scholarly journals Characterization of high frequency of recombination strains selected by integrative suppression of F’lac in dnaA, gyrA and gyrB temperature sensitive mutants

2017 ◽  
Vol 90 (1) ◽  
Author(s):  
Eugenio A. Debbia ◽  
Anna Marchese
Keyword(s):  
High Frequency ◽  
Great Majority ◽  
Dna Gyrase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Cellular Functions ◽  
Ts Mutant ◽  
Time Kill ◽  
Derivatives Of

Integration of F’lac plasmid into chromosome of both gyrA(Ts) and gyrB(Ts) cells phenotypically suppress the thermosensitive mutations of the DNA gyrase enzymes. As the comparative strains isolated from dnaA(Ts), these high frequency of recombination (Hfr) derivatives were able to transfer chromosomal markers to recipient strains, showed a growth rate of about 60 min, and developed filamentous forms when incubated at the temperature of 43°C. Conversely to dnaA(Ts) Hfr selected isolates, the great majority of Hfr derivative of gyrase mutants resulted resistant to acridine orange and rifampin. Time-kill experiments carried out at the non-permissive temperature also revealed that nalidixic acid has no antibacterial activity on these Hfr strains while derivatives of dnaA(Ts) mutant, as well as the control strain HfrH, were strongly inhibited by this drug. Therefore F plasmid induced duplication of chromosome in the mutants even if the DNA gyrase enzymes are not working. Of a certain interest is that these bacteria exhibit physiological perturbations that affect the main cellular functions, however, they do not appear essential for the survival of the strains.

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.

scholarly journals Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

1993 ◽  
Vol 4 (9) ◽  
pp. 931-939 ◽  
Author(s):  
D Feldheim ◽  
K Yoshimura ◽  
A Admon ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Functional Characterization ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Restrictive Temperature ◽  
Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

Isolation and partial characterization of a human adenovirus type 4 temperature-sensitive mutant (Mastadenovirus h 4 tsl)

1984 ◽  
Vol 30 (2) ◽  
pp. 135-141 ◽  
Author(s):  
Linda M. Mofford ◽  
R. G. Marusyk
Keyword(s):  
Selection Procedure ◽  
Temperature Shift ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Temperature Sensitive ◽  
Penton Base ◽  
Virus Stock ◽  
Ts Mutant ◽  
Viruslike Particles

A random selection procedure was used to isolate a temperature-sensitive (ts) mutant of human adenovirus type 4 (Mastadenovirus h 4 tsl) from nitrous acid mutagenized virus stock. The mutant displayed restricted growth at the nonpermissive temperature of 39 °C. Analysis of the mutant grown at 39 °C, by two-dimensional immunoelectrophoretic analysis, showed the mutant to be defective in the expression of the penton base and fibre structural components. The mutant was, however, capable of synthesizing immunologically reactive hexon components. Temperature-shift experiments revealed detectable fibre and penton to be present following shift-down from 39 to 32 °C. Time-sequence analysis of shift-down experiments suggested a possible defect in processing of the components, as indicated by an increase of immunologically detectable penton base. The ability of the mutant to assemble viruslike particles at 39 °C was confirmed by electron microscopy. Though the particles assembled appeared as mature virions, crystalline arrays of packed particles were less in number and somewhat smaller in size than those observed at 32 °C.

Temperature-sensitive mutation. IV. Induction and characterization of a ts-mutation in RNA-containing bacteriophage R17

1970 ◽  
Vol 16 (3) ◽  
pp. 165-172
Author(s):  
S. J. Igarashi ◽  
J. F. Elliott ◽  
R. P. Bissonnette
Keyword(s):  
Rna Replication ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Temperature Sensitive Mutation ◽  
Phage Multiplication ◽  
Rna Phage

Temperature-sensitive mutants of the RNA phage R17 were induced by treatment with 10−5 M 5-fluorouracil. One of the temperature-sensitive mutants, ts24, was studied and the following facts were discovered: (1) ts24 undergoes eclipse at the non-permissive temperature, 43 °C. (2) If the temperature is increased to non-permissive levels before 60 min of postinfection culture, phage multiplication ceases. (3) At 43 °C, ts24 cannot synthesize progeny viral RNA. (4) Upon decreasing the temperature to permissive levels during the first 60 min of postinfection culture, RNA replication resumes almost instantaneously. It was concluded from these facts that ts24 contains a temperature-sensitive mutation in the RNA replicating function.

