In vivo construction of linear vectors based on killer plasmids from Kluyveromyces lactis: selection of a nuclear gene results in attachment of telomeres.

Linear vectors based on plasmids pGKL1 and pGKL2 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae and selected for integration of the nuclear LEU2 gene. The linear hybrid molecules obtained had no proteins attached to their 5' ends, as is found for native pGKL plasmids. However, telomere-specific sequences were added to the ends of pGKL1. In contrast to the cytoplasmically localized pGKL plasmids, the newly obtained linear hybrid vectors probably replicate within the nucleus and provide evidence that the nuclear LEU2 gene cannot be expressed in the cytoplasm.

Genetic Mechanisms of Rare Matings of the Yeast SACCHAROMYCES CEREVISIAE Heterozygous for Mating Type.

Yeast heterozygous for mating type lacks the ability to conjugate as judged by the mass-mating technique and accordingly is designated "non-mater". However, the non-mater shows rare mating ability with a frequency of less than 10(-6). In the present study, the RD auxotroph mating method was mainly employed with the intention of examining the rare mating ability of various non-maters, using lactate ethanol minimal medium as a selective medium for hybridization. Crosses of aalphaxa, aalphaxa, aaalphaxa, aalphaalphaxa, etc. resulted in the production of respective hybrids with a relatively high frequency of about 10(-6) to 10(-7), whereas crosses of aaalphaxa, aalphaalphaxalpha, aaalphaalphaxa, aaalphaalphaxalpha, etc. resulted in hybrids with an extremely low frequency of about less than 10(-8). Genetic analyses revealed that the rare matings were mostly caused by the presence of cells derived from the non-maters in which mating type had converted to a homozygous genotype. Mitotic recombination was shown to be a likely explanation for most of the conversion, judging from associated exchange of an outside marker, thr4. By successive employment of the RD auxotroph mating method, it was possible to produce a series of polyploid yeasts, triploids to octoploids. The DNA content and the cell volume were observed to increase parallel to the elevated ploidy states.

A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride.

A novel method is described for the extraction of DNAs from fungi and yeasts. Anhydrous hydrogen fluoride (HF) selectively cleaves their cell walls under mild conditions (for 5 min at 0 degrees C), enabling the effective extraction of DNAs from organisms with a cell wall. A possible mechanism for this method concerning the selective cleavage of O-glycosidic linkages in cell walls has been described previously [(1977) Anal. Biochem. 82,289-309]. The extracted DNA is intact: in fact, the yeast DNA is directly applicable for restriction analysis and transformation of Escherichia coli.

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir.

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.

[Serum concentration of 5-FU and tegafur (FT-207) after administration of tegafur (FT-207) suppository with the colostomy].

Tegafur (FT-207) suppositories were administered at a rate of 750 mg via the artificial anus (ST Group) following surgery of rectal cancer. Comparative studies were conducted of changes in blood concentrations of 5-FU and tegafur at the time of initial administration and following one week of continuous use for the low-anterior resection cases (LA Group) and the rectal administration cases (RE Group). FT-207 concentration at initial administration was low in the ST group compared to those for both LA and RE groups which received anal administration of the drug, but only little changes were noted. Blood concentration one hour after administration was 11.1 micrograms/ml, elevated to 14.3 micrograms/ml at two hours, and remained at 10 micrograms/ml and above for six hours following administration. The ST group 5-FU concentrations at two, four and six hours after administration were significantly lower than those in the RE group but the changes were little. Blood concentrations were 0.015 microgram/ml at one hour after administration, 0.017 micrograms/ml at two hours and maintained virtually the same level thereafter. An effective concentration of 0.012 microgram/ml was maintained even at ten hours following administration. After one week of administration of the suppositories, the ST group showed the lowest concentration among three groups, but it was approximately double compared to the initial concentration; FT-207 showed nearly the same concentration in the LA group and 5-FU blood concentration was 0.025 microgram/ml at one hour after administration, reached to a maximum of 0.030 microgram/ml at two hours and maintained 0.020 microgram/ml and higher at ten hours. 5-FU concentration in the LA and RE groups after one week of continuous administration showed a dual-peaked pattern. No patient with an abnormal artificial anus was involved in this study. The artificial anus is thought to be an adequate and effective administration route of FT-207 suppositories.

Yeast DNA plasmids.

The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

Effects of elevation of strain-ploidy on transmission and recombination of mitochondrial drug resistance genes in Saccharomyces cerevisiae.

In order to study the effects of strainploidy on the transmission and recombination of the mitochondrial genes C, E and O conferring the resistance to chloramphenicol, erythromycin and oligomycin, respectively, haploids were crossed to diploids and the results of genetic analysis were compared with those from haploid X haploid crosses. All haploid X haploid crosses showed an increased transmission of diploid derived alleles, relative to haploid derived ones, but the pattern of increase differed between homosexual and heterosexual crosses. In omega-haploid X omega-diploid homosexual crosses, the increase was of roughly equal magnitude at the C, E and O LOCI: there was a polar co-transmission of the diploid derived alleles. In omega plus haploid by omega-diploid heterosexual crosses, on the contrary, a differential increase was observed at the different loci, the magnitude being the smallest at the C locus and the largest at the O locus. As a result, there was a preferential transmission in favor of the haploid derived C alleles and of the diploid derived O alleles. A near equal transmission from both parents was observed for the E alleles. A decrease and an increase in the recombination frequency were noticed in the above haploid by diploid homosexual and heterosexual crosses, respectively. The above phenomena were ascribed to different dosages of mitochrondrial genomes from parents. Experimental data were well accorded with the theoretical expectation which were obtained on the assumptions that diploids contain twice as many mitochondrial genomes as haploids, and that random pairings and recombination would occur among mitochrondrial genomes from parents. The elevation of strain-ploidy did not affect the recombination polarity which is under the control of the omega gene. It was theoretically predicted that a preferential transmission in favor of diploid derived alleles at all the C, E and O loci would be seen in omega-haploid x omega plus diploid heterosexual crosses as well as in omega plus haploid x omega plus diploid homosexual crosses, but that the magnitude of the polar transmission would vary depending upon the loci in the former crosses, while it would be the same at all the loci in the latter ones. The recombination frequency was predicted to decrease in both of these crosses.

