Intragenic location of an unstable insertion element within gene b5 of the fungus Ascobolus immersus

Summary A fine structure map of gene b5 has been established in Ascobolus immersus and the unstable mutant site b5-301 (phenotype: ascospore coloration) has been found to map within this gene. This map was constructed using seven b5 mutants induced by ICR170 and is based on the additivity of recombinant frequencies and confirmed by three point tests. The unstable site 301 is located between the induced sites. In particular, mutant 249 is located to the left of site 301, whereas sites 601 and 754 are located to the right.Previous studies showed that the inducing gene of mutant b5-301 reversions are either closely linked to the b5 locus or within it in certain strains. The study of asci resulting from reciprocal recombination between unstable mutant site and several induced mutant sites showed that neither crossovers located on the left nor the right of site 301, separate the unstable site from the inducing gene. Thus, the inducing gene was found to map within gene b5 as did the inducible site.These results constitute a genetic argument showing the presence of an insertion element. In this case, the insertion structure contains at least the integration site (inducible site) and the inducing gene which allows the excision.     

Mutational analysis of the starch utilization system in the yeast Saccharomyces cerevisiae

Seven mutants of Saccharomyces cerevisiae deficient in production of extracellular glucoamylase have been analyzed. For each of the seven a monogenic pattern of inheriting the mutant phenotype has been observed. The mutations have been shown to map within five different genetic loci, three independent mutations affecting the STA2 locus and the other four residing in four formerly unidentified genes. As expected, the sta2 mutants recover the wild phenotype when transformed with a STA2-bearing multicopy plasmid. Such reversion has also been observed for the transformed stall mutant. Unlike the others, the sta16 mutant is unable to secrete heterologous alpha-amylase encoded by a plasmid-borne DNA fragment. All the mutants have a moderately reduced ability to secrete the invertase and acid phosphatase.     

Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages

whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although bldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for bldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. bldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.     

Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.     

DNA amplification and an unstable arginine gene in Streptomyces lividans 66

Summary Streptomyces lividans 66 produced spontaneous chloramphenicol-sensitive mutants (Cml^s) at a frequency of about 1% of spores. The Cml^s mutant strains were very unstable, giving Arg^- mutants at frequencies of about 25% of spores. All the Arg^- mutants had amplified a particular 5.75 kb DNA fragment into tandem repeats of 250–500 copies per chromosome.     

Characterization of a Bacillus cereus protease mutant defective in an early stage of spore germination.

Temperature-sensitive sporulation mutants of Bacillus cereus were screened for intracellular protease activity that was more heat labile than that of the parental strain. One mutant grew as well as the wild type at 30 and 37 degrees C but sporulated poorly at 37 degrees C in an enriched or minimal medium. These spores germinated very slowly in response to alanine plus adenosine or calcium dipicolinate. During germination, spores produced by the mutant rapidly became heat sensitive, but released dipicolonic acid and mucopeptide fragments more slowly than the wild type and decreased only partially in density while remaining phase white (semirefractile). In freeze-etch electron micrographs, the mature spores were deficient in the outer cross-patched coat layer. During germination, the spore coat changes associated with wild-type germination occurred very slowly in this mutant. Although the original mutant was also a pyrimidine auxotroph, reversion to prototrophy did not alter any of the phenotypic properties discussed. Selection of revertants that germinated rapidly or sporulated well at 37 degrees C, however, resulted in restoratin of all wild-type properties (exclusive of the pyrimidine requirement) including heat-stable protease activity. The reversion frequency was consistent with an initial point mutation, indicating that a protease alteration resulted in production of spores defective in a very early stage of germination.     

Spontaneous Mutation at the Mtr Locus of Neurospora: The Spectrum of Mutant Types

We have isolated 135 strains of Neurospora which have mutations at the mtr locus resulting from independent spontaneous events. mtr is the structural gene for the neutral amino acid permease. The mutants have been characterized by their reversion behavior (both spontaneous and induced) and by hybridization studies of restriction digests of their DNA. About half of the mutants (54%) appear to result from single base-pair substitutions. Thirty-four percent have deletions, including some which extend into neighboring genes. Most of the remaining mutants have insertions. Several of the insertions are tandem duplications of 400-1000 bp and these mutants are unstable, reverting to mtr+ with a high frequency. When tandem-duplication mutants go through a cross, they are modified: the mutant progeny are fully stable. This modification is probably due to RIP (repeat-induced point mutation). This process has an important bearing on the comparison of germinal to somatic mutation.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.     

