Killer toxin production in Pichia acaciae is associated with linear DNA plasmids

Summary We have identified a strain of the yeast Pichia acaciae which produces a killer toxin active against the yeast Debaryomyces tamarii. The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs). P. acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain. A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity. Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5 protected ends. The P. acaciae system differs from that of the well-studied Kluyveromyces lactis killer system both in the range of susceptible strains and in the sizes of the plasmids involved. Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.     

Linear,non-mitochondrial plasmids of Alternaria alternata

Summary Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5 ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.     

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis

Summary Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5 ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.     

Kluyveromyces lactis killer system: ORF1 of pGKL2 has no function in immunity expression and is dispensable for killer plasmid replication and maintenance

Summary To functionally characterize the genes encoded by the larger killer plasmid pGKL2 from Kluyveromyces lactis a previously developed in-vivo recombination system was exploited. An in-vitro modified version of the cytoplasmically expressible LEU2 gene cartridge (LEU2 ^*) flanked by appropriate pGKL2 segments was used to replace the central part of the ORF1 region of pGKL2. Transformation of a Leu^- killer strain resulted in the expected disruption of ORF1 in the resident pGKL2. The Leu⁺ transformants obtained can be assigned to three classes. Class I carries both killer plasmids, pGKL1/2, and the recombinant pGKL2 derivative termed pRKL2. Class II and III additionally harbor palindrome and hairpin-like plasmids, respectively. Upon subculturing of class I transformants under selective pressure, segregation of the native pGKL2 and the recombinant pRKL2 eventually occurs resulting in total loss of pGKL2. No differences concerning killer and immunity phenotype between a pRKL2-harboring strain and the native pGKL2-carrying recipient could be detected. Thus pGKL2 ORF1 is dispensable for both expression of killer/immunity phenotypes and for the replication and maintenance of the K. lactis killer plasmids.     

Cryptic DNA plasmids of the heterothallic yeast Saccharomycopsis crataegensis

Summary Three DNA plasmids, designated pScrl-1, pScrl-2, and pScrl-3 have been found in a strain of the heterothallic yeast Saccharomycopsis crataegensis (NRRL Y-5902). pScrl-l, -2 and -3 are, respectively, 15, 7, and 5 kilobase pairs (kbp) in size. Based on the results of exonuclease digestions, all three plasmids appear to be linear molecules with blocked 5 ends. All three plasmids also have a lower buoyant density than does nuclear DNA of S. crataegensis. The two lower molecular weight plasmids hybridize strongly with one another, but only weakly to the higher molecular weight plasmid. Two of four related S. crataegensis strains surveyed were found to contain two plasmids that are of the same size as the two larger plasmids of Y-5902. Evidence is presented indicating that the plasmids in strain Y-5902 reside in the cytosol since they were found not to be located within the major organelles (mitochondria and nuclei).     

Selection and stability of yeast transformants expressing cDNA of an Ml killer toxin-immunity gene

Summary Transformants of sensitive yeast strains containing an expressed cDNA copy of the yeast killer toxin-immunity gene could be selected for by exposure to added killer toxin. For strain AH-22 the transformation frequency was approximately 10% that obtained by selection for leucine prototrophy. The procedure required time for expression of immunity prior to selection, and a screening step to remove non-transformed survivors. Under conditions where active toxin was produced, transformants containing the toxin-immunity gene were at a selective advantage, and cells losing the plasmid were killed. This resulted in self selection of transformants, and affords a way of maintaining plasmid stability in protrophic strains.     

