non-cycling state and by prior arrest in early S phase, imposed by hydroxyurea treatment. Such prevention is not allowed ts 2 cells incubated at the npt in the absence of isoleucine, a procedure which brings cells to mid-G1 arrest.

Animals↗
PubMed1985

Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

Adenoviridae↗
PubMed1983

ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity.

Nuclear novobiocin binding proteins (NBPs) from a set of mouse L cells have been extensively purified by affinity chromatography on novobiocin-Sepharose columns. The NBPs, specifically eluted with 100 micrograms of novobiocin per ml, exhibited equivalent DNA topoisomerase activities (measured as ATP-dependent relaxation or catenation of phi X174 replicative-form I DNA substrate) when extracted from equal numbers of wild-type (WT-4) mouse L cells growing logarithmically at 34 degrees C or at 38.5 degrees C, from ts A1S9 cells similarly cultivated at the low, permissive temperature or from revertant ts+ AR cells in exponential growth at either temperature. The NBPs isolated from similar numbers of ts A1S9 cells grown to midlogarithmic phase and then incubated for 24 hr at 38.5 degrees C (the nonpermissive temperature) showed no topoisomerase II activity. Preliminary NaDodSO4/polyacrylamide gel electrophoretic analysis of enzymatically active material revealed that the NBPs of WT-4 and ts+ AR cells grown at 34 degrees C comprised three major polypeptides of 76,000, 74,000, and 30,000 daltons and a number of larger molecular mass components present in trace amounts. The NBP of ts A1S9 cells grown at the permissive temperature was similar, except that the 30,000-kilodalton polypeptide was not detected. Such enzymatically active NBPs from WT-4 and ts+ AR cells were unaffected by 100 micrograms of novobiocin per ml, whereas the analogous preparation from ts A1S9 cells was totally inhibited. On the basis of these and other considerations, it is postulated that the ts A1S9 locus of mouse L cells encodes a temperature-sensitive polypeptide that is required for normal DNA topoisomerase II activity.

Animals↗
PubMed1983

Synthesis of multimeric polyoma virus DNA in mouse L-cells: role of the tsA1S9 gene product.

Several different forms of progeny viral DNA can be identified in polyoma virus (Py)-infected mouse L-cells. The majority comprise mature form I superhelical DNA and the circular, double-stranded "theta" replicating intermediates in which the progeny DNA strands never exceed the unit genome length of the template. There is formed, in addition, a minority fraction of multimeric, linear, double-stranded Py DNA molecules that sediment heterogeneously at 28 to 35S and greater than 35S. Restriction enzyme analysis of these large Py DNA molecules reveals them to be tandem arrays of multiple unit genome lengths, covalently linked head to tail. It is estimated that the 28 to 35S multimeric DNA has an average size of about 20 megadaltons, made up of 6 to 20 Py genome units. The greater than 35S Py DNA is, of course, larger. Kinetic analysis indicates that formation of the monomeric progeny viral DNA and the 28 to 35S multimeric Py DNA reaches a peak at about 35 to 36 h postinfection. Synthesis of the very large linear molecules of greater than 35S is first detected after this interval and continues thereafter. The de novo synthesis of all of these progeny Py DNA molecules proceeds apparently normally in Py-infected tsA1S9 mouse L-cells incubated at 38.5 degrees C under conditions which restrict normal cellular DNA replication. These findings suggest that the cellular DNA topoisomerase II activity, encoded in the tsA1S9 locus (R. W. Colwill and R. Sheinin, submitted for publication), is not required for de novo formation of any form of Py DNA. However, the total amount made and the rate of synthesis of the large molecular weight Py DNA are affected very late in temperature-inactivated tsA1S9 cells.

Animals↗
PubMed1981

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis.

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).

Animals↗
PubMed1980

Protein synthesis and degradation during expression of the temperature-sensitive defect in ts A1S9 mouse L-cells.

The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins.

Animals↗