The fission yeast bromodomain protein Bdf2 is required for growth of cells with circular chromosomes

2021 ◽  
Author(s):  
Misaki Yasuda ◽  
Ahmed G K Habib ◽  
Kanako Sugiura ◽  
Hossain Mohammad Shamim ◽  
Masaru Ueno
Keyword(s):  
Fission Yeast ◽  
High Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
M Phase ◽  
Ts Mutant ◽  
Synthetically Lethal ◽  
Circular Chromosomes ◽  
Synthetic Growth

Abstract Circular chromosomes have frequently been observed in tumors of mesenchymal origin. In the fission yeast Schizosaccharomyces pombe, deletion of pot1+ results in rapid telomere loss, and the resulting survivors have circular chromosomes. Fission yeast has two bromodomains and extra-terminal (BET) proteins, Bdf1 and Bdf2; both are required for maintaining acetylated histones. Here, we found that bdf2, but not bdf1, was synthetically lethal with pot1. We also obtained a temperature-sensitive bdf2-ts mutant, which can grow at high temperatures but becomes camptothecin sensitive. This suggests that Bdf2 is defective at high temperatures. The cell cycle of the pot1 bdf2-ts mutant was delayed in the G2 and/or M phase at a semi-permissive temperature. Furthermore, a temperature-sensitive mutant of mst1, which encodes histone acetyltransferase, showed a synthetic growth defect with a pot1 disruptant at a semi-permissive temperature. Our results suggest that Bdf2 and Mst1 are required for the growth of cells with circular chromosomes.

scholarly journals Functional Analysis of the Signal Recognition Particle in Escherichia coli by Characterization of a Temperature-Sensitive ffh Mutant

2002 ◽  
Vol 184 (10) ◽  
pp. 2642-2653 ◽  
Author(s):  
Sei-Kyoung Park ◽  
Fenglei Jiang ◽  
Ross E. Dalbey ◽  
Gregory J. Phillips
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Signal Recognition ◽  
E Coli ◽  
Elevated Expression ◽  
Type Gene ◽  
Shock Proteins

ABSTRACT The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42°C as well as show reduced growth at the permissive temperature of 30°C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42°C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.

scholarly journals Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 499-506
Author(s):  
A Sakai ◽  
Y Shimizu ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Temperature Sensitive ◽  
Cellular Functions ◽  
Isolation And Characterization ◽  
Gene Coding ◽  
Complementation Groups ◽  
Homozygous Diploid ◽  
Diploid Cells

Abstract We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.

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 Serotypes and Antibiotic Susceptibilities of Pseudomonas aeruginosa Isolates from Single Sputa of Cystic Fibrosis Patients

1979 ◽  
Vol 9 (1) ◽  
pp. 72-78
Author(s):  
Thomas W. Seale ◽  
Hilary Thirkill ◽  
Martha Tarpay ◽  
Marinus Flux ◽  
Owen M. Rennert
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
High Frequency ◽  
Susceptibility Testing ◽  
Phenotypic Characterization ◽  
Single Patient ◽  
Antibiotic Susceptibilities ◽  
Different Strains ◽  
Derivatives Of

A phenotypic characterization of Pseudomonas aeruginosa from single sputum samples of 21 typical cystic fibrosis patients indicated a high frequency of heterogeneity among isolates on the basis of differences in antibiotic resistance, colony morphology, pigmentation, and serotype. Two or more isolates with different but stable susceptibilities to carbenicillin, gentamycin, streptomycin, tetracycline, chloramphenicol, and sulfamethoxazole plus trimethoprim were detected in 38% of the sputa. Differences generally were independent of the mucoid state of the strain. O-antigen group determination with the Difco typing set showed that two or more serologically distinct strains were present in 10/21 sputum specimens. Nonmucoid derivatives of mucoid isolates almost always retained both the antibiotic susceptibilities and serotype of their parent strain. These data suggest that cystic fibrosis patients may be cocolonized/coinfected by different strains of P. aeruginosa more frequently than generally believed. Alternatively, phenotypically distinct strains from a single patient might arise as phenotypic dissociants from a single infecting strain. Because of the frequency and multiplicity of phenotypically distinct P. aeruginosa isolates which we obtained from our cystic fibrosis patients, it is important to select multiple isolates from sputum cultures for antimicrobial susceptibility testing so as to assess adequately the susceptibility of this organism to antibiotic therapy in cystic fibrosis. We recommend that several colonies of each distinguishable colony type of P. aeruginosa be pooled for the antibiogram.

Induction of G1- cells at high frequency

1979 ◽  
Vol 40 (1) ◽  
pp. 33-42
Author(s):  
P.M. Naha
Keyword(s):  
Dna Synthesis ◽  
Ultraviolet Irradiation ◽  
High Frequency ◽  
Generation Time ◽  
G1 Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Experimental Conditions ◽  
G1 Cells

Experimental conditions are reported on the induction of G1- cells at high frequency by ultraviolet irradiation of temperature-sensitive cells arrested in G1 phase at the restricted temperature. Neither the wild type cells of Balb/C-3T3 nor the temperature-sensitive derivative of tsA83 grown at the permissive temperature could be ‘mutated’ under similar conditions. One such ‘mutant’ (RI8) was ‘inducible’ from a G1+ phenotype at 33 degrees C to G1- state at 38 degrees C within one cell generation time. The ‘inducible’ property of RI8 from G1+ to G1- lends support to the theory that the duration of the G1+ phenotype is determined by the level of precursors for DNA synthesis. Since the G1- variants were isolated from revertants, the frequency of ‘mutagenesis’ appeared to be artifically high.

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