Structure of a linear plasmid of the yeast Kluyveromyces lactis; Compact organization of the killer genome

Summary Some strains of the yeast Kluyveromyces lactis contain a pair of linear DNA plasmids, k1 and k2, 8.8 and 13.8 kilobase pairs long, respectively. Simultaneous presence of the two plasmids confer a killer phenotype on the cell by producing a toxin which blocks the growth of sensitive yeast species. Previous genetic studies have suggested that the toxin protein is coded by the k1 plasmid. We have now determined the total nucleotide sequence of k1 DNA. The genome is 8,874 base pairs in length. It contains four protein-coding reading frames, three transcribed from one strand and the fourth transcribed from the complementary strand and has terminal inverted repeats of 202 base pairs. Nuclease S1 mapping confirmed this arrangement and showed that these genes are transcribed. The terminal repeats and the four genes form an extremely compact genome, with some overlapping of genes. All four genes use highly biased codons, 86% of them having A or T at the wobble position, reminiscent of yeast mitochondrial genes. Three genes share a very similar 5 leader sequence. The nature of gene products is discussed in the light of what is known of the excreted toxin protein.     

A mutant of Saccharomyces cerevisiae with impaired maintenance of centromeric plasmids

Summary A mutant with unstable maintenance of hybrid plasmids containing either one of the centromeric loci CEN3, CEN6, CEN11 and arsl or the replicator of the 2 plasmid has been obtained. The frequency of loss of hybrid plasmids in the mutant was up to 3 · 10^–1 per one generation versus 10^–2 in the original strain. The unstable maintenance of minichromosomes in the mutant is controlled by a recessive nuclear gene, named SMC for stability of minichromosomes. Loss of some minichromosomes is connected with impairment of their segregation in cell division. In diploids homozygous for smc mitotic chromosomal segregation is not affected but sporulation is impaired. The question of adequacy of usage of minichromosomes for selection of mutants with impaired function of centromeric loci is discussed.     

Killer DNA plasmids of the yeast Kluyveromyces lactis

Summary We have studied the structure of the two linear DNA plasmids, kl and k2, present in killer strains of Kluyveromyces lactis. Two killer strains of different origins, CBS 2359 and IFO 1267 were examined. For both strains, identical restriction maps of kl and k2 DNA were obtained. Several restriction sites previously reported for the kl DNA of the strain IFO 1267 have been confirmed. The molecular weights of these double-stranded DNAs were 8.8 kilobase pairs for kl and 13.4 for k2, as determined by electrophoresis of restriction fragments. The plasmid DNA from a nonkiller mutant, NK2/1, was also examined. In this mutant, the kl DNA was replaced by a smaller DNA (5.9 kilobase pairs), the k2 DNA being normal. Restriction enzyme analysis showed that the new plasmid DNA was also linear. Hybridization experiments demonstrated that it was derived from the kl DNA by deletion of a 2.9 kilobase pair segment from the central part of the kl DNA. The deleted segment carries a gene involved in toxin production, but is not related to immunity since the mutant is resistant to killers. The plasmid DNA of K. lactis showed no detectable sequence homology with the double stranded RNA of the killer system of Saccharomyces cerevisiae. Neither was any homology found with nuclear and mitochondria) DNA.     

Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani

Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1–5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300–1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and-5, indicating that the plasmids do not play any role in pathogenicity.     

Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo

Mitochondrial RNA was isolated from the morel strain Morchella conica 3 harbouring the linear plasmid pMC3-2 and subjected to gel electrophoresis followed by a Northern analysis using cloned fragments of the plasmid pMC3-2 as probes. Hybridization was obtained only with central parts of pMC3-2 and specific bands of mtRNA. The hybridization bands (2.8 kb and 1.0 kb) correspond in size to the length of the two ORFs of pMC3-2 which were deduced from nucleotide-sequence data. Thus, both ORFs, one encoding a DNA polymerase and the other a yet unknown protein, are transcribed in the mitochondria of the plasmid-bearing Morchella conica strain.     