Reversion of nonsense mutants induced by 4-nitroquinoline-1-oxide in Schizosaccharomyces pombe.

We have studied the reversion of 8 nonsense alleles located in 7 different genes of Schizosaccharomyces pombe using 4-nitroquinoline-1-oxide (NQO) as a mutagenic agent. The nonsense mutants of S. pombe have been classified according to their suppressibility by defined opal and ochre suppressors into a class of efficiently suppressed opal and a class of inefficiency suppressed ochre mutants. The UGA alleles tested all revert consistently with NQO, in agreement with the high specificity of this mutagen for G-residues reported for bacteria and yeast. The UAA alleles show a lack or a low level of reversion with NQO. This low level of reversion is due to the low level of non-G-specific transversions at A sites of the UAA triplet. Within each class of nonsense mutants the extent of induction is site-dependent. We conclude that NQO acts predominantly on G-residues in S. pombe.     

GENETICS OF SORDARIA FIMICOLA. IX. LINKAGE GROUP II

Ten mutant sites have been located in linkage group II of Sordaria fimicola, nine of them affecting fertility. Two different regions of the chromosome are marked with allelic mutations or mutant sites in closely linked loci involved in sexual reproduction. Mutants of one of these two regions also have an arginine deficiency. Crosses between the self-sterile mutants of each region yield hybrid perithecia containing asci with four spores of each parental genotype and few or no asci with recombinant spores. The bearing that these findings have upon the evolution of heterothallism is discussed.     

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.     

EMS-induced reversion studies in the white locus of Drosophila melanogaster

5 white-locus mutants of Drosophila melanogaster, representing 5 different sub-sites, were treated with EMS and tested for reversion to wild-type. 4 of them were genuine mutants and one was not. Moreover, the ability of the 4 mutants to revert to wild-type differed from one another which therefore reflects a qualitatively distinct alteration in the genetic material delimited by each mutant.     

High-Frequency RecA-Dependent and -Independent Mechanisms of Congo Red Binding Mutations in Yersinia pestis

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar. The hmsHFRS genes required for CR binding (Crb(+)) are genetically linked to virulence-associated genes encoding a siderophore uptake system. These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb(+) phenotype. We constructed a recA mutant strain of Y. pestis KIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flanked pgm locus. Two Pgm-associated phenotypes (Crb(+) and pesticin sensitivity [Pst(s)]) were used as markers for the presence of the pgm locus in the RecA(+) KIM10+ and RecA(-) YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 x 10(-3)) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb(+) phenotype was still lost at a high frequency (4.5 x 10(-5)), although the loss of the Pst(s) phenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 x 10(-7)). These RecA-independent Crb(-) mutants were caused by mutations in both the hmsHFRS locus and in a newly identified gene, hmsT. Nonpigmented Yersinia pseudotuberculosis and Escherichia coli strains transformed with both hmsT and hmsHFRS became Crb(+). This study demonstrates that in a laboratory culture, the Crb(+) phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y. pestis in vitro may contribute to the mutation frequencies observed at the hmsHFRS and hmsT loci.     

