Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.

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Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.902-905.1985 ◽

1985 ◽

Vol 5 (4) ◽

pp. 902-905

Author(s):

M Narkhammar ◽

R Hand

Keyword(s):

Cell Line ◽

Nuclear Extract ◽

Cell Extract ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Whole Cell ◽

Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

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Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.902 ◽

1985 ◽

Vol 5 (4) ◽

pp. 902-905 ◽

Cited By ~ 2

Author(s):

M Narkhammar ◽

R Hand

Keyword(s):

Cell Line ◽

Nuclear Extract ◽

Cell Extract ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Whole Cell ◽

Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

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A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1171 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1171-1183 ◽

Cited By ~ 93

Author(s):

T Hirano ◽

Y Hiraoka ◽

M Yanagida

Keyword(s):

Schizosaccharomyces Pombe ◽

Gradient Centrifugation ◽

Metaphase Plate ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

In The Wild ◽

Spindle Elongation ◽

Mutant Cells

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

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Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.1191-1199.1989 ◽

1989 ◽

Vol 9 (3) ◽

pp. 1191-1199

Author(s):

M Bernstein ◽

F Kepes ◽

R Schekman

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Product ◽

Carboxypeptidase Y ◽

Secretory Proteins ◽

Restrictive Temperature ◽

Wild Type ◽

Partial Restoration ◽

Alpha Factor ◽

Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

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Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.1191 ◽

1989 ◽

Vol 9 (3) ◽

pp. 1191-1199 ◽

Cited By ~ 18

Author(s):

M Bernstein ◽

F Kepes ◽

R Schekman

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Product ◽

Carboxypeptidase Y ◽

Secretory Proteins ◽

Restrictive Temperature ◽

Wild Type ◽

Partial Restoration ◽

Alpha Factor ◽

Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.165 ◽

1995 ◽

Vol 15 (1) ◽

pp. 165-172 ◽

Cited By ~ 13

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.

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Cell culture of individual Drosophila embryos

Development ◽

10.1242/dev.45.1.173 ◽

1978 ◽

Vol 45 (1) ◽

pp. 173-187

Author(s):

David P. Cross ◽

James H. Sang

Keyword(s):

Cell Cultures ◽

Fat Body ◽

Subsequent Development ◽

Cell Types ◽

Mixed Cultures ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type

Results are reported from the culturing in vitro of cells from individual early gastrulae of the following four groups of X-linked embryonic lethal mutants of Drosophila melanogaster. (1) Notch lethals. Five Notch mutants were studied which have been reported to give similar abnormalities in whole embryos: the nervous system displays a three-fold hypertrophy as part of a shift in the pattern of differentiation within ectodermal derivatives, and mesodermal derivatives do not differentiate. An hypertrophy of nerve was found in cell cultures prepared from embryos of all five mutants. In addition, four of the five alleles consistently gave abnormalities of muscle differentiation: when compared to controls, Notch cultures had a reduced frequency of myotubes, and displayed unusual clusters of myocytes which had either failed to fuse or had fused incompletely. Results from mixed cultures prepared from two embryos were consistent with the autonomous expression of nerve and muscle abnormalities by Notch-8 cells in the presence of wild-type cells. It is argued that the Notch locus has a direct role in the differentiation of both nerve and muscle. (2) white deficiencies. Cells carrying either of two deficiencies gave a clear-cut pattern of abnormalities: initial cellular differentiations were normal, but nerve, muscle and fat-body cells progressively deteriorated during the culture period. Mixed cultures showed that wild-type cells could not ‘rescue’ mutant muscle and fat-body cells; however, the status of the autonomy of mutant nerve abnormalities in these cultures was unclear. Both white deficiencies remove cytological band 3C1, and this permits a comparison of results with those from cultures of cells from Notch-8 embryos (also deficient for 3C1). Abnormalities displayed in cultures of the two types of mutant show no overlap. Therefore no consistent cellular abnormality can be attributed to absence of band 3C1. (3) lethal{l)myospheroid. In contrast to earlier observations on in vitro cell cultures (Donady & Seecof, 1972) muscle was seen to differentiate, though its morphology was extremely abnormal. Observations indicated that all cell types within the cultures had poor properties of adhesion to a glass substrate. It is argued that the observed abnormalities are not consistent with a mutant lesion which is restricted to the basement membrane (contra Wright, 1960), and that all cell types carry a basic defect which may reside in the cell membrane. (4) shibirets alleles. Cultures of two temperature-sensitive lethal shibire alleles (shits1, shits3) were normal at the permissive temperature of 22 °C. At the restrictive temperature (29 °C) early cell differentiation was normal but subsequent development was blocked. This blockage could be partially reversed by shifting cultures to the permissive temperature after as much as 10 days exposure to the high temperature. It is suggested that shits cells are mutant in a process which is basic to several cell types.

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RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽

10.1093/genetics/166.4.1631 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1631-1640 ◽

Cited By ~ 4

Author(s):

Janet R Donaldson ◽

Charmain T Courcelle ◽

Justin Courcelle

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Dna Lesions ◽

Replication Forks ◽

Wild Type ◽

Replication Machinery ◽

Dna Junctions ◽

Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4594-4601.1986 ◽

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.

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