Mutational blockage of DNA synthesis in Paramecium tetraurelia

1976 ◽  
Vol 54 (12) ◽  
pp. 2089-2097 ◽  
Author(s):  
E. L. Peterson ◽  
J. D. Berger
Keyword(s):  
Dna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Paramecium Tetraurelia ◽  
Selection System ◽  
Replication Process ◽  
Temperature Sensitive Mutants ◽  
Fold Enrichment ◽  
Comparison Of Experiments

One hundred and ninety-eight temperature-sensitive mutants of Paramecium tetraurelia were isolated after nitrosoguanidine mutagenesis. In some experiments, mutants were recovered with the aid of a bromouracil (BU) selection system. Fifty-six mutants showed cessation of cell division within one cell cycle at the restrictive temperature and were designated ts-0. Fourteen of the ts-0's showed a greater than 90% reduction in rnacronuclear deoxyribonucleic acid (DNA) synthesis at the restrictive temperature. Two ts-0. DNA-defective lines continued protein synthesis at greater than 50% the normal rate after arrest of DNA synthesis. Hence, these two mutants may be directly affected in the replication process itself. The two mutants are allelic and, in addition, a third 'leaky' allele was recovered. Comparison of experiments in which either BU selection or no selection was employed shows that a greater than 10-fold enrichment for ts mutants resulted from BU selection.

scholarly journals Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants.

1976 ◽  
Vol 71 (1) ◽  
pp. 242-260 ◽  
Author(s):  
J Frankel ◽  
L M Jenkins ◽  
L E DeBault
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Synthesis ◽  
Tetrahymena Pyriformis ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Developmental Pathway ◽  
Temperature Sensitive Mutants ◽  
Oral Development ◽  
Macronuclear Division

Utilization of temperature-sensitive mutants of Tetrahymena pyriformis affected in cell division or developmental pathway selection has permitted elucidation of causal dependencies interrelating micronuclear and macronuclear replication and division, oral development, and cytokinesis. In those mutants in which cell division is specifically blocked at restrictive temperatures, micronuclear division proceeds with somewhat accelerated periodicity but maintains normal coupling to predivision oral development. Macronuclear division is almost totally suppressed in an early acting mutant (mola) that prevents formation of the fission zone, and is variably affected in other mutants (such as mo3) that allow the fission zone to form but arrest constriction. However, macronuclear DNA synthesis can proceed for about four cycles in the nondividing mutant cells. A second class of mutants (psm) undergoes a switch of developmental pathway such that cells fail to enter division but instead repeatedly carry out an unusual type of oral replacement while growing in nutrient medium at the restrictive temperature. Under these circumstances no nuclei divide, yet macronuclear DNA accumulation continues. These results suggest that (a) macronuclear division is stringently affected by restriction of cell division, (b) micronuclear division and replication can continue in cells that are undergoing the type of oral development that is characteristic of division cycles, and (c) macronuclear DNA synthesis can continue in growing cells regardless of their developmental status. The observed relationships among events are consistent with the further suggestion that the cell cycle in this organism may consist of separate clusters of events. with a varying degree of coupling among clusters. A minimal model of the Tetrahymena cell cycle that takes these phenomena into account is suggested.

scholarly journals A search for temperature-sensitive mutants ofUstilago maydisblocked in DNA synthesis

1970 ◽  
Vol 15 (2) ◽  
pp. 157-169 ◽  
Author(s):  
P. Unrau ◽  
R. Holliday
Keyword(s):  
Dna Synthesis ◽  
Nuclear Division ◽  
Ustilago Maydis ◽  
Temperature Treatment ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Temperature Sensitive Mutants ◽  
Dna And Rna

SUMMARYOver 400 temperature-sensitive mutants ofUstilago maydishave been tested for DNA synthesis at the restrictive temperature of 32°C by measuring14C adenine incorporation into DNA and RNA. Five mutants were defective in DNA synthesis but none was completely blocked. One mutant,tsd-1, which is unlinked to the others, forms long uninucleate filaments at 32°C which die exponentially after 4h temperature treatment. The phenotype is comparable to that of thymine-starved bacteria, but it is possible that rather than being specifically defective in DNA synthesis the mutant is blocked in nuclear division.

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.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Induced cellular DNA synthesis by ‘early’ and ‘late’ temperature-sensitive mutants of polyoma virus

1971 ◽  
Vol 177 (1046) ◽  
pp. 59-63 ◽  
Keyword(s):  
Dna Synthesis ◽  
Polyoma Virus ◽  
Viral Gene ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Virus Growth ◽  
Temperature Sensitive Mutants ◽  
Cellular Dna

Temperature-sensitive mutants of polyoma virus have been examined to determine whether they are able to induce the synthesis of cellular DNA under conditions where viral gene products are defective. Two ‘early’ mutants, and one ‘late’ mutant of polyoma induce cellular DNA synthesis normally under conditions where virus growth is inhibited because viral gene products are defective.

scholarly journals AN ENRICHMENT FOR TEMPERATURE-SENSITIVE MUTANTS IN TETRAHYMENA THERMOPHILA

Genetics ◽  
1979 ◽  
Vol 92 (4) ◽  
pp. 1079-1092
Author(s):  
Duane W Martindale ◽  
Ronald E Pearlman
Keyword(s):  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Macromolecular Synthesis ◽  
Treatment Results ◽  
Uv Treatment ◽  
Temperature Sensitive Mutants ◽  
Near Ultraviolet ◽  
Treatment Data ◽  
Resistant Mutants

ABSTRACT The parameters for the killing of Tetrahymena by 5-bromodeoxyuridine (BUdR) and near-ultraviolet light have been determined. Significant preferential killing by UV* of cells that have incorporated BUdR was obtained when the cells were irradiated in a nonnutrient buffer. UV alone was found to be toxic to cells irradiated in growth medium. Mutants defective in division at a restrictive temperature were isolated from mutagenized cultures that had been treated with BUdR and UV and from mutagenized cultures that had no such treatment. Results indicate that the number of temperature sensitive (ts) growth mutants can be increased five to six times using the BUdR/UV treatment. Data are presented that indicate differences in the frequency of occurrence of various types of ts mutants, with and without enrichment. A mutant that immediately stopped macromolecular synthesis and cell division upon being placed at the restrictive temperature was more resistant to BUdR/UV treatment than wild type by 1000-fold. Using the above techniques, BUdR-resistant mutants altered in the phosphorylation of thymidine have been isolated.

scholarly journals Functional Analysis of the Interaction between VPg-Proteinase (NIa) and RNA Polymerase (NIb) of Tobacco Etch Potyvirus, Using Conditional and Suppressor Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8732-8740 ◽  
Author(s):  
José-Antonio Daròs ◽  
Mary C. Schaad ◽  
James C. Carrington
Keyword(s):  
Rna Polymerase ◽  
Proteolytic Activity ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Genome Replication ◽  
Selection System ◽  
Mutant Proteins ◽  
Suppressor Mutants ◽  
Tobacco Etch Potyvirus ◽  
Two Hybrid

ABSTRACT The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred ats-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.

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