Search PubMed⌕ Search

Biomedical subjects

A Weissbach

Publications and source records attributed to A Weissbach.

At least 55 records · Page 3Linked to original sources
PubMed1977

Study of DNA synthesis in chromatin isolated from HeLa cells.

When incubated in vitro, HeLa cell chromatin can synthesize DNA at rate comparable to that observed with isolated nuclei. The in vitro DNA synthetic activity of chromatin reflects DNA synthesis in intact cells since chromatin from cells in S phase are several times more active thatn preparations derived from mitotic cells. The requirements for the synthesis of DNA by chromatin preparations are also similar to those of isolated nuclei and the size of the DNA pieces made in both systems is roughly comparable. The chromatin system offers several advantages not available with isolated nuclei. Chromatin will synthesize DNA for a much longer time than isolated nuclei so that larger amounts of DNA can be synthesized in vitro In addition, although chromatin has its own endogenous ability to synthesize DNA, it is markedly stimulated by the presence of exogenously added HeLa cell DNA polymerase alpha, beta, and gamma, and, thus, may provide a new template system for the study of DNA synthesis.

Cell Nucleus↗
PubMed1977

HeLa cell DNA polymerases: the effect of cycloheximide in vivo and detection of a new form of DNA polymerase alpha.

Blockage of protein synthesis in HeLa cells by cycloheximide leads to selective effects on the levels of DNA polymerases alpha, beta, and gamma in the cell. The total activity of DNA polymerase alpha remains unchanged after 7 h exposure of cells to cycloheximide but drops to 50% of its original level after 24 h. The level of the beta-polymerase falls rapidly in the cell and is reduced to less than 30% of its initial value by 7 h after treatment of the cells with cycloheximide. The gamma-polymerase level is diminished by 30--40% during the 7 h cycloheximide treatment and reaches 50% of its original level after 24 h. Cells which have been exposed to cycloheximide for 7 h will regain normal levels of the beta- and gamma-polymerases within 90 min after removal of the drug. The cycloheximide-treated cells also show the presence of a new form of the alpha-polymerase, designated alpha1, which can be clearly detected as a separate entity in column chromatography. The level of alpha1 in the nucleus increases during the period that the cells are treated and cycloheximide so that after 24 h it represents almost 50% of the nuclear DNA polymerase activity. The presence of alpha1 in the cytoplasmic fraction can also be demonstrated in both cycloheximide-treated and normal, growing cells.

Cell Nucleus↗
PubMed1977

DNA polymerase of mitochondria is a gamma-polymerase.

Mitochondria isolated from rat liver cells or mycoplasma-free HeLa cells contain a single DNA polymerase activity which is closely related to, or identical to, the DNA polymerase gamma activity found in the homologous cell. In rat liver cells, about 16% of the total cytoplasmic gamma-polymerase activity is found associated with mitochondria and in HeLa cells about 20% of the total cellular gamma-polymerase is mitochondria associated. Since mitochondria possess no unique DNA polymerase activity, the number of DNA polymerases now known in mammalian cells is reduced, from the previously proposed four enzymes, to three--DNA polymerases alpha, beta, and gamma.

Animals↗
PubMed1977

Evidence of a repetitive sequence in vaccinia virus DNA.

Analysis of vaccinia DNA by reassociation kinetics revealed that 7% of the genome contains a sequence repeated 10 times. This sequence does not contain any host cell DNA, is viral specific, and is found in virions passed at either high or low multiplicities of infection.

Base Sequence↗
PubMed1976

HeLa cell DNA polymerase gamma: further purification and properties of the enzyme.

DNA polymerase gamma has been purified over 60 000-fold from HeLa cells which contain no detectable type C viral particles. This purified enzyme shows a specific activity of 25 000 units/mg of protein which is comparable to the known specific activity of homogeneous preparations of human alpha and beta polymerases. The isolated enzyme shows apparent molecular weights ranging from 160 000 to 330 000 according to the method of analysis. The enzyme exhibits optimal activity for copying poly(A) in the presence of 50 mM KPO4 and 130 mM KCl and, under these conditions, copies poly(A) 20 times more rapidly than activated DNA. These assay conditions permit a clear distinction between the gamma-polymerase and DNA polymerase beta which is markedly inhibited by phosphate at this concentration. A comparison of the copying of activated DNA, poly(dA) and poly(A) by DNA polymerases alpha, beta, and gamma under optimal assay conditions for each enzyme is presented. Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.

