Vegetative incompatibility in Neurospora: its effect on horizontal transfer of mitochondrial plasmids and senescence in natural populations

We have investigated the horizontal transfer of two mitochondrial plasmids and the Kalilo senescence phenotype in the fungus Neurospora without the use of heterokaryon-forcing markers. The Kalilo senescent state was only transferred between fully-compatible N. crassa strains, but not between strains differing at any of the loci het-c, het-d, het-e or mating-type. However, the linear plasmid kalDNA and the circular plasmid Han-2 were transferred following incompatible vegetative interactions. Our data suggest that vegetative incompatibility due to allelic differences at het-c is more effective in preventing transfer than that due to het-d, het-e or mating-type. Based on these observations we have developed a novel test for assessing vegetative incompatibility between Kalilo and non-Kalilo field isolates of N. intermedia. In this procedure combinations of Kalilo and non-Kalilo field isolates of N. intermedia were grown together and tested for senescence. Compatibility is inferred if the young non-Kalilo strain dies along with the senescent Kalilo strain, whereas incompatibility is inferred when the Kalilo strain dies without imposing its senescent state onto the non-Kalilo strain. Our results suggest that each of the nine Kalilo strains tested is incompatible with each of 20 non-Kalilo isolates from the same N. intermedia population of the Hawaiian island of Kauai. However, the observed incompatibility did not completely prevent cytoplasmic exchange, and in several cases plasmid transfer could be detected.     

The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.     

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.     

Development of breeding stocks of the yeast Saccharomycopsis lipolytica by methods of moderate inbreeding

Sporulation parameters of genetically labelled strains, derived from a wild strain of the alkane-utilizing yeast Saccharomycopsis lipolytica were improved by a breeding program using brother-ssister crosses. Sporulation frequency, the number of four-spored asci and viability of ascospores could be significantly enhanced. To date a number of genetically well-defined strains is available that have good sporulation parameters and show a 1:1 segregation pattern of markers suitable for genetic analysis.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed “naked” nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Suppressor specificity in Aspergillus nidulans

Summary Seven allele specific gene unspecific suppressors mapping at four loci have been described previously (Roberts et al. 1979). Three new suppressors mapping in suaA are characterised, and the spectrum of suppression of all the suppressors with respect to seventeen suppressible mutations in eight different genes is described. Two distinct classes of suppressor are defined. The diversity of suppression of five suaA alleles, and the temperature sensitivity of some suaA suppressor mutant combinations but not others, suggests that suppressors at this locus are acting via ribosomal protein alteration. suaC109, a mutation that results in cold-sensitivity for growth shows a similar broad spectrum of suppression. Suppressors at the suaA and suaC loci suppress mutations that have the properties of chain termination mutations as well as missense mutations. suaB111, and suaD103 and suaD108 have a very restricted range of suppression. These suppressors may be mutations in tRNA genes.     

Genetic analysis of apomictic wine yeasts

The Saccharomyces cerevisiae wine yeast IFI256 was selected because of its high fermentative capacity and tolerance to ethanol. Sporulation of the IFI256 strain produced two-spore asci unable to conjugate, but able to sporulate again and the spores produced two-spore asci in all cases. That process was studied for at least five generations. The electrophoretic karyotype showed a pattern of 21 chromosomal bands, which was identical both in the parental and in all the descendants analyzed, from the first to the fifth generation. The DNA content of the parental and the descendants was of 1.7 n, which indicates that the capacity for sporulation shown by all descendants was due to apomixis rather than homothallism of the strain. Different concentrations of glucose and acetate and the addition of zinc salts to the presporulation and sporulation media increased the frequency of four-spore asci by up to 9%. However, the tetrads formed were in fact two dyads that resulted from induced endomitosis. Crosses of IFI256 with laboratory strains produced hybrids giving four-spore asci after sporulation, thus indicating the mutation to be recessive. Transformation of IFI256 with plasmids carrying either SPO12 or SPO13 functional genes and crosses with strains carrying functional or mutated SPO12 and/or SPO13 genes indicated that IFI256 carries several mutations, one of which was located to the SPO12 gene. Parasexual cycles and chromosome loss induced after crossing IFI256 with cir⁰ strains indicated that apomictic mutations were exclusively located at chromosome VIII. The high frequency of wine strains which are apomictic suggests apomixis to be an advantageous phenotype which allows the formation of stress-resistant asci but prevents the loss of favored chromosomal rearrangements.Communicated by S. Hohmann     