Genetic analysis of unequal transmission of the mitochondrial markers in Saccharomyces cerevisiae.

The presence of mitochondrial sex factor, omega, was demonstrated in haploid strains of yeast Saccharomyces cerevisiae which came from our laboratory. Transmission and recombination of the mitochondrial genes (CR/CS, ER/ES and OR/OS), conferring the resistance/sensitivity to chloramphenicol, erythromycin and oligomycin, respectively, were non-polar in homosexual crosses and highly polar in heterosexual crosses. Different results were obtained in crosses involving an erythromycin resistant mutant G706E11 (CSEROS) which was found to contain cellular DNA of diploid level. This strain was omega- and showed no alleles from G706E11 (CS, ER and OS) were transmitted to the zygote progeny in preference to the CR, ES and OR alleles. When crossed to omega+ haploid strains, there was a highly polar recombination, but no transmission was seen for the E and O alleles. Polar transmission of markers from omega+ haploid parental strain, characteristic of heterosexual crosses, was noticed only for the C allele. The crosses of G706E11 to omega+ haploids featured an increase in the recombination frequency. The values of % suppressiveness of sigma- petite mutants were relatively low when determined by crossing to G706E11 or to sigma+ diploid strain M2-8C rather than by crossing to sigma+ haploid strains, indicating that there is a positive correlation between the polar transmission of drug resistance markers and the suppressiveness degrees. Genetic mechanism of the anomalous behaviors if mitochondrial genes in crosses involving G706E11 was discussed and interpreted as due to an unbalanced supply of mitochondrial genomes from parental strains.

Replication and maintenance of the Kluyveromyces linear pGKL plasmids.

Two new linear plasmids, pK192L (4.9 kb) and pK192S (2.4 kb), were isolated from a Kluyveromyces lactis killer strain carrying pGKL1 and pGKL2. pK192L was a deletion plasmid of pGKL1, derived from a part of the ORF1, and had a palindrome structure of a 215 bp unique sequence flanked by 2.35 bp inverted repeats. pK192S was a hairpin plasmid produced by self-annealing of a single-stranded pK192L DNA. In genetic analysis, pK192L and pK192S always coexisted and replicated in cells harboring pGKL2 and pGKL1, in contrast to other pGKL1-derived deletion plasmids, such as F1, F2 and pGKLIS, which could replicate in cells carrying pGKL2 only. Based on these and other lines of evidence, it was concluded that the reason for the pGKL1 dependent replication of the pK192L/S plasmids was the absence of the intact pGKL1-ORF1 gene and that the ORF1 function was necessary for the replication of the pGKL1 genome. This finding is in good agreement with a recent view reporting that ORF1 may encode a DNA polymerase of pGKL1. In a separate experiment, four new linear plasmids were isolated from a Saccharomyces cerevisiae strain carrying pGKL1 and pGKL2. Structural analysis showed that they consisted of two pairs of hairpin-palindrome type plasmids, each derived from different parts of pGKL2, respectively. pGKL1 stability replicated in cells carrying both these pGKL2 derived deletion plasmids.

Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1.

The yeast Kluyveromyces lactis harboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes. Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation. pK192S was always accompanied by pK192L in subclones of KUV192. Both plasmids were derived from pGKL1 by deletion of the large right part of it. pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence. Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L. The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1Sin addition to pGKL2 and completed with pFKL1 or pGKL1S for their maintenance. Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.

Intergeneric transfer of deoxyribonucleic acid killer plasmids, pGKl1 and pGKl2, from Kluyveromyces lactis into Saccharomyces cerevisiae by cell fusion.

Two novel linear deoxyribonucleic acid plasmids, pGKl1 and pGKl2, were isolated from the yeast Kluyveromyces lactis. K. lactis strains harboring the pGK1 plasmids killed a certain group of yeasts, including Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces rouxii, K. lactis, Kluyveromyces thermotolerans, Kluyvermyces vanudenii, Torulopsis glabrata, Candida utilis, and Candida intermedia. In this experiment, the pGKl1 and pGKl2 plasmids were intergenerically transferred from a K. lactis killer strain into a non-killer (killer-sensitive) strain of S. cerevisiae by the use of a protoplast fusion technique. Both of the pGKl plasmids replicated autonomously and stably in the new host cells of S. cerevisiae and could coexist with the resident 2-micrometers deoxyribonucleic acid plasmid. The S. cerevisiae cells which accepted the pGKl plasmids expressed the same killer phenotype as that of the donor K. lactis killer and became resistant to the K. lactis killer. The pGKl plasmids existing in the S. cerevisiae cells were cured by treatment with ethidium bromide, and the killer and resistance characters were simultaneously lost. From there results, it was concluded that both the killer and the resistance genes are located on the pGKl plasmids.

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability.

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.