scholarly journals INVOLVEMENT OF GENE 49 IN RECOMBINATION OF BACTERIOPHAGE T4

Genetics ◽  
1983 ◽  
Vol 104 (1) ◽  
pp. 1-9
Author(s):  
Junichi Miyazaki ◽  
Yeikou Ryo ◽  
Teiichi Minagawa
Keyword(s):  
Dna Replication ◽  
Gene Function ◽  
Bacteriophage T4 ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Recombinant Frequency ◽  
Defective Mutant ◽  
Ts Mutant

ABSTRACT The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvs X, but recombination between rII markers was not.

scholarly journals The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

2007 ◽  
Vol 189 (24) ◽  
pp. 8793-8800 ◽  
Author(s):  
Christopher J. Rosario ◽  
Mitchell Singer
Keyword(s):  
Dna Replication ◽  
Developmental Process ◽  
Myxococcus Xanthus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Temperature Growth ◽  
Developmental Program

ABSTRACT Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaBA116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaBA116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaBA116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

scholarly journals Dual Role of the Mitochondrial Chaperone Mdj1p in Inheritance of Mitochondrial DNA in Yeast

1999 ◽  
Vol 19 (12) ◽  
pp. 8201-8210 ◽  
Author(s):  
Marlena Duchniewicz ◽  
Aleksandra Germaniuk ◽  
Benedikt Westermann ◽  
Walter Neupert ◽  
Elisabeth Schwarz ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Optimal Growth ◽  
Polymerase Activity ◽  
Mitochondrial Genomes ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Active Conformation ◽  
Nonpermissive Temperature ◽  
Chaperone Protein

ABSTRACT Mdj1p, a homolog of the bacterial DnaJ chaperone protein, plays an essential role in the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. We analyzed the role of Mdj1p in the inheritance of mitochondrial DNA (mtDNA). Mitochondrial genomes were rapidly lost in a temperature-sensitive mdj1 mutant under nonpermissive conditions. The activity of mtDNA polymerase was severely reduced in the absence of functional Mdj1p at a nonpermissive temperature, demonstrating the dependence of the enzyme on Mdj1p. At a permissive temperature, the activity of mtDNA polymerase was not affected by the absence of Mdj1p. However, under these conditions, intact [rho +] genomes were rapidly converted to nonfunctional [rho −] genomes which were stably propagated in an mdj1 deletion strain. We propose that mtDNA polymerase depends on Mdj1p as a chaperone in order to acquire and/or maintain an active conformation at an elevated temperature. In addition, Mdj1p is required for the inheritance of intact mitochondrial genomes at a temperature supporting optimal growth; this second function appears to be unrelated to the function of Mdj1p in maintaining mtDNA polymerase activity.

scholarly journals Role of deoxycytidylate deaminase in deoxyribonucleotide synthesis in bacteriophage T4 DNA replication

1977 ◽  
Vol 252 (23) ◽  
pp. 8603-8608 ◽  
Author(s):  
C.S. Chiu ◽  
T. Ruettinger ◽  
J.B. Flanegan ◽  
G.R. Greenberg
Keyword(s):  
Dna Replication ◽  
Bacteriophage T4

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.

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation

1986 ◽  
Vol 6 (6) ◽  
pp. 2027-2032
Author(s):  
R Kai ◽  
M Ohtsubo ◽  
M Sekiguchi ◽  
T Nishimoto
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Human Gene ◽  
Chromosome Condensation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Hindiii Fragment ◽  
Human Dna ◽  
Ts Mutant ◽  
Tsbn2 Cells

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

1986 ◽  
Vol 6 (6) ◽  
pp. 2027-2032 ◽  
Author(s):  
R Kai ◽  
M Ohtsubo ◽  
M Sekiguchi ◽  
T Nishimoto
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Human Gene ◽  
Chromosome Condensation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Hindiii Fragment ◽  
Human Dna ◽  
Ts Mutant ◽  
Tsbn2 Cells

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.

scholarly journals Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis.

1982 ◽  
Vol 92 (3) ◽  
pp. 629-633 ◽  
Author(s):  
D J Scharff ◽  
A M Delegeane ◽  
A S Lee
Keyword(s):  
Cell Line ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Molecular Weights ◽  
A Cell ◽  
Ts Mutant ◽  
Spontaneous Revertant

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.

An Alternatively Spliced Survivin Variant Is Positively Regulated by p53 and Sensitizes Leukemia Cells to Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3497-3497
Author(s):  
Ningxi Zhu ◽  
Lubing Gu ◽  
Harry W. Findley ◽  
Fengzhi Li ◽  
Muxiang Zhou
Keyword(s):  
Cell Line ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Doxorubicin Treatment ◽  
Cryptic Exon ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Abstract Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53–143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53–143. When EU-4/p53–143 cell culture was shifted from 37.5°C to the wt-p53-permissive temperature (32.5°C), the expression of survivin and survivin-DEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a pro-apoptotic factor and its expression is upregulated by p53.

A ts mutant isolated from CHL cells: Inhibition of DNA replication at nonpermissive temperature

1979 ◽  
Vol 5 (1) ◽  
pp. 97-115 ◽  
Author(s):  
D. J. Roufa ◽  
S. M. McGill ◽  
J. W. Mollenkamp
Keyword(s):  
Dna Replication ◽  
Nonpermissive Temperature ◽  
Ts Mutant
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