Functional nuclear suppressor of mitochondrialoxi2 mutations in yeast

Summary A semidominant nuclear suppressor, callednam6, ofoxi2-V276 mitochondrial mutation has been isolated and characterized. The nuclear character ofnam6 was proved by its retention inrho° strains, lack of mitotic segregation in diploids and meiotic 2:2 segregation in tetrads. The specificity ofnam6 was tested on 315mit ^– mutations of four mitochondrial genes (oxi1, oxi2, oxi3, andcob-box). It suppresses clearly only three mutations in theoxi2 gene, restoring partially or completely cytochrome aa₃ formation. The results suggest a functional character of the suppression.     

Mutant alleles of tRNAThr genes suppress the hisG46 missense mutation in Salmonella typhimurium

Extragenic suppressors of the hisG46 missense mutation were mapped to the 71 and 88 min regions of the Salmonella typhimurium chromosome, positions that in Escherichia coli contain the thrV (tRNA^Thr1) and thrT (tRNA^Thr3) genes, respectively. The suppressor loci were identified as mutant alleles of thrV and thrT, using allelespecific colony hybridization. An oligomer, based on the conserved 5′ sequence of the thrT and thrV genes in E. coli and designed to contain the putative mutant anticodon, discriminated between suppressor-containing and wild-type strains. Similarly, probes specific for the thrV-[SuGGG] and thrT[SuGGG] were used to differentiate the two suppressors. To date, all extragenic suppressors of hisG46 have been identified as either thrV[SuGGG] or thrT[SuGGG]. A near equal distribution of thrV[SuGGG] and thrT[SuGGG] suppressors was found among 29 spontaneous and 43 mutagen-induced hisG46 extragenic suppressor revertants. It was concluded, therefore, that mutant alleles of thrV and thrT are predominantly, if not solely, responsible for intergenic suppression of the hisG46 mutation. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Mitochondrial and nuclear mitoribosomal suppressors that enable misreading of ochre codons in yeast mitochondria

Summary We describe studies on the action spectra of the mitochondrial suppressor mim3-1 and the three alleles of nuclear suppressor nam3. Their specificity of action was tested on 516 mit ^– mutations located in different mitochondrial genes. The degree of suppression was quantified by the extent of cytochrome oxidase and cytochrome b synthesis. We show that the four suppressors are allele-specific gene-nonspecific informational suppressors. They would act by changing the structure of the small mitoribosomal subunit which would decrease fidelity of translation enabling misreading of some but not all ochre codons. The implications of the results on the role of intron encoded maturases are discussed.     

Crosses between Saccharomyces cerevisiae and Saccharomyces bayanus generate fertile hybrids

Crossings between strains of Saccharomyces cerevisiae and Saccharomyces bayanus were carried out. Genetic, molecular and electrophoretic karyotyping data indicated that interspecific hybrids were obtained. The hybrid cells segregated "grande" and "petite" colonies, and the latter ranged between 20 and 50%; unlike "grande" colonies, "petite" colonies did not sporulate and did not ferment maltose. In the hybrids, the extent of sporulation varied between 10 and 20%; only very rare asci (around 10(-4)) held viable ascospores. Clones from the viable ascospores sporulated and produced asci with viable ascospores able to give mating with spores from both hybrid derivatives and parental species. Fertile asci could derive from allotetraploid cells generated by endomitotic events in allodiploid cells, a mechanism that enables overcoming the species barrier between S. cerevisiae and S. bayanus.     