Molecular mapping of 36 soybean male-sterile, female-sterile mutants

Mutability of the w ( 4 ) flower color locus in soybean [Glycine max (L.) Merr.] is conditioned by an unstable allele designated w ( 4 ) -m. Germinal revertants, purple-flower plants, recovered among self-pollinated progeny of mutable flower plants were associated with the generation of necrotic root, chlorophyll-deficiency, and sterility mutations. Thirty-seven male-sterile, female-sterile mutant lines were generated from 37 independent reversion events at the w ( 4 ) -m locus. The first germinal revertant study had one male-sterile, female-sterile mutant (st8, T352), located on Molecular Linkage Group (MLG) J. The second study had 36 germinal-revertant derived sterility mutants descended from four mutable categories of w ( 4 ) -m. The mutable categories were designated; (1) low frequency of early excisions, (2) low frequency of late excisions, (3) high frequency of early excisions, and (4) high frequency of late excisions. The objectives of the present study were to; (1) molecularly map the 36 male-sterile, female-sterile mutants, and to (2) compare map locations of these mutants with T352 (st8), identified from the first germinal revertant study. Thirty-three of 36 male-sterile, female-sterile mutations were derived from germinal reversions that were classified in the late excision categories. Thirty-five male-sterile mutants mapped to the st8 region on MLG J. The only exception mapped to MLG G. Most likely mutants were generated through insertion of a putative transposon that was excised from the w ( 4 ) locus. The location of 36 of 37 mutations to a single chromosomal region suggests preference for sequence-dependent insertion.     

Modification of the high frequency of reversion of the nicotonic-2 mutant of Coprinus radiatus. II. Polymorphism of strains with low frequencies of reversion (author's transl)]

The nic2 mutation of Coprinus radiatus is unstable at meiosis. Strains derived from the initial mutant can be divided intto two classes: the "neutral genomes" which all revert at meiosis with different frequencies but in an autonomous way; and the "aggressive genomes", which inhibit the reversion of neutral genomes, present a large polymorphism in their own reversion and lose their aggressiveness at meiosis, becoming neutral genomes. The characteristics and the relationships of these two strains are presented in this paper.     

Isolation and characterization of respiration-deficient mutants inAspergillus ochraceus

Two respiration-deficient mutants (rd) were isolated from the acetate-nonutilizing mutants (acu) induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) inAspergillus ochraceus. A complementation analysis of the tword mutants indicated that MNNG had caused a mutation at a single locus. The diameter of the tword mutant colonies in glucose medium was found to be small in comparison to that of the wild type and the otheracu mutants; the diameter of the isolated mutant colonies in acetate medium was very small. The grown zone ofrd mutants remained colorless up to 20 h incubation in 2,3,5-triphenyltetrazolium-overlaid solid Czapek-Dox medium and it turned pink after prolonged incubation, whereas the wild type and the otheracu mutants became pink within 30 min in the same medium. Therd mutants were further characterized by measuring the respiratory activities of intact mycelia in the presence of glucose.     

Identification of genes associated with natural competence in Helicobacter pylori by transposon shuttle random mutagenesis.

To identify genes involved in DNA transformation, we generated 1500 insertion mutants of a Helicobacter pylori strain by transposon shuttle mutagenesis. All mutant strains were screened for their frequency of natural transformation. A total of 20 mutant strains were found to exhibit a significantly decreased transformation frequency. DNA sequencing revealed seven genetic loci, including the reported comB locus, HP0017 (a putative virB4 homologue) and five loci without database match (HP0015, HP1089, HP1326, HP1424, and HP1473) from the 20 mutants. Reknockout of HP1326 revealed no impairment in natural transformation, while the other 5 mutants showed the same defective in natural transformation. Mutation of HP0017 severely impaired natural transformation both chromosome and plasmid DNA. Slot blot analysis revealed that some noncompetent strains had decreased virB4 RNA expression levels compared with competent strains. Nineteen ORFs had decreased expression levels in virB4 knockout mutant by microarray. Therefore, our data indicate that HP0017 is a virB4 homologue and is essential in the natural competence of H. pylori. HP0015, HP1089, HP1424, and HP1473 genes could be also involved in natural transformation.     

Specificity in the Photoreactivation of Premutational Damage Induced in Neurospora crassa by Ultraviolet

Summary Photoreactivation of the events which lead to ultraviolet-induced reversion of sixteen mutants has been studied. Reversion of one mutant, the inositol allele 37401, is photoreactivated far less than lethal damage. Reversion of the remaining 15 mutants, alleles at three different loci, is photoreactivated to the same extent as lethal damage. Various explanations of this finding are considered and the implications of repair specificity in modifying mutagen specificity are discussed briefly.