• Journal of Microbiology
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• v.35 no.4
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• pp.264-270
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• 1997

The structure of plasmid mlp1, a linear 10.2kb mitochondrial plasmid of Pleurotus ostreatus NFF A2 was determined by restriction enzyme mapping and partial sequencing. The plasmid encodes at least two proteins; a putative RNA polymerase showing homology to yeast mitochondrial RNA polymerase and to viral-encoded RNA polymerases, and a putative DNA polymerase showing significant homology to the family B thpe DNA polymerases. It also contains terminal inverted repeat sequences at both ends which are longer than 274 bp. A 1.6 kb EcoRI restriction fragment of m1p1 containing the putative RNA polymerase gene did not hybridize to the nuclear or motochondrial genomes from P. ostreatus, suggesting that it may encode plasmidspecific RNA polymerase. The gene fragment also did not hybridize with the RNA polymerase gene (RPO41) from Saccaromyces cerevisiae. The relationship between genes in m1p1 and those in another linear plasmid pC1K1 of Claviceps purpurea was examined by DNA hybridization. The result indicates that the genes for DNA and RNA polymerases are not closely related with those in C. purpurea.

• Journal of Microbiology
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• v.37 no.4
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• pp.229-233
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• 1999

The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.37-41
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• 2001

Two linear plasmid-like DNAs, 10.2 kb and 7.2 kb were found in the mitochondria of P. ostreatus. They have covalently linked 5'-terminal proteins in both ends. Two continuous fragments of 4.7 kb and 2.3 kb from 7.2 kb DNA were cloned and sequenced. Two long open reading frames (ORF1; 2982 bp, 993 a.a and ORF2; 2703 bp, 900 a.a) and one short open reading frame(ORF3; 771 bp, 256 a.a) were found in the 7.2 kb plasmid. The putative ORF1 and ORF2 have conserved motifs of DNA polymerases and RNA polymerases, respectively, while the ORF3 has homologous regions with phosphatase from Plasmodium, and also with adhesine from Mycoplasma.

• Korean Journal of Microbiology
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• v.31 no.2
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• pp.141-147
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• 1993

Plasmid DNAs were detected from the mitochondrial fraction of four strains of whiterot fungus, Pleurotus ostreatus. The size of the plasmids were 10.2 and 7.2 kb in strain NFFA 2, 10.2 kb in NFFA 4001, 11.2 kb in NFFA 4501, and 10.2 and 11.2 kb in KFCC 11635. The two strains,NFFA 2ml and NFFA 2m2, which are mutant derivatives of NFFA 2, did not contain any plasmids. The cleavage by proteinase K indicated that these plasmids have DNA ends associated with proteins. In digestion with proteinase K all the plasm ids remained resistant to lambda exonuclease which hydrolyzes DNA from 5' ends and were sensitive to exonuclease III which hydrolyzes DNA from 3' ends. This suggests that the plasmids are linear double-stranded DNA and the terminal proteins are covalently linked to 5' ends of plasm ids. In order to find relationship between these plasmids, hybridization of plasm ids by each separate plasmid DNA was done. The result indicated that the plasmids can be classified into at least 3 groups. Plasmids of group I were present in all the P ostreatus. More mitochondrial plasmids were detected in P cornucopiae. P ,florida, P pulmonarius, P sajor-caju, and P spodoleucus. The size of plasmids ranged between 7.2 kb and 14 kb. All the species except P cornucopiae contained plasmids of approximately 10 kb which hybridized with the 10.2 kb plasmid (group I) of P ostreatus NFFA 2.

• Journal of Microbiology
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• v.35 no.3
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• pp.228-233
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• 1997

The MTF1 gene of Saccharomyces cerevisiae encodes a 43 kDa MITOCHONDRIAL RNA polymerase specificity factor which recognizes mitochondrial promoters to initiate correct transcription. To better understand structure-function of the MTF1 gene as well as the transcription mechanism of mitochondrial RNA polymerase, two cold-sensitive alleles of the MTF1 mutation were isolated by plasmid shuffling method after PCR-based random mutagenesis of the MTF1 gene. The mutation sites were analyzed by nucleotide sequencing. These cs phenotype mtf1 mutants were respiration competent on the nonfermentible glycerol medium at the permissive temperature, but incompetent at 13.deg.C. The cs phenotype allele of the MTF1, yJH147, encoded an L146P replacement. The other cs allele, yJH148, contained K179E and K214M double replacements. Mutations in both alleles were in a region of Mtflp which is located between domains with amino acid sequence similarities to conserved regions 2 and 3 of bacterial s factors.