Ssb1 chaperone is a [PSI +] prion-curing factor

Yeast SUP7 or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI ⁺] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI ⁺]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI ⁺] prion. Received: 9 August 2000 / Accepted: 20 October 2000     

Every ribosomal suppressor mutation in Aspergillus nidulans has a unique and highly pleiotropic phenotype

Summary 18 suppressors of alcR125 have been selected in Aspergillus nidulans. They have been located in genes as follows: 12 in suaA, 1 in suaB and 5 in suaC. Suppressors have been examined to see whether their phenotype is diagnostic for their genotype. Several new traits are described: conidial viability, cycloheximide resistance, fertility, suppression of niaD500, naaD501 and fWA1. These tests, added to those already in use, provide a battery of tests suitable for assigning suppressor mutations to physiological type (tRNA or ribosomal), and in one case to a specific gene since only suaA mutations suppressed fWA1. A very broad range of phenotypes was associated with suppressors such that every mutation had a unique phenotype. This indicates that the ribosomal suppressor mutations are in genes which code directly for ribosomal proteins, rather than genes which code for modifying enzymes.     

Mitochondrial release factor in yeast: interplay of functional domains

The MRF1 gene encodes the only class I release factor found in Saccharomyces cerevisiae mitochondria, mRF1. The previously isolated point mutation mrf1-13 caused respiratory deficiency due to inhibition of mitochondrial translation. In this study, we have isolated second-site suppressors of mrf1-13. Among over 200 respiratory positive suppressor colonies, ten nuclear dominant suppressors had a new mutation in the MRF1 gene. The suppressors in combination with the original mrf1-13 revealed increased levels of mitochondrially synthesized proteins, Cox2 and Atp6. One of the suppressor alleles was cloned on a plasmid and was found to support weaker respiratory competence than in combination with mrf1-13. Finally, the possible effects of the suppressor mutations are discussed based on a structural model of mRF1 protein built for its “open” and “closed” forms using known crystal structures of prokaryotic release factor RF1 as templates. The 3D models suggest that at least some suppressors switch the structure of mRF1 from the “closed” to a permanently “open” form causing stronger binding of the mRF1 protein to the ribosome and increasing the time of ribosome occupation. This explains how the suppressor mutants may facilitate translation termination despite a defect in decoding of the stop signal.     

Meiotic segregation of circular plasmid-minichromosomes from intact chromosomes in Saccharomyces cerevisiae

Summary Distributive disjunction is defined by first meiotic division segregation of either two nonhomologous chromosomes that lack homologous pairing partners, or of two homologous chromosomes that have failed to undergo crossing-over. In the yeast Saccharomyces cerevisiae, plasmid minichromosomes, synthetic linear chromosomes and a fragment of a real chromosome have been observed to segregate from nonhomologous DNA species at the first meiotic divisions. Suggesting that this organism may have a distributive mechanism for chromosome segregation. However, it is not known whether intact chromosomes also participate in a distributive process. To determine whether intact, full length, S. cerevisiae chromosomes could segregate from nonhomologous chromosomal species, the meiotic behavior of an unpaired intact copy of chromosome I has been analyzed with respect to several centromere-containing circular plasmid minichromosomes. Strains monosomic or trisomic for chromosome I were transformed with centromere plasmids containing either homologous or nonhomologous inserts, sporulated, and analyzed genetically both for the presence of plasmid and for the number of copies of chromosome 1. Each plasmid segregated from an intact unpaired copy of chromosome I at the first meiotic division in a significant majority (63–93%) of the asci examined. These results suggest that intact chromosomes from S. cerevisiae are capable of distributive disjunction.     

The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe

Summary Meiotic and mitotic fine-structure maps of two efficient UGA suppressors of Schizosaccharomyces pombe which are known (sup3-e) or inferred (sup9-e) to code for two serine tRNAs carrying the mutant anticodon U*CA (Kohli et al. 1979a, b, Rafalski et al. 1979) are presented. Maps based on spontaneous meiotic, spontaneous mitotic and MMS induced mitotic recombination between the primary site of the anticodon mutation and a number of inactivating second-site mutations are similar. Specific marker effects, which drastically increase the frequency of spontaneous meiotic and mitotic recombination in crosses involving one or the other of four exceptional sites (including the anticodon sites of both sup3-e and sup9-e), disappear when mapping is based on MMS induced mitotic recombination. The meiotic marker effect characterizing the anticodon site of one of the two efficient UGA suppressors (sup3-e) also disappears upon further mutation to an inefficient UAA suppressor allele (sup3-i), as shown by its absence in a fine-structure map based on meiotic recombination between the anticodon mutation of this ochre suppressor allele and a new set of inactivating second-site mutations derived from it.     

