THE CHARACTERISTICS AND GENETIC MAP LOCATION OF A TEMPERATURE SENSITIVE DNA MUTANT OF E. COLI K12

Genetics ◽  
1972 ◽  
Vol 72 (4) ◽  
pp. 569-593
Author(s):  
Beverly Wolf
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chain Elongation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Bacterial Dna ◽  
E Coli ◽  
Dna Chain ◽  
Inhibition Of Dna Synthesis ◽  
Dna Mutant

ABSTRACT A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40°C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40°C; (2) upon a shift down to 30°C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30°C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40°C. These three observations indicate that rounds of DNA replication were completed at 40°C. Also (4) infective λ particles can be made at 40°C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40°C since (1) a limited amount of DNA synthesis does occur at 40°C after prior alignment of the chromosomes by amino acid starvation at 30°C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

scholarly journals MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1978 ◽  
Vol 90 (1) ◽  
pp. 49-68
Author(s):  
Yona Kassir ◽  
Giora Simchen
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Division ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Essential Function ◽  
Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

scholarly journals Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster.

1980 ◽  
Vol 85 (3) ◽  
pp. 777-785 ◽  
Author(s):  
M Zannis-Hadjopoulos ◽  
M W Taylor ◽  
R Hand
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Cell Line ◽  
Growth Medium ◽  
Replication Fork ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Auxotrophic Mutant ◽  
Chain Elongation ◽  
Dna Chain

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.

Relationship between nucleoside triphosphate pools and DNA replication in the cell cycle of Escherichia coli

1974 ◽  
Vol 20 (5) ◽  
pp. 747-750 ◽  
Author(s):  
George G. Khachatourians ◽  
Lydia Huzyk
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Specific Inhibitor ◽  
Replication Cycle ◽  
Nucleoside Triphosphate ◽  
E Coli ◽  
Synchronous Cultures ◽  
Inhibition Of Dna Synthesis

The correlation between DNA replication and the nucleoside triphosphate pool fluctuation in the cell cycle of Escherichia coli B/r was examined. 32P-labelled endogenous nucleoside triphosphates in normal synchronous cultures of E. coli B/r and those in which the chromosome replication cycle was inhibited by nalidixic acid, a specific inhibitor of DNA synthesis, were compared. No marked accumulation or depletion of nucleoside triphosphate pools was observed during the inhibition of DNA synthesis in the cell cycle. We suggest that changes in the pool levels during the cell cycle of E. coli occur independently of the DNA replication cycle.

The punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral DNA synthesis

Microbiology ◽  
2000 ◽  
Vol 81 (5) ◽  
pp. 1231-1235 ◽  
Author(s):  
Arban Domi ◽  
Georges Beaud
Keyword(s):  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Chain Elongation ◽  
Dapi Staining ◽  
Viral Dna ◽  
Virus Particles ◽  
Early Proteins ◽  
Infected Cells ◽  
Dna Chain ◽  
Inhibition Of Dna Synthesis

Several vaccinia virus early proteins (encoded by genes B1R, H5R and I3L) synthesized in the presence of an inhibitor of DNA synthesis localize, at least in part, to punctate inclusions that are visible by immunofluorescence in the cytoplasm of poxvirus-infected cells. It is shown that these inclusions contain DNA (visualized by DAPI staining of the infected cells) and that the number of inclusions is proportional to the amount of input virus. Their mean diameter (about 680 nm) was larger than that of purified vaccinia virus particles. When the inhibition of DNA synthesis was reversed, incorporation of BrdU into the B1R particles was demonstrated after labelling for 30 min, suggesting that these cytoplasmic focal sites correspond to viral DNA replication complexes that have initiated normally but are inhibited at the step of DNA chain elongation. These experiments suggest strongly that these inclusions are the precursors of the virosomes.

scholarly journals Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

1995 ◽  
Vol 15 (1) ◽  
pp. 165-172 ◽  
Author(s):  
M Seki ◽  
T Kohda ◽  
T Yano ◽  
S Tada ◽  
J Yanagisawa ◽  
...  
Keyword(s):  
Dna Replication ◽  
Atpase Activity ◽  
Dna Synthesis ◽  
Dna Helicase ◽  
Chain Elongation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Dependent Atpase ◽  
Mutant Cells

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

scholarly journals Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601 ◽  
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

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