Specificity and transformations of the trisporic acid series of fungal sex hormones

Chromatographic and spectroscopic characterizations and comparative bioassay data are given for trisporic acid A, the separate 9-cis- and 9-trans-isomers of trisporic acids B and C, and trisporol C, all obtained from 'mated' (plus and minus) cultures of Blakeslea trispora. All five acids show comparable levels of hormone activity on both the mating types of Mucor mucedo, whereas natural trisporol C more specifically affects a plus strain and the laboratory-derived methyl esters are minus-specific. Similarly plus and minus strains of B. trispora convert trisporol C and the esters into trisporic acids at different rates, and they effect different transformations of administered methyl ¹⁴C-trisporate C.     

The induction of protein X in DNA repair and cell division mutants of Escherichia coli

Certain temperature-sensitive Escherichia coli cell division mutants and DNA repair mutants were treated in several ways to alter DNA synthesis or cell division. The bacteria were pulsed with [³⁵S]methionine; then membrane proteins were prepared and examined using sodium dodecyl sulfate/polyacrylamide slab gels. Autoradiography was performed on the slab gels so that the rate of synthesis of protein X could be determined by microdensitometry.Several changes in the rate of synthesis of the 40,000 molecular weight protein X were found in the different mutants. The wild-type (rec⁺ and lex⁺) strains synthesized protein X in response to DNA synthesis inhibition. However, neither recA^? strains nor lex^? strains synthesized protein X.Both the filament forming, temperature-sensitive mutants tif^? and tsl^? (which was derived from lex^?) synthesized protein X when DNA synthesis was inhibited, but at rates different from the wild-type strains. Moreover, these strains also produced protein X at their non-permissive temperature, even though DNA synthesis was not inhibited. In the tif^? mutant, the rate of synthesis of protein X was influenced by the addition of nucleic acid precursors.A double mutant tsl^?recA^? produced protein X when DNA synthesis was inhibited, or at the non-permissive temperature (although DNA synthesis was normal). This was the only strain carrying a recA^? mutation capable of synthesizing protein X.From these results it is suggested that the genes lex, recA and tif comprise a system that controls DNA repair and limits DNA degradation by the recBC nuclease. The inducer of this control system might be a DNA degradation product.     

Sex specificity of hormone synthesis in Mucor mucedo

Sex specificity is observed in the mating types of the fungus Mucor mucedo with respect to the production of 4-hydroxy methyltrisporates (plus mating type) and trisporins (minus mating type), and in the conversion of these metabolites to trisporic acids by the mating partner. These compounds induce zygophores on the opposite mating type only.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

Cultures of separated mating types of Blakeslea trispora make D and E forms of trisporic acids

Trisporic acids are end products of the sex-specific pheromones in mucoraceous fungi. We have found three new trisporic acids in cultures of Blakeslea trispora in which (+) and (-) mating types were separated by a membrane with 0.45-microns pores. Two of the trisporic acids were new compounds; the structure of the third [previously described by Miller and Sutter [(1984) J. Biol. Chem. 259, 6420] as methyl trisporate-E with a hydroxyl group at C-2] was revised. Trisporic acid-E(3R), trisporic acid-E(3S), and trisporic acid-D(2S) were in a 1:1:2 ratio, accounted for 9% of the total trisporic acids, and differed by the position and configuration of a hydroxyl group on the ring at C-2 or C-3, the conformation of the ring, the extent of rotation of the side chain relative to the ring, and either a carbonyl or hydroxyl group on the side chain at C-13. These three compounds accounted for only 0.5% of the total trisporic acids in combined mating type cultures. Since the combined cultures did not metabolize trisporic acid-E(3R), its biosynthesis apparently ceases when opposing mating types contact each other physically. We speculate that B. trispora and Phycomyces blakesleeanus utilize different pheromones to regulate an early event (possibly zygotropism) in sexual development.     

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

Breeding and characterization of amino acid-analogue-resistant mutants of Arthrospira platensis

To obtain amino acid-analogue-resistant mutants the wild strain A9 of Arthrospira platensis was mutated by ethylmethane sulfonate (EMS). Mutagenic effects of strain A9 by EMS were studied. The experimental results indicated that the survival rate curve of strain A9 took a typical “exponential shape” with lethal dosage of EMS being 1 %. The survival of A9 strain was 13.2 % when treated with 0.4 % of EMS, and the resistant mutation rates to two amino acid analogues, ρ-fluorophenylalanine (FPA) and L-canavanine sulphate (CS), were greatly increased with the highest rates being at 4.9 × 10^?4 and 3.24 × 10^?4, respectively. By repeated screening, two stable mutants resistant to amino acid analogues, A9f resistant to FPA and A9c resistant to CS, were obtained. Resistances of the two mutants to corresponding amino acid-analogues were both significantly increased. Compared with their parent strain A9, A9f appeared larger than A9 performance in filament diameter, spiral diameter, spiral pitch, filament length and spiral number, and A9c showed much longer length and spiral pitch than those of the initial strain. Analysis results on amino acids compositions and contents showed that both two mutants accumulated quite higher concentration of amino acids in cells. The two mutants might be excellent high amino acids producing strain. By this means two useful mutants with stable genetic makers for further genetic study of A. platensis were obtained, which laid a good foundation for further study on the transformation of A. platensis.     

