Suppression of methionine mutants in Coprinus

Summary The met-1-1 mutation in Coprinus lagopus is known to be suppressed by five recessive non-allelic suppressor genes (sup-1 to sup-5). Two of these genes complement normally in heterozygotes but the other three fail to complement each other in any combination. Four of the suppressor genes, sup-1, sup-2, sup-3 and sup-5, were tested for ability to suppress met-1-2 a second met-1 mutation. Non-identity of the two met-1 alleles was first confirmed by demonstrating intragenic recombination. The complementing suppressors, sup-1 and sup-2, proved to be allele unspecific and suppressed both met-1 mutations. The non-complementing suppressors, sup-3 and sup-5, were allele specific and could only suppress the met-1-1 mutation. This is interpreted to mean that sup-1 and sup-2 act indirectly to circumvent the metabolic lesion caused by any met-1 mutation whereas sup-3 and sup-5 are missense informational suppressors involving modified tRNA species which specifically mistranslate the met-1-1 mutant codon.     

Chromosome dynamics in rad12 mutants of Coprinus cinereus

We have characterized the phenotypes of three rad12 mutants of the basidiomycete Coprinus cinereus, which were isolated on the basis of sensitivity to ionizing radiation. Electron microscopic studies of meiotic nuclear spreads showed that all three rad12 mutants are defective in chromosomal synapsis. For rad12-1 and rad12-4, very limited assembly of the synaptonemal complex occurs. The phenotype of rad12-15 is less severe and longer stretches of synapsed chromosomes are formed. However, for all three alleles mutant nuclei arrest in a diffuse state with little synaptonemal complex structure. Observations made of spreads of acridine orange-stained meiotic nuclei correlated with the electron microscopic data. In rad12 strains, chromosomes condense but do not pair, and they later arrest in a decondensed state; very few rad12 cells enter metaphase I. Homozygous dikaryons of rad12 mutants produce fruiting bodies with significantly fewer basidiospores than are found in wild-type dikaryons. The viability of these spores is greatly reduced: all spores produced by rad12-1 and rad12-4 mushrooms fail to germinate, while only 16% of rad12-15 spores are viable. Recombination within the tract of the ribosomal RNA gene repeats was not significantly different in the mutants when compared with a wild-type congenic control. Quantitative measurements of oidial survival indicate that all three rad12 alleles are sensitive to gamma radiation but insensitive to UV radiation relative to wild-type strains.     

White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus

Among many white-cap mutants of Coprinus cinereus, four distinct classes have been identified cytologically. Mutants of one class progress through meiosis normally but fail to sporulate; the defect is post-meiotic and it triggers apoptosis in the tetrad stage. Mutants of the other three classes have defects in meiotic prophase and these are: (1) those that assemble synaptonemal complexes (SCs) normally; (2) those that assemble axial elements (AEs) but not SCs; and (3) those that assemble neither AEs nor SCs even though the chromosomes are condensed and also paired. All three meiotic mutant classes arrest at meiotic metaphase I and the arrest triggers meiosis-specific apoptosis showing characteristic chromatin condensation, DNA fragmentation as shown by the TUNEL assay, cytoplasmic shrinkage, and finally total DNA degradation. Apoptosis is very cell-type specific; it occurs only in the basidia while the neighboring somatic cells are perfectly healthy and the mushroom continues to develop and mature with very few basidiospores produced. The meiotic apoptosis in C. cinereus is under strict cell cycle control rather than at any time after defect; apoptosis is triggered only after entry to meiotic metaphase. It is intriguing to note that C. cinereus has two checkpoints for arrest and entry to apoptosis: one is meiotic at the metaphase I spindle checkpoint regardless of the time of defects, and one is post-meiotic at the tetrad stage. This is in striking contrast to multiple checkpoint arrests and entries to meiotic apoptosis found in the mouse.     

Kinetic stability of designed glycosylation mutants of Coprinus cinereus peroxidase

The effect of glycans and surface mutations on protein unfolding induced by heat or urea has been studied. Removal of the only native high mannose type glycan in the N142P, N142T, and N142D CIP mutants reduced the lifetime to half of that of wtCIP at irreversible conditions of unfolding. The effect was moderate at reversible conditions. Five glycomutants designed to have 0, 1, 2, 4 and 6N glycans showed a correlation between increased carbohydrate mass and increased stability toward irreversible unfolding. The results are in agreement with a dampening effect of glycans on backbone fluctuation in both the native and the unfolded states. However, experiments in reversible conditions were less clear because of additional effects of an increasing number of amino acid substitutions and aggregation. Examples of strong effects from minor surface changes were also observed.     

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

In order to improve the methionine yield of the isolate B. heali, attempts were made to isolate mutants resistant to the methionine analogue DL-ethionine after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The minimum inhibitory concentration (MIC) of ethionine for B. heali was found to be 2 mM. After mutagenesis and screening, five mutants resistant to 50 mM of ethionine were isolated. The yield of the best ethionine resistant mutant, B. heali Br EthR, was 13 mg/l of methionine medium under optimum cultivation conditions.     

