Simple identification of mutant clones.
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Cell Count↗
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[Identification of mutant gene responsible for cotyledons necrosis in developing seedlings of Arabidopsis thaliana].
Genetic and molecular analyses of an Arabidopsis thaliana mutant with necrotic cotyledons from the collection of insertion mutants obtained earlier were conducted. The mutation under study showed incomplete dominance and represented a single insertion of the T region of pLD3 vector used for transformation of germinating seeds to the plant genome during the creation of the collection. Using TAIL-PCR, a fragment of the mutant DNA adjacent to the left border of the T-DNA insertion was isolated and sequenced. Computer r-aided analysis showed that the insertion was located on the left arm of chromosome 1. The open reading frame containing the insertion has one exon and encodes a protein of 446 amino acids, whose functions are unknown.
Amino Acid Sequence↗
[The study of actinomyces viscosus adhere to teeth. III. The identification of mutants of A. viscosus and preparation of rabbit IgG against type 1 and type 2 fimbriae].
The authors prepared rabbit antisera against 5519 and 5951. The IgGs against type 1, type 2 fimbriae were purified from the antisera using sera absorbed with the other strain and IgG purification method. It was identified by SDS-PAGE and ELISA. This result suggested the purified proteins were the IgG against type 1 and type 2 fimbriae.
Actinomyces viscosus↗
Study of isolation of fluoroquinolone-resistant Ureaplasma urealyticum and identification of mutant sites.
OBJECTIVE: To study the resistance mechanism of clinical isolates of Ureaplasma urealyticum resistant to fluoroquinolones. METHODS: Thirteen isolates of Ureaplasma urealyticum resistant to six fluoroquinolones were selected out of 184 clinical isolates and their QRDRs (quinolone resistance-determining region) gyrA, gyrB, parC and parE were amplified by PCR. Sequencing results were compared to those susceptible reference strains and a comparison of deduced amino acid sequences were performed. RESULTS: Sequence comparison revealed a C to A change at 87nt of gyrA QRDR leading to the substitution of Asp95 with glutamic acid and a C to T change at 50nt of parC QRDR leading to the substitution of Ser80 with leucine. CONCLUSION: These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with fluoroquinolone resistance of Ureaplasma urealyticum.
Amino Acid Substitution↗
[Gd- allele distribution patterns in Azerbaijan. III. The identification of mutant forms of glucose-6-phosphate dehydrogenase].
In 28 families with G6PD deficiency living in 3 settlements of Shekii district of Azerbaijan 11 G6PD variants of II and III classes differing by kinetic properties were identified according WHO program. 9 of them are characterized with the same electrophoretic mobility. Comparison of G6PD spectra in two subpopulations and in a mixed group permits to make a conclusion about existence of common and rare G6PD alleles in examined population. They distribute by gene drift supported by natural selection. Among 7 samples of G6PD with normal and increased activity two new variants of IV class -- Nukha and Bash-Kungut -- were found.
Alleles↗
[Problems in hemolytic anemias with insufficient pyruvate kinase. II. Identification of mutants of pyruvate kinase].
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Anemia, Hemolytic↗
Identification of mutants of pyruvate kinase from red blood cells by means of trypsinization, electrophoresis, kinetic properties and immunological methods.
Enzymopathies of pyruvate kinase (PK) are characterized by polymorphism. 11 different mutants of PK were detected in 12 analysed cases. The frequency of double heterozygotes of the PK deficiency is very high. The mutant forms have been identified by electrophoretic, kinetic and immunologic methods in red blood cells of patients and members of their families. The effect of trypsin has greatly increased the sensitivity of the differentiation procedures. Differences in activity and quality of the proteolytic systems in liver and kidney in comparison to red blood cells are responsible for inactivation and degradation of PK mutants in both organs. Homozygotes and double heterozygotes show clinic manifestations. The rigidity of red blood cells increases with the severity of the nonspherocytic haemolytic anaemia.
Erythrocytes↗
A rapid screening procedure for the isolation of nonconditional replication mutants of Mason-Pfizer monkey virus: identification of a mutant defective in pol.
A rapid, sensitive, and reproducible method for the isolation of human cell clones containing nonconditional, replication-defective (rd) mutants of Mason-Pfizer monkey virus (M-PMV), the prototype of the D-type retroviruses is described. The two mutants, rd1 and rd2, thus far isolated have been analyzed for virus particle production (using radiolabeled precursors and by electron microscopy) and for the status of intracellular viral precursors. Thin sections of rd1 and rd2 infected cells showed typical M-PMV particles when observed under electron microscope. A more direct assay of virus production, by labeling the mutant cell clones with [3H]uridine, also showed a distinct virus peak at an approximate density of 1.16 g/ml when culture fluids from rd1 and rd2 were analyzed. Analyses of these two mutants showed no defect in either gag or env gene products, however, further analysis of rd1 showed that the Pr180gag-pol was altered in its migration on SDS-polyacrylamide gel electrophoresis and no reverse transcriptase activity could be detected in rd1 virions. Mutant rd2, on the other hand, assembles noninfectious virus particles that are otherwise indistinguishable from those produced by wild-type cell clones. The biochemical basis for the defect in this mutant remains to be established.
