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PubMed1976

RNA polymerase mutants of Escherichia coli. III. A temperature-sensitive rifampicin-resistant mutant.

Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1973). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for RNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the beta subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of beta subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both beta and alpha subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the beta subunit, beside the primary mutation affecting the beta subunit. The hypothetical beta subunit mutation seems to modify quantitatively the rifampicin resistance caused by the beta subunit mutation.

DNA-Directed RNA Polymerases↗

PubMed1977

Extrachromosomal inheritance in Schizosaccharomyces pombe. IV. Isolation and genetic characterization of mutants resistant to chloramphenicol and erythromycin using the mutator properties of mutant anar-8.

Spontaneous chloramphenicol (capr)- and erythromycin (eryr)-resistant mutants were isolated from strain ade7-50 h- and the antimycin-resistant mutant anar-8 ade 7-50 h- of Schizosaccharomyces pombe (Sch. p.). By mitotic segregation analysis all 154 capr- and 120 eryr-mutants derived from ade 7-50 h- proved to be recessive chromosomal, whereas all 108 capr- and 200 eryr-mutants originating from anar-8 were extrachromosomally inherited. The rate of spontaneous capr- and eryr-mutants was about hundredfold in anar-8 compared to ade 7-50 h-. Growth of capr- and eryr-mutants was not inhibited by chloramphenicol or erythromycin, respectively, in glucose-medium and only slightly in glycerol-medium at concentrations which completely inhibited anar-8. By mitotic segregation-, tetrad-, and mitotic haploidization-analysis the extrachromosomal inheritance of mutants derived from anar-8 was established. Segregational patterns of capr- and eryr-determinants during mitosis, meiosis, and mitotic haploidization of diploids are discussed.

Ascomycota↗

PubMed1978

A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain.

NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.

Enzyme Induction↗

PubMed1976

LambdacI mutants: intragenic complementation and complementation with a cI promoter mutant.

Complementation for the maintenance of lysogeny was studied by superinfecting lambdacIts lysogens at 34 degrees C. and then heating to 43 degrees C. With certain exceptions, ts mutants with defects in the left half of the repressor complemented ts mutants with defects in the right half to produce a less heat-labile repressor (Fig. 3). All cI amber mutants failed to complement cIts mutants. The cI mutant c50 complements all ts mutants. Mutations in Pre (cy) or genes cII and cIII do not significantly affect the expression of cI by a superinfecting lambda genome in an immune lysogen. Mutants with very heat-labile repressors failed to complement lambdacy42 for the establishment of lysogeny at elevated temperatures, while those with less heat-sensitive repressors apparently did complement cy. According to a suggested model, the left side of the cI product is concerned primarily with subunit aggregation, while operator binding is the function of the right side of the oligomer.

Coliphages↗

PubMed2000

Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA.

Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO2 assimilation measured in individual leaves was substantially diminished only under conditions of high CO2 and low O2. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod.

Alleles↗

PubMed2002

Mutant pso8-1 of Saccharomyces cerevisiae, sensitive to photoactivated psoralens, UV radiation, and chemical mutagens, contains a rad6 missense mutant allele.

A novel mutant isolate of Saccharomyces cerevisiae, sensitive to photoactivated mono- and bi-functional psoralens, to UV at 254 nm (UVC), and to nitrosoguanidine, was found to complement the photoactivated psoralen-sensitivity phenotype conferred by the pso1- pso7 mutations and was therefore named pso8-1. A constructed pso8-1 rad4-4 double mutant was super-sensitive to UVC, thus indicating a synergistic interaction of the two mutant alleles. Molecular cloning via complementation of the pso8 mutant's sensitivity phenotype and genetic studies revealed that pso8 is allelic to RAD6. While a pso8-1 mutant had low mutagen-induced mutability, homoallelic diploids showed nearly wild-type sporulation. Sequence analysis of the mutant allele showed pso8-1 to contain a novel, hitherto undescribed T-->C transition in nucleotide position 191, leading to a substitution by leucine of a highly conserved proline at position 64, Rad6-[P64L], which may have severe consequences for the tertiary structure (and hence binding to Rad18p) of the mutant protein.

Alleles↗

PubMed1986

The nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by gamma-rays or ethyl methanesulphonate.