DNA Polymerase I↗
PubMed1976

Chloramphenicol-induced loss of mitochondrial DNA polymerase activity in HeLa cells.

HeLa cells exposed to chloramphenicol for approximately one cell generation were found to contain a mitochondria-associated DNA polymerase with a significantly lower specific activity than that of control cells. This observation was not due to the presence of inhibitors in mitochondrial DNA polymerase preparations of chloramphenicol-treated cell cultures. In addition, there was no accumulation of a typical mitochondrial DNA polymerase in the post-mitochondrial supernatant of drug-treated cells.

Chloramphenicol↗
PubMed1975

RNA-primed DNA synthesis: specific catalysis by HeLa cell DNA polymerase alpha.

We have analyzed and compared the responses of the three major HeLa cell DNA polymerases (alpha, beta, and gamma) to a HeLa DNA template with short RNA or DNA primers hybridized to it. Only DNA polymerase alpha is able to synthesize DNA covalently bonded to the RNA primer via a 3' yields 5' phosphodiester bond. 32P transfer experiments showed that all combinations of ribo- and deoxyribonucleotides are represented in the RNA-DNA linkages but their distribution is nonrandom. The RNA-DNA linked molecules base-paired to a HeLa DNA template strand represent a possible "natural" in vitro primer-template for DNA polymerases and can be extended by all three DNA polymerases (alpha, beta, and gamma). These findings indicate that DNA polymerases beta and gamma are capable of DNA-primed but not RNA-PRIMED DNA synthesis, while DNA polymerase alpha is capable of both RNA-primed and DAN-primed DNA synthesis.

Base Sequence↗
PubMed1975

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.

Cell Nucleus↗
PubMed1975

Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

The replication of vaccinia virus is thought to take place exclusively in the cytoplasm of host cells. However, using DNA-DNA hybridization techniques, it can be shown that a significant fraction of the synthesis of vaccinia DNA takes place in the nucleus as well as the cytoplasm. The (3H) thymiding pulse-labeled vaccinia DNA synthesized in the nucleus reaches a maximum at about 3 h after infection, corresponding to the time of maximal DNA synthesis in infected cells. At this time host DNA synthesis drops to about 25% of the rate of the uninfected cells. Even with short labeling times (2 min) the nucleus is found to contain 60% of the incorporated (3H)thymidine, much of which is in vaccinia DNA. Prior inhibition of host nuclear DNA synthesis with mitomycin C, followed by removal of the antibiotic causes a subsequent inhibition of vaccinia DNA synthesis and complete suppression of mature virus. Purified nuclei, isolated from vaccinia-infected cells, also synthesize vaccinia DNA in vitro. Over 90% of the DNA synthesized in vitro by isolated nuclei contain vaccinia-specific sequences.

Cell Fractionation↗
PubMed1975

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.

Adenoviridae↗
PubMed1975

Presence of a herpes simplex virus type 1 genome fragment in HSV-transformed cells.

LTK-cells infected with UV-irradiated HSV produce transformants that contain a thymidine kinase (TK) activity not found in the parental LTK-line (Munyon et al., 1971). One of these (TK+) transformants (clone 139) has been analysed for the presence of the HSV genome. Reassociation kinetics studies with iodinated HSV DNA of specific activity of about 9 x 107 cpm/mug have established that there are approximately six copies of a fragment comprising about 15% of the HSV genome in HSV-transformed clone 139. Neither the parental LTK-nor a "revertant" cell line (clone 139 BUDR) obtained from clone 139 showed any detectable HSV-specific sequences. Analysis of data on RNA-125I-HSV DNA reassociation kinetics indicates that perhaps 5% of the HSV genome is transcribed in HSV-transformed clone 139. These results indicate that transformation is probably maintained by the presence of only a fraction of the HSV genome in the TK+ clones.

Cell Line↗