De-novo generation of mitochondrial DNA plasmids following cytoplasmic transmission of a degenerative disease in Ophiostoma novo-ulmi

A mitochondrial DNA plasmid was detected in an isolate of Ophiostoma novo-ulmi with a degenerative disease. The DNA plasmid was shown to be derived from the mitochondrial DNA and to map to a region corresponding to the large ribosomal RNA coding region. The DNA plasmid was not transmitted into sexual (ascospore) progeny, irrespective of whether the diseased isolate acted as the female or male parent. Transmission of the disease to healthy, plasmid-free, recipient isolates by hyphal anastomosis was not accompanied by transfer of mitochondrial DNA or DNA plasmid from the diseased donor isolate, but resulted in de-novo generation of different plasmids, derived from the recipient's mitochondrial DNA.     

Titration of replication activity by increasing ARS dosage in yeast plasmids

The rep1 region of the yeast mitochondrial genome, a putative replication origin, contains a weak autonomously replicating sequence (ARS). Nucleotidesequence and deletion analyses have identified two 11-base pair ARS consensus sequences, numerous near matches to the ARS core, and a region of curvature that may contribute to ARS function. Based on the amplified nature of petite-derivative mitochondrial DNA encompassing this locus, we have constructed plasmids containing an increasing dosage of ARS elements. The rep1 ARS element can have an additive effect on plasmid stability when present either as a tandem dimer or as an unlinked pair. However, the presence of a third ARS copy does not further enhance plasmid stability. These results indicate that measurable dosage effects can be defined only in circumstances where weak ARS elements are employed, and that plasmid maintenance within yeast cells is saturable and varies among the different sequences promoting replication.     

Genetic control of cell type and complex organization of the mating type locus in the yeast Pichia pinus

Summary Genetic mechanisms of switching the mating type locus MAT1 in the homothallic yeast Pichia pinus were studied. By analysis of mutations affecting MAT1 complex structural and functional organization of this locus was shown. The existence of two functional regions in MAT1 is postulated. Region I controls mating ability of haploid cells, determines the neutrality of heterozygous cells and regulates the work of Region II. Region II controls meiosis and/or sporulation in the cultures heterozygous for Region I as well as controls switching MAT1 MAT1 in haploid cells.     

A mitochondrial plasmid specifically associated with male sterility and its relation with other mitochondrial plasmids in Vicia faba L.

Summary Small supercoiled DNA molecules of Vicia faba were cloned and characterized by restriction mapping and molecular hybridization. The 1700 by S plasmid has been found to be specifically associated with the male sterile cytoplasms and exhibited large homologies with the other 1700 by F plasmid as shown by molecular cross hybridization. In one line bearing the male sterile 350 cytoplasm, a deleted mitochondrial plasmid of 1540 bp was observed and this plasmid derives from the 1700 bp S plasmid. Our results indicate therefore that three of the four plasmids analysed are closely related between each other.     

Effect of chlorpromazine on changes in cortical electrical activity caused byClostridium perfringens type a toxin

Experiments on cats showed that injection of chlorpromazine (3 mg/kg, intramuscularly) 1 h before injection ofClostridium perfringens type A toxin prevents the desynchronization of cortical electrical activity which usually arises in the first phase of poisoning, delays the phase of depression of electrical activity in the second phase, and increases by 50–100% the duration of survival of the animals. The effect of chlorpromazine is evidently connected with blocking of adrenergic structures of the reticular formation of the brain stem.Department of Microbiology and Department of Pathological Physiology, N. I. Pirogov Odessa Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 10, pp. 412–416, October, 1977.     

Hybridization of Pichia stipitis with its presumptive imperfect partner Candida shehatae

Summary A triauxotrophic strain of the D-xylose fermenting yeast Pichia stipitis, was hybridized with diauxotrophic strains of its presumptive imperfect partner Candida shehatae through polyethylene glycol (PEG) induced protoplast fusion. A small fraction of prototrophic clones, selected on specific media, appeared to be partial hybrids as determined by cellular diphenylamine DNA quantitations after three passages on a complex medium. The hybrid nature of the Pichia-resembling fusants was confirmed by cell volume estimation, analysis of nuclear condition and the isolation of a variety of mutant recombinant phenotypic segregants by meiotic segregation, as well as induced and spontaneous mitotic segregation.