A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells (Brown and Thacker, 1984), were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from gamma-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT (cross-reacting material, CRM), using an antibody raised against partially purified V79-4 HPRT enzyme. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the gamma-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency, either spontaneously or after treatment with the powerful mutagen ethyl nitrosourea (ENU). All except 2 of the EMS-induced mutants reverted with ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of gamma-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. The EMS-induced mutations are likely to be mostly point mutations, with at least 40% of the missense type, while gamma-ray-induced mutations may arise mostly through larger genetic changes.

Animals↗

PubMed1985

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12.

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.

Bacterial Proteins↗

PubMed2006

Gain of function of mutant p53: the mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation.

This article investigates the mechanistic aspects of mutant p53 "gain of function" in response to DNA damage. We show that mutant forms of p53 protein interact with NF-Y. The expression of cyclin A, cyclin B1, cdk1, and cdc25C, as well as the cdk1-associated kinase activities, is upregulated after DNA damage, provoking a mutant p53/NF-Y-dependent increase in DNA synthesis. Mutant p53 binds NF-Y target promoters and, upon DNA damage, recruits p300, leading to histone acetylation. The recruitment of mutant p53 to the CCAAT sites is severely impaired upon abrogation of NF-YA expression. Endogenous NF-Y, mutant p53, and p300 proteins form a triple complex upon DNA damage. We demonstrate that aberrant transcriptional regulation underlies the ability of mutant p53 proteins to act as oncogenic factors.

Base Sequence↗

PubMed1999

Single-chain 434 repressors with altered DNA-binding specificities. Isolation of mutant single-chain repressors by phenotypic screening of combinatorial mutant libraries.

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA--6 base-pairs--NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10-50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.

Base Sequence↗

PubMed1995

Characterization of rhodopsin mutants that bind transducin but fail to induce GTP nucleotide uptake. Classification of mutant pigments by fluorescence, nucleotide release, and flash-induced light-scattering assays.

The photoreceptor rhodopsin is a seven-transmembrane helix receptor that activates the G protein transducin in response to light. Several site-directed rhodopsin mutants have been reported to be defective in transducin activation. Two of these mutants bound transducin in response to light, but failed to release the bound transducin in the presence of GTP (Franke, R. R., König, B., Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990) Science 250, 123-125). The present study was carried out to determine the nucleotide-binding state of transducin as it interacts with rhodopsin mutants. Five mutant bovine opsin genes were prepared by site-specific mutagenesis. Three mutant genes had deletions from one cytoplasmic loop each: AB delta 70-71; CD delta 143-150; and EF delta 237-249. Two additional loop CD mutant genes were prepared: E134R/R135E had a reversal of a conserved charge pair, and CD r140-152 had a 13-amino acid sequence replaced by a sequence derived from the amino-terminal tail. Three types of assays were carried out: 1) a fluorescence assay of photoactivated rhodopsin (R*)-dependent guanosine 5'-O-(3-thiotriphosphate) uptake by transducin, 2) an assay of R*-dependent release of labeled GDP from the alpha-subunit of transducin holoenzyme (Gt alpha).GDP, and 3) a light-scattering assay of R*.Gt complex formation and dissociation. We show that the mutant pigments, which are able to bind transducin in a light-dependent manner but lack the ability to activate transducin, most likely form R*.Gt alpha beta gamma.GDP complexes that are impaired in GDP release.

Amino Acid Sequence↗

PubMed1996

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants.

The EcoRV DNA methyltransferase (M.EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M.EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M.EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M.HhaI and M.TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M.EcoRV (Val2-Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M.EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant. According to the structure prediction, Trp231 is located in a loop at the putative active site of M.EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M.EcoRV confirming the importance of these regions.

Amino Acid Sequence↗

PubMed1990

Polyamines and Flower Development in the Male Sterile Stamenless-2 Mutant of Tomato (Lycopersicon esculentum Mill.) : II. Effects of Polyamines and Their Biosynthetic Inhibitors on the Development of Normal and Mutant Floral Buds Cultured in Vitro.

