Nature of Mutations Conferring Resistance to 8-Azaguanine in Mouse Cell Lines

1974 ◽  
Vol 14 (2) ◽  
pp. 235-251
Author(s):  
SEUNG-IL SHIN
Keyword(s):  
Rabbit Antiserum ◽  
Low Frequency ◽  
Mouse Cell ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Wild Type ◽  
Mouse Tissues ◽  
In The Wild ◽  
Normal Enzyme ◽  
Mutant Phenotypes

Two stable mouse cell mutants A9 and RAG, which are resistant to 8-azaguanine and deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT), have been studied in order to establish the nature of molecular changes conferring the mutant phenotypes. A specific precipitating rabbit antiserum was prepared against the normal HGPRT purified from mouse tissues, and used to test for cross-reacting material (CRM) in the mutant lysates. Neither mutants contained detectable cross-reacting material, as demonstrated by precipitation in-hibition tests. However, the L cell-derived mutant A9 was shown to have a low but significant level of HGPRT activity which was clearly different from that of the normal enzyme in the wild-type parental cell line. Compared to the wild-type enzyme, the HGPRT in A9 is extremely heat labile, and has an elevated substrate-binding constant in addition to distinct antigenic differences. Both A9 and RAG have been shown previously to revert to the normal phenotype with low frequency, thus ruling out gene deletions as a possible cause of the 8-azaguanine resistance. It is suggested that RAG could involve a recessive regulatory mutation, while A9 may contain a structurally altered HGPRT as a result of a missense mutation within the structural gene for this enzyme.

Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line

1985 ◽  
Vol 5 (10) ◽  
pp. 2527-2532
Author(s):  
C H Jenh ◽  
P K Geyer ◽  
L F Johnson
Keyword(s):  
Cell Line ◽  
Thymidylate Synthase ◽  
Northern Blot Analysis ◽  
Mouse Cell ◽  
Parental Cell ◽  
S Phase ◽  
Parental Cell Line ◽  
Mouse Cell Line ◽  
Isolated Nuclei ◽  
Mrna Content

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that over produces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20- to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)- RNA species.

scholarly journals Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line.

1985 ◽  
Vol 5 (10) ◽  
pp. 2527-2532 ◽  
Author(s):  
C H Jenh ◽  
P K Geyer ◽  
L F Johnson
Keyword(s):  
Cell Line ◽  
Thymidylate Synthase ◽  
Northern Blot Analysis ◽  
Mouse Cell ◽  
Parental Cell ◽  
S Phase ◽  
Parental Cell Line ◽  
Mouse Cell Line ◽  
Isolated Nuclei ◽  
Mrna Content

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that over produces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20- to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)- RNA species.

Role of the Tectorial Membrane Revealed by Otoacoustic Emissions Recorded From Wild-Type and Transgenic TectaΔENT/ΔENT Mice

2004 ◽  
Vol 91 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Andrei N. Lukashkin ◽  
Victoria A. Lukashkina ◽  
P. Kevin Legan ◽  
Guy P. Richardson ◽  
Ian J. Russell
Keyword(s):  
Otoacoustic Emissions ◽  
Low Frequency ◽  
Tectorial Membrane ◽  
Local Minima ◽  
Wild Type ◽  
Distortion Product ◽  
Growth Functions ◽  
In The Wild ◽  
Surrounding Fluid

Distortion product otoacoustic emissions (DPOAE) were recorded from wild-type mice and mutant TectaΔ ENT/Δ ENT mice with detached tectorial membranes (TM) under combined ketamine/xylaxine anesthesia. In TectaΔ ENT/Δ ENT mice, DPOAEs could be detected above the noise floor only when the levels of the primary tones exceeded 65 dB SPL. DPOAE amplitude decreased with increasing frequency of the primaries in TectaΔ ENT/Δ ENT mice. This was attributed to hair cell excitation via viscous coupling to the surrounding fluid and not by interaction with the TM as in the wild-type mice. Local minima and corresponding phase transitions in the DPOAE growth functions occurred at higher DPOAE levels in wild-type than in TectaΔ ENT/Δ ENT mice. In less-sensitive TectaΔ ENT/Δ ENT mice, the position of the local minima varied nonsystematically with frequency or no minima were observed. A bell-like dependence of the DPOAE amplitude on the ratio of the primaries was recorded in both wild-type and TectaΔ ENT/Δ ENT mice. However, the pattern of this dependence was different in the wild-type and TectaΔ ENT/Δ ENT mice, an indication that the bell-like shape of the DPOAE was produced by a combination of different mechanisms. A nonlinear low-frequency resonance, revealed by nonmonotonicity of the phase behavior, was seen in the wild-type but not in TectaΔ ENT/Δ ENT mice.

scholarly journals Identification of a Fourth Formate Dehydrogenase in Methylobacterium extorquens AM1 and Confirmation of the Essential Role of Formate Oxidation in Methylotrophy