Defective Viruses↗
Carbon dioxide modulation of peroxynitrite-induced mutagenesis of the supF gene in pSP189.
Peroxynitrite (ONOO(-)), a potent oxidant formed by the reaction of nitric oxide with superoxide anion, may play a significant role as an intermediate in NO(*)-related cytotoxicity and genotoxicity. In addition to causing other types of toxicity, peroxynitrite damages DNA and induces mutations in genetic targets following in vitro or in vivo exposure. It has recently been established that the reaction of CO(2) with ONOO(-) significantly changes its chemistry in biological media. The objective of this investigation was to characterize impacts of CO(2) on peroxynitrite-induced mutagenesis of the supF gene in the shuttle vector pSP189. The dose-response relationship between ONOO(-) concentration and mutation frequency was determined following bolus exposure of the plasmid suspended in 150 mM phosphate buffer at pH 7.4, with and without addition of 25 mM sodium bicarbonate. After treatment, plasmids were replicated in Escherichia coli MBL50 cells for mutant identification. CO(2) significantly reduced the mutagenic potency of peroxynitrite, in that increases in mutant fraction were reduced by 47-77% at ONOO(-) doses ranging from 0 to 4 mM. We also characterized the spectrum of mutations induced under these conditions and found mutational hotspots at positions 110, 113, 116, 141, 156, 168, and 172 in mutants induced in the presence of CO(2) and at positions 113, 124, 126, 141, and 156 in those induced in its absence. Among the 22 guanines present in the 84 nucleotide supF sequence, 18 were sites of mutation, including four mutational hotspots (G113, G116, G141, and G156), in plasmids exposed to ONOO(-) in the presence of bicarbonate. After exposure in the absence of bicarbonate, 14 of the 22 guanines were mutation sites, with hotspots located at five (G113, G124, G126, G141, and G156). Remaining mutations were located almost exclusively at cytosine residues. It is evident from these data that reactive intermediates formed through reaction of ONOO(-) with CO(2) play an important role in the mutagenicity of ONOO(-).
Base Sequence↗
Shuttle-vector mutagenesis by aflatoxin B1 in human cells: effects of sequence context on the supF mutational spectrum.
Rat-liver microsomes were used to activate aflatoxin B1 for in vitro modification of the pS189 shuttle vector and the related signature vector pSP189, both of which carry the Escherichia coli supF gene as a mutational target. Plasmid degradation was minimized by carrying out the in vitro incubations in the absence of Mg2+ ions. Modified plasmids were transfected into human Ad293 cells, then recovered and electroporated into E. coli MBM7070 for mutant identification. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background level. Mutant plasmids were characterized by DNA-sequence analysis. The vast majority of aflatoxin B1-induced mutations were base substitutions, mostly G:C to T:A transversions. The spectrum of aflatoxin B1-induced mutations in the pS189 supF gene was very similar to that observed previously for the pS189 supF gene with aflatoxin B1 activated by cytochrome P450 1A2 (CYP1A2) synthesized from a cDNA expression vector within transfected Ad293 cells. However, the spectrum for pSP189, which carries the same supF gene as pS189, but with different surrounding sequences, exhibited some notable differences from that of pS189; this suggests that sequence context effects on mutagenic specificity can operate over distances of tens of base pairs.
Aflatoxin B1↗
Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.
A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 in E. coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.
Alleles↗
Physical and partial genetic map of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genome.
A Nicaraguan isolate of Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) is undergoing field trials for control of this pest in the Americas. This isolate is composed of multiple genotypes, some of which are deletion mutants. Identification of the genetic changes in deleted genotypes cannot be accomplished without the construction of a detailed physical map. In the present study, combinations of restriction endonuclease analysis and Southern blot analysis was performed. This map was refined by sequencing the termini of cloned restriction fragments. The SfMNPV genome was estimated to be 129.3 kb, 8 kb larger than the previously characterized Sf-2 variant from the United States, due to a deletion between 14.8 and 21.0 m.u. in the physical map described in this study. A total of 27.92 kb were sequenced, which represented 21.5% of the whole genome and included 38 ORFs. Comparison with other sequenced baculoviruses revealed that SfMNPV displayed the highest sequence identity (66%) and gene arrangement (78%) with Spodoptera exigua MNPV, sharing 36 putative ORFs. In addition, the genome organization was similar to that of SeMNPV, with minor differences. Phylogenetic analysis confirmed the close relatedness between SeMNPV and SfMNPV, suggesting they evolved from a common ancestor.