Structural Genes of ATP-dependent Deoxyribonuclease of <Emphasis Type="Italic">Escherichia coli</Emphasis>

RECOMBINATION-deficient (Rec^?) mutants of E. coli express pleiotropic alterations of various phenotypes such as increased ultraviolet light sensitivity, altered patterns of DNA degradation after irradiation, inability to support growth of certain λ phage mutants and many others in addition to reduced recipient ability in mating with Hfr bacteria¹. Yet the primary function of any one of the genes responsible for these alterations has not been elucidated. In this paper, the characteristics of recB and recC mutants having temperature-sensitive functions are described. Particular attention is paid to the properties of the ATP-dependent deoxyribonuclease which is known to be missing in recB and recC mutants^2–5.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Relationship Between Sexuality and Carotene Synthesis in Blakeslea trispora

When stimulated by equivalent amounts of progametangia-inducing hormones, cultures of the minus mating type of Blakeslea trispora produce about the same quantities of carotenoids as mated cultures of the fungus, which suggests that the stimulation of carotene synthesis during the sexual activity of mated cultures is the result of hormonal action. These hormones were isolated and purified. From spectroscopic analysis of purified samples, it appears that the hormones are identical with trisporic acids B and C. When both mating types of B. trispora were cultivated in one vessel but were kept apart by membrane filters, the formation of sex hormones was not inhibited. Physical contact between the mating types is obviously not required for the induction of sexual activity. The sex hormones also formed in combined cultures of B. trispora-plus and Zygorhynchus moelleri (a homothallic species), but not in combined cultures of B. trispora-minus and Z. moelleri. This is evidence for the hypothesis that the hormones are produced by B. trispora-plus only.     

The correlation between the synthesis of β-carotene and zygote formation by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> heterothallic strains

It was demonstrated for the first time that the level of carotenogenesis by the heterothallic Blakeslea trispora strains intensively forming zygospores decreased under conditions of a surface cocultivation during their sexual interaction as compared with the strains grown separately. On the contrary, carotenogenesis was stimulated during a sexual interaction of the strains incapable of forming zygotes. In a submerged culture, the zygote-forming pairs of strains synthesized a considerably larger amount of trisporic acids but a smaller amount of carotenoids than the strains not forming zygospores. The discovered inverse dependence between zygote formation and carotenogenesis allowed us to suggest the inability to form zygotes as a criterion for selecting carotenogenic strain pairs.     

Trisporic Acid Biosynthesis in Separate Plus and Minus Cultures of Blakeslea trispora: Identification by Mucor Assay of Two Mating-Type-Specific Components

Separate plus and minus cultures of Blakeslea trispora synthesize small amounts of trisporic acids under specific conditions. These amounts are expressed as a percentage of the trisporic acids (50 mg/liter of medium) synthesized by mixed plus-minus cultures in 5 days. Plus cultures, without additives from minus cultures, synthesize 0.1% trisporic acids. Plus cultures synthesize 0.4% trisporic acids when stimulated by M-factor, a mating-type-specific component synthesized by minus cultures. Minus cultures, without additives from plus cultures, do not synthesize even 0.0001% trisporic acids. Minus cultures synthesize 1% trisporic acids when stimulated by P-factor, a mating-type-specific component synthesized by plus cultures. Minus cultures synthesize M-factor when stimulated by pi, a component synthesized by plus cultures. We speculate that (i) minus cultures synthesize a component, mu, which stimulates P-factor synthesis in plus cultures, and (ii) both M-factor and P-factor are precursors of trisporic acids.     

Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants.

Toxoplasma gondii, strain RH, produced plaques in human fibroblast tissue cultures over the temperatures 30–41 C. Muta?enesis with N-methyl-N′-nitro-N-nitrosoguanidine yielded seven temperature-sensitive mutants that had lost the ability to form plaques at 40 C but still grew well at 33 C. No spontaneous mutants were detected. The temperature-sensitive mutants were not markedly thermolabile and adsorbed normally to tissue culture cells at 40 C. Three mutants differed from one another in their temperatures for optimal growth, and in their ability to remain infectious within cells incubated at 40 C. Both mutants that were tested were found to be markedly less virulent for mice than was the wild type RH strain.     

Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales

A study was made of some late reactions in the trisporic acid biosynthetic pathway in Mucor mucedo. Trisporic acids induce sexual reproduction in several Mucorales.Two enzymes involved in these reactions, a NADP-dependent dehydrogenase and an esterase, appeared to be highly specific for the minus mating type.The synthesis of these enzymes is stimulated by trisporic acids, indicating a positive control of these hormones upon their own synthesis.The dehydrogenase was histochemically shown to be concentrated in the zygophores of Mucor mucedominus. In the homothallic Zygorhynchus moelleri the copulating main branch (which is known to have a minus character) appeared to be the major site of dehydrogenase activity.     