2007 ◽  
Vol 189 (24) ◽  
pp. 9076-9081 ◽  
Author(s):  
Ludmila Chistoserdova ◽  
Gregory J. Crowther ◽  
Julia A. Vorholt ◽  
Elizabeth Skovran ◽  
Jean-Charles Portais ◽  
...  
Keyword(s):  
Formate Dehydrogenase ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Methanol Metabolism ◽  
Reading Frame ◽  
Formate Oxidation ◽  
In The Wild ◽  
Mutant Phenotypes ◽  
Small Open Reading Frame

ABSTRACT A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [13C]formate, as well as 14C-labeling experiments, demonstrated production of labeled CO2 in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO2 formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.

scholarly journals His20 Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

2007 ◽  
Vol 189 (7) ◽  
pp. 2825-2833 ◽  
Author(s):  
Ray A. Larsen ◽  
Gail E. Deckert ◽  
Kyle A. Kastead ◽  
Surendranathan Devanathan ◽  
Kimberly L. Keller ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Protonmotive Force ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
In The Wild ◽  
Mutant Phenotypes

ABSTRACT The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser16 and His20, the resultant “all-Ala Ser16 His20” TMD TonB retained 90% of wild-type iron transport activity. Ser16Ala in the context of a wild-type TonB TMD was fully active. In contrast, His20Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser16 His20 TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser16 His20 TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser16 His20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser16 His20 TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

scholarly journals Antibacterial Activity of Gatifloxacin (AM-1155, CG5501, BMS-206584), a Newly Developed Fluoroquinolone, against Sequentially Acquired Quinolone-Resistant Mutants and thenorA Transformant of Staphylococcus aureus

1998 ◽  
Vol 42 (8) ◽  
pp. 1917-1922 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Satoshi Hori ◽  
Keiichi Hiramatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Low Frequency ◽  
Dna Gyrase ◽  
Second Step ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Sequential Selection ◽  
In The Wild ◽  
Fourth Step ◽  
Resistant Mutants

ABSTRACT Alternate mutations in the grlA and gyrAgenes were observed through the first- to fourth-step mutants which were obtained from four Staphylococcus aureus strains by sequential selection with several fluoroquinolones. The increases in the MICs of gatifloxacin accompanying those mutational steps suggest that primary targets of gatifloxacin in the wild type and the first-, second-, and third-step mutants are wild-type topoisomerase IV (topo IV), wild-type DNA gyrase, singly mutated topo IV, and singly mutated DNA gyrase, respectively. Gatifloxacin had activity equal to that of tosufloxacin and activity more potent than those of norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin against the second-step mutants (grlA gyrA; gatifloxacin MIC range, 1.56 to 3.13 μg/ml) and had the most potent activity against the third-step mutants (grlA gyrA grlA; gatifloxacin MIC range, 1.56 to 6.25 μg/ml), suggesting that gatifloxacin possesses the most potent inhibitory activity against singly mutated topo IV and singly mutated DNA gyrase among the quinolones tested. Moreover, gatifloxacin selected resistant mutants from wild-type and the second-step mutants at a low frequency. Gatifloxacin possessed potent activity (MIC, 0.39 μg/ml) against the NorA-overproducing strain S. aureus NY12, thenorA transformant, which was slightly lower than that against the parent strain SA113. The increases in the MICs of the quinolones tested against NY12 were negatively correlated with the hydrophobicity of the quinolones (correlation coefficient, −0.93;P < 0.01). Therefore, this slight decrease in the activity of gatifloxacin is attributable to its high hydrophobicity. Those properties of gatifloxacin likely explain its good activity against quinolone-resistant clinical isolates of S. aureusharboring the grlA, gyrA, and/ornorA mutations.

Mutations in the clk-1 gene of Caenorhabditis elegans affect developmental and behavioral timing.

Genetics ◽  
1995 ◽  
Vol 139 (3) ◽  
pp. 1247-1259 ◽  
Author(s):  
A Wong ◽  
P Boutis ◽  
S Hekimi
Keyword(s):  
Cell Cycle ◽  
Caenorhabditis Elegans ◽  
Embryonic Development ◽  
Postembryonic Development ◽  
Cycle Period ◽  
Biological Clocks ◽  
Wild Type ◽  
Individual Variations ◽  
In The Wild ◽  
Mutant Phenotypes

Abstract We have identified three allelic, maternal-effect mutations that affect developmental and behavioral timing in Caenorhabditis elegans. They result in a mean lengthening of embryonic and postembryonic development, the cell cycle period and life span, as well as the periods of the defecation, swimming and pumping cycles. These mutants also display a number of additional phenotypes related to timing. For example, the variability in the length of embryonic development is several times larger in the mutants than in the wild type, resulting in the occasional production of mutant embryos developing more rapidly than the most rapidly developing wild-type embryos. In addition, the duration of embryonic development of the mutants, but not of the wild type, depends on the temperature at which their parents were raised. Finally, individual variations in the severity of distinct mutant phenotypes are correlated in a counterintuitive way. For example, the animals with the shortest embryonic development have the longest defecation cycle and those with the longest embryonic development have the shortest defecation cycle. Most of the features affected by these mutations are believed to be controlled by biological clocks, and we therefore call the gene defined by these mutations clk-1, for "abnormal function of biological clocks."

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

Sign in / Sign up

Export Citation Format

Share Document