Modifiers of ochre suppressors in Saccharomyces cerevisiae that exhibit ochre suppressor-dependent amber suppression

Summary Mutants of Saccharomyces cerevisiae were selected that would interact with ochre (UAA) suppressors so as to allow ochre -suppressor dependant amber (UAG) suppression, but which do not exhibit opal (UGA) suppression. Strains mutant at four distinct loci were isolated, and two of these are recessive mutations while the other two behave as dominants or semidominants. MOS3 has some suppressor activity in the absence of a resident SUP4-o gene and shares other characteristics with previously described omnipotent suppressors. MOS4, mos1 and mos2, on the other hand, exhibit no suppressor activity in the absence of a resident SUP4-o gene but do exhibit suppression of UAG alleles when there is a resident SUP4-o gene. These latter modifier strains do not interact with a SUP4-o gene to suppress UGA alleles. By genetic and physiological criteria the MOS4, mosl, and most mutations appear to be different than previously described allosuppressors or modifiers of suppression.     

Indirect intergenic suppression of a radiosensitive mutant of Sordaria macrospora defective in sister-chromatid cohesiveness

Summary Six ultra violet (UV) mutageneses were performed on the spo76 UV-sensitive mutant of Sordaria macrospora. Spo76 shows an early centromere cleavage associated with an arrest at the first meiotic division and therefore does not form ascospores. Moreover, it exhibits altered pairing structure (synaptonemal complex), revealing a defect in the sister-chromatid cohesiveness. From 37 revertants which partially restored sporulation, 34 extragenic suppressors of spo76 were isolated. All suppressors are altered in chromosomal pairing but, unlike spo76, show a wild type centromere cleavage. The 34 suppressors were assigned to six different genes and mapped. Only one of the suppressor genes is involved in repair functions.     

Podospora anserina mutations inhibiting several developmental alternatives and growth renewal

Summary A mutation (modG) was selected on the basis of the suppression of a defect resulting from a mutation of modD gene. The modG mutation shows the same developmental consequences as modD mutations: the absence of protoperithecia and aerial hyphae, the non-renewal of growth from stationary cells and the non-germination of ascospores. Investigations of heterokaryotic mycelia showed that the action of modD and modG mutations on the formation of protoperithecia and aerial hyphae is autonomous. Furthermore, taking advantage of the thermosensitivity of a modD mutation, it was shown that the mutations have no incidence on cell differentiation prior to the achievement of stationary physiology. These findings lead to suggest the existence, in Podospora anserina, of a stage of cell totipotency, which we call the switch stage, that derives from the stationary state through the action of modD and mode genes. Accordingly, the switch stage would be the intermediate in the process of growth renewal from stationary cells and the basic stage for the selection of the developmental alternatives (aerial hyphae, protoperithecia ...) of Podospora mycelia.     

Allele-specific suppression of temperature-sensitive mutations of theSaccharomyces cerevisiae RAD52 gene

We screened forrad52 suppressors against temperature-sensitive (ts), missense, nonsense, and deletionrad52 mutations. Except for the deletion strain all mutants yielded suppressor candidates, indicating that suppressors completely bypassing the need forRAD52 are rare. Characterization of seven, recessive extragenic suppressors from our screen and two previously identified suppressors revealed that nearly all exhibit allele specificity. The allele specificity is positional in that suppressors that suppress a is mutation in the C-terminal third of the coding region do not suppress three is mutations in the N-terminal third. Conversly, suppressors against one of the three N-terminal mutations suppress more than one of these mutations but not the C-terminal mutation.