Temperature-sensitive nonfusing myoblast variant and spontaneous revertant: Isolation and characterization

1985 ◽  
Vol 11 (4) ◽  
pp. 325-338 ◽  
Author(s):  
Leslie C. Engel ◽  
John D. David
Keyword(s):  
Temperature Sensitive ◽  
Isolation And Characterization ◽  
Spontaneous Revertant

scholarly journals Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient

2020 ◽  
Author(s):  
Daniel J Wolter ◽  
Alison Scott ◽  
Catherine R Armbruster ◽  
Dale Whittington ◽  
John S Edgar ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Lipid A ◽  
Clinical Laboratory ◽  
Membrane Phospholipid ◽  
Growth Defect ◽  
Genetic Mutations ◽  
Temperature Sensitive ◽  
Isolation And Characterization

Abstract Background Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. Objectives We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. Methods Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. Results P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. Conclusions A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

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.

Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation

1988 ◽  
Vol 8 (6) ◽  
pp. 2335-2341
Author(s):  
R J Akhurst ◽  
N B Flavin ◽  
J Worden
Keyword(s):  
Cell Line ◽  
Progenitor Cell ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Isolation And Characterization ◽  
New Variant ◽  
Myoblast Cell Line ◽  
Myoblast Cell ◽  
Progenitor Cell Line

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.

scholarly journals Mutations in Bartonella bacilliformis gyrB Confer Resistance to Coumermycin A1

1998 ◽  
Vol 42 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
James M. Battisti ◽  
Laura S. Smitherman ◽  
D. Scott Samuels ◽  
Michael F. Minnick
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Extract ◽  
Natural Resistance ◽  
Temperature Sensitive ◽  
Bartonella Bacilliformis ◽  
Reading Frame ◽  
E Coli ◽  
Isolation And Characterization ◽  
Spontaneous Mutants

ABSTRACT This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of ∼77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants ofB. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124→Ser), Arg184→Gln, and Thr214→Ala or Thr214→Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes fromBorrelia burgdorferi, E. coli,Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.

Isolation and characterization of further defective clones of a temperature sensitive mutant (ts-1) of respiratory syncytial virus

1977 ◽  
Vol 54 (1-2) ◽  
pp. 53-60 ◽  
Author(s):  
Linda S. Richardson ◽  
T. J. Schnitzer ◽  
R. B. Belshe ◽  
Ena Camargo ◽  
D. A. Prevar ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Isolation And Characterization ◽  
Syncytial Virus

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly

1982 ◽  
Vol 300 (1099) ◽  
pp. 207-223 ◽  
Keyword(s):  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Oligosaccharide Processing ◽  
Biochemical Studies ◽  
The Common ◽  
Specific Lipid ◽  
Temperature Sensitive Phenotype

The formation of N -glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

scholarly journals Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.

1988 ◽  
Vol 8 (6) ◽  
pp. 2335-2341 ◽  
Author(s):  
R J Akhurst ◽  
N B Flavin ◽  
J Worden
Keyword(s):  
Cell Line ◽  
Progenitor Cell ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Isolation And Characterization ◽  
New Variant ◽  
Myoblast Cell Line ◽  
Myoblast Cell ◽  
Progenitor Cell Line

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.

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