PubMed Health⌕ Search

Biomedical subjects

N Gunge

Publications and source records attributed to N Gunge.

At least 19 recordsLinked to original sources
PubMed1994

ATP1 and ATP2, the F1F0-ATPase alpha and beta subunit genes of Saccharomyces cerevisiae, are respectively located on chromosomes II and X.

Southern blot analysis showed that ATP1 and ATP2 map on chromosomes II and X, respectively. Physical mapping of ATP1 and ATP2 by chromosome fragmentation showed that ATP1 is at the left end of chromosome II and ATP2 is at the right end of chromosome X. Both are located close to telomere sequences of each chromosome; ATP1 and ATP2 being approximately 30 kb and 85 kb from the respective telomeres.

Chromosome Mapping↗
PubMed1994

UV hypersensitivity of yeast linear plasmids.

The Kluyveromyces linear plasmids pGKL1 and pGKL2, encoding killer activity, were efficiently cured by UV irradiation. This event was investigated in more detail by the use of the terminal protein (TP)-associated cytoplasmic linear plasmids, pJKL1 and pRKL2, with a selectable marker LEU2. This observation was compared with the UV effect on the nuclear plasmids pLS1 (telomere-associated linear form) and YCp121 (centromere-integrated circular form), indicating that the UV hypersensitivity was specific to the cytoplasmic plasmids. Using rad4 and wild-type strains of S. cerevisiae, both pJKL1 and the nuclear plasmids were found to respond not only to photoreactivation repair but also to excision repair of UV-induced DNA damage. Thus these DNA repair systems were functional for both the nuclear and cytoplasmic plasmids in yeast, and it was suggested that the UV hypersensitivity of cytoplasmic plasmids might have been caused by a defect in other repair systems or in the TP-primed replication. Possibly TP-associated Debaryomyces linear plasmids were also UV hypersensitive.

DNA Damage↗
PubMed1992

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

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.

Immunity↗
PubMed1991

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

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.

Base Sequence↗
PubMed1989

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.

Base Sequence↗
PubMed1988

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.

Base Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1987

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.

DNA Restriction Enzymes↗
PubMed1987

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomous replication of hybrid plasmids in Saccharomyces cerevisiae.

By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.

Base Sequence↗
PubMed1987

[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.

Adult↗
PubMed1985

Hairpin plasmid--a novel linear DNA of perfect hairpin structure.

The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double-stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non-killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5-kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5' terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.

Base Sequence↗
PubMed1985

Linear plasmids with terminal inverted repeats obtained from Streptomyces rochei and Kluyveromyces lactis.

Linear plasmids with inverted terminal repeats of 614 bp were obtained from Streptomyces rochei which produced lankacidin. The 5' ends were blocked by the association of a terminal protein. A DNA model of racket frame-like structure is presented implying the juxtaposition of 2 double-stranded DNAs of the same sequence through the binding of cohesive proteins which recognize and bind to the DNA. Two linear plasmids with the inverted terminal repeats of 202 and 184 bp were obtained from a yeast, Kluyveromyces lactis. A killer toxin was produced from the shorter plasmid. The protein toxin inhibited the adenylatecyclase activity of the yeast membrane.

Anti-Bacterial Agents↗
PubMed1985

Transfer of DNA killer plasmids from Kluyveromyces lactis to Kluyveromyces fragilis and Candida pseudotropicalis.

Killer plasmids pGKL1 and pGKL2 of double-stranded linear DNAs were transferred from Kluyveromyces lactis to strains of Kluyveromyces fragilis and Candida pseudotropicalis. The resultant killer strains produced 17-fold and 6-fold larger amounts of killer toxin than K. lactis did, respectively. The killer toxin produced by each species appeared to be a glycoprotein.

Bacterial Toxins↗
PubMed1984

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.

Base Sequence↗
PubMed1984

Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis.

A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK l-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320 000-fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below -20 degrees C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl alpha-D-mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2-35-fold by the presence of 4-60% glycerol.

Ascomycota↗
PubMed1984

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.

Ascomycota↗