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.276.1-276
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• 1997

No Abstract, See Full Text

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6977-6982
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• 2014

Background: The aim of this study was to investigate the effect of a Lipofectamine2000 (Life2000) Transfection Reagent transfected psiRNA-STAT3 plasmid on 4T1 breast cancer cells. Materials and Methods: MTT was used to detect the cell proliferation of breast cancer 4T1 cells at different periods (0h, 6h, 8h, 10h); the cell cycle was assessed by flow cytometry; variation of apoptosis and mitochondrial membrane potential was observed under a fluorescence microscope; immunohistochemical staining was used to determine the expression of caspase-3 and cyclin-D1 protein. Results: An obvious effect of inhibition to 4T1 cancer cells could be observed at 8h after the psiRNA-STAT3 was transfected. Typical alterations of apoptotic morphological features were visible in the psiRNA-STAT3 treatment group. Mitochondrial membrane potential decreased significantly, the number of cells was increased in G0/G1 phase, and the number of cells was decreased in S phase, and the data were statistically significant (p<0.05), compared with the Scramble and Mock groups. Expression of caspase-3 protein was increased significantly, while that of cyclin D1 was significantly decreased. Conclusions: Life2000 transfected psiRNA-STAT3 plasmid can inhibit 4T1 tumor cell proliferation and promote apoptosis of 4T1 tumor cells, which process depends on the regulation of expression of cyclin D1 and caspase-3 protein.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.365-368
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• 2006

Yeast mitochondrial transformation has been confirmed by cell death and CFP expression (CDF: cell death factor gene). Expression vector containing CDF and CFP driven by one TPI (Triose-phosphate isomerase) promoter (called artificial operon type) was bombarded to Yeast. Interestingly, yeast cells were progressively deformed into unusual shapes and lysed inner cytoplasm resulting in ell death after all after bombarding with expression vector (CDC and GFP). Since there is no report about more than one gene expression simultaneously in a single mitochondria, this report is very important to novel type of eukaryotic gene expression. Successful yeast cell transformation in this report implies possible eukaryotic mitochodrial transformation including plants and animals and moreover two or more gene expression which can be excellent applicable protocols to pharmaceutical field including antibody production.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.189-194
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• 2006

Sperm-mediated gene transfer(SMGT) can be used to transfer exogenous DNA into the oocyte at fertilization. The main objective of this study was to assess efficiency of transferring mitochondrial DNA(mtDNA) fragment into boar spermatozoa in either presence or absence of liposome and quality of transfected spermatozoa. The mtDNA of chicken liver was isolated and purified by phenol and alkaline lysis extraction, and it was inserted to plasmid. The genome of transfected spermatozoa treated with DNase I was purified by alkaline lysis, and then amplified by the PCR analysis. After electrophoresis, DNA quantitation of each well was calculated by comparison of the band intensity with standard. As a result, exogenous DNA was composed of mtDNA fragment(1.2 kb) and plasmid(2.7 kb). On the other hand, efficiency of transfection by liposome($9.0{\pm}0.34ng/{\mu}l$) in SMGT was higher than that by DNA solution($6.9{\pm}0.53ng/{\mu}l$). However, there was no significant difference. Transfering exogenous DNA into spermatozoa was completed within 90 min of incubation. In another experiment, there were significant (p<0.05) differences between transfected spermatozoa using both DNA solution and DNA/liposome completes with unheated spermatozoa for viability ($70.8{\pm}1.80$ and $68.0{\pm}2.16%$ vs. $83.3{\pm}1.69%$, respectively) and motility($78.7{\pm}1.59$ and $79.3{\pm}2.14%$ vs. $86.7{\pm}1.59%$, respectively). This study indicates that exogenous mtDNA can be efficiently transferred into boar spermatozoa regardless of the presence of liposome, and transfected spermatozoa can also use insemination and in vitro fertilization to generate transgenic pig.

• Journal of Microbiology
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• v.34 no.1
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• pp.38-39
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• 1996

The internal regions of nuclear small subunit rRNA from 6 plaeurotus species and 5 Pleurotus ostreatus strains were amplified by PCR and sequenced. The DNA sequences of 8 Pleurotus strains (P. ostreatus NFFA2, NFFA4501, NFFA4001, KFFA4001, KFCC11635, P florida, P. florida, P. sajor-cuju, P. pulmonarius, and P. spodoleucus) were idential, but P. cornucopiae differed from them in two bases out of 605 bases. However, p[hylogenetic analysis of the sequences by DNA-distance matrix and UPGMA methods showed that P. ostreatus NFFA2m1 and NFFA2m2, known as mutants of P. ostreatus NFFA2, belonged to anther group of Basidiomycotina, which is close to the genus Auricularia. The difference of the SSU rDNA sequences of P. cornucopiae from other Pleurotus species tested corresponds to the difference of mitochondrial plasmid type present in Pleurotus species as observed by Kim et al. (1993, Korean J. Microbiol. 31, 141-147).