Analysis of rpsD mutations in Escherichia coli. I. Comparison of mutants with various alterations in ribosomal protein S4.

Summary Streptomycin-independent revertants were selected from streptomycin-dependent mutants. Twenty-five out of 150 such revertants were temperature sensitive. Ribosomal proteins from 18 temperature-sensitive and 10 temperature-insensitive revertants were analysed by SDS-polyacrylamide gel electrophoresis. Seventeen of the former but none of the latter category showed an alteration of protein S4. The mutated rpsD allele of 6 temperature-sensitive revertants was transduced into a rpsL ⁺ strain. In all cases an increased suppressibility of T4 amber phages was observed. Such suppressibility was not observed in the original rpsD, rpsL strains. All 18 temperature-sensitive mutants were disturbed in the processing of 17s to 16s RNA at non-permissive temperature and the accumulated 17s RNA was degraded. Temperature-insensitive rpsD revertants could be isolated, which had gained a second alteration in S4. Such revertants, which had lost the temperature-sensitive property, were also unable to suppress growth of T4 amber phages.It is concluded that temperature-sensitive growth, inability to process 17s RNA and to assemble 30S ribosomes at non-permissive temperature as well as increased translational ambiguity are highly correlated properties in rpsD mutants.     

Effect of the recC gene in Escherichia coli on frequencies of ultraviolet-induced mutants

UV induction of Lac^? mutations was compared with UV induction of Mal⁺ mutations in E. coli B/r strains differing in the recC gene. The frequency of Lac^? mutants per survivor induced by the same dose was not significantly affected by the recC gene but the percentage of pure rather than sectored Lac^? colonies was greater when the recC gene was present. On the other hand, as reported previously, frequencies of Mal⁺ mutants induced by the same UV dose were lower when the strain was recC. The reduction factor was the same as for spontaneous Mal⁺ mutants. The difference in the effect of the recC gene on the yields of Lac^? and Mal⁺ mutants can be explained by taking into account the influence of lethal sectoring, which introduces an artifact when mutants arising in the recC strain are scored selectively as in the case for Mal⁺ mutants, but not when the scoring is non-selective as for Lac^? mutants. Lethal sectoring as indicated by a discrepancy between total cell counts and numbers of colony-formers, was observed for the recC strain growing in liquid minimal medium corresponding to the agar medium used to score Mal⁺ mutants but was not observed for the rec⁺ strain. Both strains showed lethal sectoring in the liquid medium corresponding to the agar medium to score Lac^? mutants. The hypothesis concerning the role of lethal sectoring in the selective scoring of mutants arising in a recC background is supported by evidence concerning the UV induction of mutants in a polA1 background. Like the recC gene, the polA1 gene did not affect yields of Lac^? mutants. However, unlike the recC gene, the polA1 gene has previously been shown not to influence UV yields of prototrophic mutations (scored selectively) and not to cause lethal sectoring except under irrelevant conditions.     

Blakeslea trispora mutants with a disrupted sexual process

Three groups of Blakeslea trispora (+) and (-) mutants were obtained and their phenotypical characteristics were studied as well as biochemical changes in the course of mating and the ability to synthesize carotenoids when the sexual process of these mutants was disordered. The first group of mutants synthesized carotenoids at the control level, the second group produced them below the control level, and the third group accumulated less than 1% of carotenoids as compared to the control. The difference in the yields of carotenoids among the three groups of mutants in the mated culture is attributed to the presence (or absence) of the ability to synthesize trisporic acids in them.     

Deletion mutants of nitrogen fixation in Klebsiella pneumoniae: Mapping of a cluster of nif genes essential for nitrogenase activity

Deletions of the nitrogen fixation (nif) region of the Klebsiella genome were isolated by selecting for resistance to virulent phages whose resistance loci are adjacent to nif. The extent of the various deletions was monitored by assaying several different enzymes or gene products coded for by this segment of DNA. Three classes of deletion mutants were detected: (1) gluconate-6-phosphate dehydrogenase minus (gnd^?), histidine minus but histidinol dehydrogenase plus (his^?, his D⁺), nitrogenase plus (nif⁺), shikimate utilization plus (shu⁺); (2) gnd^?, his D^?, nif^?, shu⁺; (3) gnd^?, his D^?, nif^?, shu^?. From these studies we conclude that the cluster of nif genes essential for nitrogenase activity is located on the genetic linkage map of Klebsiella between his and shu; the gene order in this region in thus phage-resistance locus (rfb?), gnd, his operon, nif, shu. Genetic analysis substantiates the finding that the nif cluster is located proximally to the operator end of the his operon.     

Identification of a new mating group and reproductive isolation in the <Emphasis Type="Italic">Closterium peracerosum–strigosum–littorale</Emphasis> complex

Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to compare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracerosum–strigosum–littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt^?) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt^? cells of mating group G was induced in the presence of mt⁺ cells of mating group I-E. In fact, the sex pheromone, protoplast-release-inducing protein of mating group I-E induced the release of protoplasts from mt^? cells of mating group G. When mt⁺ and mt^? cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E.