The floral organs of the male sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.) contain significantly higher level of polyamines than those of the normal (R Rastogi, VK Sawhney [1990] Plant Physiol 93: 439-445). The effects of putrescine, spermidine and spermine, and three different inhibitors of polyamine biosynthesis on the in vitro development of floral buds of the normal and sl-2/sl-2 mutant were studied. The polyamines were inhibitory to the in vitro growth and development of both the normal and mutant floral buds and they induced abnormal stamen development in normal flowers. The inhibitors of polyamine biosynthesis also inhibited the growth and development of floral organs of the two genotypes, but the normal flowers showed greater sensitivity than the mutant. The inhibitors also promoted the formation of normal-looking pollen in stamens of some mutant flowers. The effect of the inhibitors on polyamine levels was not determined. The polyamine-induced abnormal stamen development in the normal, and the inhibitor-induced production of normal-looking pollen in mutant flowers support the suggestion that the elevated polyamine levels contribute to abnormal stamen development in the sl-2/sl-2 mutant of tomato.

Journal Article↗

PubMed2006

Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response.

New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

Bryopsida↗

PubMed1980

Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis.

A single-gene nuclear choline-requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of dimethylethanolamine, monomethylethanolamine or ethanolamine. DL-Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth-arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild-type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild-type cells. The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant. In cell-free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild-type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.

Cell Membrane↗

PubMed2006

A rapid mutant screening technique for detection of technetium [Tc(VII)] reduction-deficient mutants of Shewanella oneidensis MR-1.

Microbial metal reduction forms the basis of alternate bioremediation strategies for reductive precipitation and immobilization of toxic metals such as the radionuclide technetium [Tc(VII)]. A rapid mutant screening technique was developed to identify Shewanella oneidensis MR-1 respiratory mutants unable to reduce Tc(VII) as anaerobic electron acceptor. The Tc(VII) reduction-deficient (Tcr) mutant screening technique was based on the observation that wild-type S. oneidensis produced a black Tc(IV) precipitate on its colony surface during growth on Tc(VII)-amended agar, while colonies arising from mutagenized cells did not. Tcr mutants unable to produce the black precipitate were subsequently tested for anaerobic growth on an array of three electron donors and 13 alternate electron acceptors. The Tcr mutants displayed a broad spectrum of anaerobic growth deficiencies, including several that were unable to reduce Tc(VII) with hydrogen or lactate as electron donor, yet retained the ability to reduce Tc(VII) with formate. This report describes the development of a novel Tcr mutant screening technique and its application to identify the first set of Tcr mutants in a metal-reducing member of the genus Shewanella.

Anaerobiosis↗

PubMed2006

The anti-methicillin-resistant Staphylococcus aureus quinolone WCK 771 has potent activity against sequentially selected mutants, has a narrow mutant selection window against quinolone-resistant Staphylococcus aureus, and preferentially targets DNA gyrase.

WCK 771 is a broad-spectrum fluoroquinolone with enhanced activity against quinolone-resistant staphylococci. To understand the impact of the target-level interactions of WCK 771 on its antistaphylococcal pharmacodynamic properties, we determined the MICs for genetically defined mutants and studied the mutant prevention concentrations (MPCs), the frequency of mutation, and the cidality against the wild type and double mutants. There was a twofold increase in the MICs of WCK 771 for single gyrA mutants, indicating that DNA gyrase is its primary target. All first- and second-step mutants selected by WCK 771 revealed gyrA and grlA mutations, respectively. The MICs of WCK 771 and clinafloxacin were found to be superior to those of other quinolones against strains with double and triple mutations. WCK 771 was also cidal for high-density double mutants at low concentrations. WCK 771 and clinafloxacin showed narrow mutant selection windows compared to those of the other quinolones. Against a panel of 50 high-level quinolone-resistant clinical isolates of staphylococci (ciprofloxacin MIC > or = 16 microg/ml), the WCK 771 MPCs were < or =2 microg/ml for 68% of the strains and < or =4 microg/ml for 28% of the strains. Our results demonstrate that gyrA is the primary target of WCK 771 and that it has pharmacodynamic properties remarkably different from those of quinolones with dual targets (garenoxacin and moxifloxacin) and topoisomerase IV-specific quinolones (trovafloxacin). WCK 771 displayed an activity profile comparable to that of clinafloxacin, a dual-acting quinolone with a high affinity to DNA gyrase. Overall, the findings signify the key role of DNA gyrase in determining the optimal antistaphylococcal features of quinolones.

Anti-Bacterial Agents↗

PubMed2007

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications.

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

Anti-Bacterial Agents↗