Animals↗
Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA.
The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H-ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA). The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences were amplified by PCR. When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.
Adenosine Triphosphatases↗
Molecular cloning and characterization of the yeast gene for squalene synthetase.
Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway. The structural gene for this enzyme was cloned from the yeast Saccharomyces cerevisiae by functional complementation of a squalene synthetase-deficient erg9 mutant. Identification of this ERG9 clone was confirmed by genetic linkage analysis in yeast and expression of enzyme activity in Escherichia coli. The predicted squalene synthetase polypeptide of 444 amino acids (Mr, 51,753) lacks significant homology to known protein sequences, except within a region that may represent a prenyl diphosphate (substrate) binding site. The ERG9-encoded protein contains a PEST consensus motif (rich in proline, glutamic acid, serine, and threonine) present in many proteins with short cellular half-lives. Modeling of the protein suggests that it contains at least one, and possibly two, membrane-spanning domains. Disruption of the chromosomal squalene synthetase coding region by insertional mutagenesis indicates that ERG9 is a single copy gene that is essential for cell growth in yeast.
Amino Acid Sequence↗
Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73.
To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10(-6)). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.
Amino Acid Sequence↗
Genetic Analysis of Genomic and Methylomic Variation and Identification of Multi-Trait Mutants in Rice Carried on Chang'e-5.
Global food security is facing challenges from population growth to diminishing arable land. Space mutation breeding holds promise for overcoming the variation limitations in conventional breeding; however, the mutagenic effects of the deep-space environment on rice and the transgenerational inheritance patterns of induced variations remain unclear. In this study, rice seeds carried by the Chang'e-5 spacecraft were used as materials. Whole-genome sequencing and whole-genome bisulfite sequencing were performed on the first (SP1) and second generations (SP2) of space-mutagenized plants after their return to Earth. The results showed that the number of genomic variants in the SP2 generation increased significantly compared with SP1, and SNPs, homozygous sites, and variants in coding regions were more heritable. The genome-wide methylation level was elevated in the SP2 generation, and among differentially methylated cytosines, those in the CG context exhibited the highest heritability. Furthermore, large-scale screening for nitrogen efficiency, tolerance to PEG-induced stress, and germination-stage cold resistant mutants was conducted in the SP2 generation, and phenotypic validation was performed in the third generation (SP3). By integrating multi-omics analyses of representative mutants to mine candidate genes, a number of heritable elite mutants were obtained, and seven candidate genes for key traits were identified. This study systematically elucidates the transgenerational inheritance patterns of deep-space-induced variation in rice. The multi-trait mutants obtained provide valuable germplasm resources for gene cloning and breeding applications in rice.
DNA methylation↗
Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes.
Hb Hasharon has an electrophoretic mobility similar to that of Hb S in cellulose acetate and a mobility between Hb S and C at acid pH. In high-performance liquid chromatography, Hb Hasharon shows a distinct chromatographic profile and retention time. The origin of this variant is a mutation in codon 47 (GAC --> CAC) of the alpha2-globin gene, resulting in the replacement of asparagine by histidine during the translation process. Ten blood samples from individuals suspected of being Hb Hasharon carriers were analyzed. In addition to classic laboratory tests and high-performance liquid chromatography, molecular analysis by polymerase chain reaction with restriction fragment length polymorphism designed in the laboratory was performed to confirm this mutation. The study of these cases showed that a combination of classical and molecular methodologies is necessary in the diagnosis of hemoglobinopathies for a correct hemoglobin mutant identification. The accurate identification of hemoglobin variants is essential for genetic counseling and choice of therapy.
Adolescent↗
Identification of yeast mutants with altered telomere structure.
The chromosomes of the yeast Saccharomyces cerevisiae terminate in a tract of simple-sequence DNA [poly(C1-3A)] that is several hundred base pairs long. We describe the identification of mutant yeast strains that have telomeric tracts that are shorter than normal. A genetic analysis of these strains indicates that these short telomeres are the result of single nuclear recessive mutations and that these mutations can be classified into two different complementation groups. The full expression of the mutant phenotype shows a very long lag (approximately equal to 150 cell divisions). From our analysis of these mutants as well as other data, we suggest that the duplication of the telomeric poly(C1-3A) tract involves two processes, semiconservative replication and untemplated terminal addition of nucleotides.
Base Sequence↗