Different types of temperature-sensitive mutants ofEscherichia coli requiring methionine

Путем обратных мутаций абсолютно зависимого метионинового мутанта Escherichia coli были получены З различных типа температурных мутантов. Температурные максимумы роста двух из них, по-видимому, очень близки друт к другу, но оба типа отличаются друт от друта но температурным максимумам эндотенното синтеза метионина, по зависимости от метионина в момент температурното максимума роста и по частоте случаев обратных мутаций. Третий тип отличается от двух других по температурному максимуму роста. Температурный максимум роста температурных мутантов не тождествен с температурным максимумом эндогенното синтеза метионина.     

Regulation of the methionine feedback-sensitive enzyme in mutants of Salmonella typhimurium

Assay of the first enzyme unique to methionine biosynthesis, homoserine-O-transsuccinylase, in metJ and metK regulatory mutants of Salmonella typhimurium showed that synthesis of the enzyme was derepressed seven- and fourfold, respectively. The possibility of noncoordinate regulation of the methionine enzymes is discussed. In metA feedback-resistant mutants, the enzyme activity can be inhibited in vitro by 10 mmS-adenosylmethionine but not by 10 mm l-methionine; hence, the synergistic inhibition found for the wild-type enzyme is not effective in these latter mutants.     

S-adenosyl methionine requiring mutants in Saccharomyces cerevisiae: evidences for the existence of two methionine adenosyl transferases.

Summary Mutants requiring S-adenosyl methionine (SAM) for growth have been selected in Saccharomyces cerevisiae. Two classes of mutants have been found. One class corresponds to the simultaneous occurrence of mutations at two unlinked loci SAM1 and SAM2 and presents a strict SAM requirement for growth on any medium. The second class corresponds to special single mutations in the gene SAM2 which lead to a residual growth on minimal medium but to normal growth on SAM supplemented medium or on a complex medium like YPGA not containing any SAM. These genetic data can be taken as an indication that Saccharomyces cerevisiae possesses two isoenzymatic methionine adenosyl transferases (MAT). In addition, SAM1 and SAM2 loci have been identified respectively with the ETH-10 and ETH2 loci previously described.Biochemical evidences corroborate the genetic results. Two MAT activities can be dissociated in a wild type extract (MATI and MATII) by DEAE cellulose chromatography. Mutations at the SAM1 locus lead to the absence or to the modification of MATII whereas mutations at the SAM2 locus lead to the absence or to the modification of MATI. Moreover, some of our results seem to show that MATI and MATII are associated in vivo.     

Ultraviolet light sensitive mutants of Coprinus lagopus. I. Isolation and characterization

4UV-sensitive mutants have been isolated from the wild type strain BC9/66 of Coprinus lagopus by following a new replica plating technique. Complementation and recombination studies between these mutants suggest 3 gene loci uvs1, uvs2 and uvs3, two of the mutants being allelic (uvs3-1 and uvs3-2). The mutants uvs2, uvs3-1 and uvs3-2 show photoreactivation whereas the mutant uvs1 appears to be deficient in this respect. None of the mutants of the wild type showed significant recovery after dark holding.     

Further genetic characteristics of methionine mutants and their suppressors in Aspergillus nidulans

Summary At least eight methF55 suppressor loci have been identified (Tables 1-4). All suppressors tested were effective towards methA, methB and methG mutants, while some of them were effective also towards methD10, methH2 and probably methE53 mutants (Tables 5 and 6).In the progenies of non-leaky methionine mutants leaky segregants were found (Table 7).Double mutant strains carrying methE31 and methA34 or methF55 or methD10 mutants reverted with approximately the same frequencies as did strains carrying a single methE31 mutant (Table 8).The possible implications of these findings, together with the other data on methionine mutants and their suppressors in A. nidulans, are discussed.     

Intraspecific heterokaryon and fruit body formation in Coprinus macrorhizus by protoplast fusion of auxotrophic mutants

Summary Fusion of protoplasts of Coprinus macrorhizus mutants with different amino acid requirements resulted in the production of prototrophic clones at frequencies of 1–4% of the protoplasts surviving the fusion treatment. The frequencies were at least 200 times higher than those of the appearance of revertants. Few prototrophic colonies appeared also when the mutant protoplasts were individually subjected to fusion treatment, or when they were mixedly cultured without fusion treatment. It was thus concluded that intraspecific heterokaryons were formed by protoplast fusion.The auxotrophic mutants did not form fruit bodies when cultured singly or mixedly with each other. In contrast, the heterokaryons produced by protoplast fusion between the mutants of compatible mating types developed into fruit bodies with intermediate morphology of those of the strains from which the mutants were derived. Heterokaryons were also formed by fusion of mutant protoplasts with identical mating genotype, but they failed to form fruit bodies.Abbreviations PEG polyethyleneglycol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid