• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.476-483
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• 1988

Yeat-Escherichia coli shuttle vectors were constructed by combination of various functional segments such as autonomous replicating sequence (ARS1), centromere region (CEN3), origin of replication of 2 $\mu$m plasmid (2 $\mu$m OR). Transformation efficiency, stability and copy number of constructed vectors were analyzed in yeast strains, SHY4(cir$^+$) containing 2 $\mu$m plasmid and NNY1(cir$^{\circ}$) without it. The results showed that centromere containing plasmids were very stable and existed at one copy per cell; fused replication system (2$\mu$m OR and ARS1) containing Plasmids were more stable and higher copy number than one replicon containing plasmids ; presence of endogenous 2$\mu$m plasmid influenced on stability and copy number of 2 $\mu$m based plasmids.

• Biomedical Science Letters
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• v.23 no.4
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• pp.372-379
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• 2017

This study was investigated to test whether paternal DNA that was destined for degradation was properly licensed by testing for the presence of mini-chromosome maintenance protein (MCM) 7 and origin recognition complex (ORC) 2 in the paternal pronuclei. ORC2 is one of the first licensing protein to come on and MCM7 is one of the last licensing protein to come on. Zygotes were prepared by injection of control and treated sperm injection (ICSI). To control for DNA breakage, epididymal spermatozoa were treated with DNase I to fragment the DNA, then injected into oocytes. The presence of MCM7 and ORC2 in the pronuclei of mouse zygotes was tested by immunohistochemistry, just before the onset of DNA synthesis, at 5 h after fertilization, and after DNA synthesis began, at 9 h post fertilization. We found that in all cases, both MCM7 and ORC2 were present in both pronuclei at 5 h after sperm injection, just before DNA synthesis began. This indicates that no matter how extensive the DNA damage, recruitment of licensing proteins to the origins of replication was not inhibited. Sperm DNA fragmentation does not prevent licensing of DNA replication origins. Furthermore, the embryo recognizes DNA that is damaged by nucleases. Our data indicate that the one-cell embryo does harbor a mechanism to prevent the replication of severely damaged DNA from spermatozoa, even though the embryos do not undergo classical apoptosis.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.403-408
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• 2009

A number of bifidobacterial species of human origin were screened for the presence of cryptic plasmids. One strain, Bifidobacterium longum FI10564, harbored plasmids of approximately 2.2 kb, 3.6 kb, and 4.9 kb in size. The smallest plasmid, pFI2576(2,197 bp), was studied in detail and its complete nucleotide sequence was determined. Computer-assisted analysis of this novel plasmid(G+C content 62%) identified 9 putative open reading frames(orfs), 3 of which were shown to be probable genes. These putative genes are arranged in an operon-like structure, in which the overlapping orfs 1 and 2 encode putative Rep proteins and are highly homologous to the rep genes of the B. longum plasmid pMBI(1,847 bp). The mechanism of replication of pFI2576 was investigated using Southern blot analysis of whole cell lysates, with and without S1 nuclease treatment, and atomic force microscopy(AFM). The results indicate that pFI2576 is likely to use the theta mode of replication.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.53-57
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• 2008

From the extensive screening for small cryptic plasmid among about 23 lactic acid bacteria (LAB), 2.4 kb of cryptic plasmid was isolated from Lactobacillus farciminis strain KCTC 3681 and named as pLF24. The plasmid pLF24 was a circular molecule of 2,396 base-pairs in length with a G+C content of 38%. Two protein-coding sequences could be predicted. ORF1 and ORF2 showed homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and the plus origin, were similar to replication regions of other gram-positive bacteria as shown in plasmids such as pLH2, pLS141-1 and pLC2. The nucleotide sequence of pLF24 was deposited into Genbank data base with an accession number of EU429343. The newly isolated plasmid can be used for construction of shuttle vector in Lactobacillus bacteria.

• Journal of Life Science
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• v.23 no.2
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• pp.290-298
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• 2013

The genus Mycobacterium includes crucial animal and human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium bovis. Although it is important to understand the genetic basis for their virulence and persistence in host, genetic analysis in mycobacteria was hampered by a lack of sufficient genetic tools. Therefore, many functional vectors as molecular genetic tools have been designed for understanding mycobacterial biology, and the application of these tools to mycobacteria has accelerated the study of mechanisms involved in virulence and gene expression. To overcome the pre-existing problems in genetic manipulation of mycobacteria, this paper reports new vector systems as effective genetic tools in Mycobacterium smegmatis. Three vectors were developed; pKOTs is a suicide vector for mutagenesis containing a temperature-sensitive replication origin (TSRO) and the sacB gene encoding levansucrase as a counterselectable marker. pMV306lacZ is an integrative lacZ transcriptional fusion vector that can be inserted into chromosomal DNA by site-specific recombination. pTnMod-OKmTs is a minitransposon vector harboring the TSRO that can be used in random mutagenesis. It was demonstrated in this study that these vectors effectively worked in M. smegmatis. The vector systems reported here are expected to successfully applicable to future research of mycobacterial molecular genetics.

• Journal of Microbiology
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• v.40 no.2
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• pp.140-145
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• 2002

The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMHl turned out to have a replication origin and two open reading frames (ORFs) of the putative γ-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMHl all constructed vectors were stable over 100 generations In a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a navel secretion vector, Using the $\alpha$-amylase promoter/signal sequence of B. subtilils the novel plasmid pJSN was constructed. When $\beta$-glucosidase was expressed using pJSN, we found $\beta$-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2005.11a
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• pp.58-58
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• 2005

• Microbiology and Biotechnology Letters
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• v.25 no.6
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• pp.566-570
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• 1997

During sporulation, Bacillus thuringiensis strains produce crystals consist of toxin proteins highly specific against insect pests. Their host specificities are desirable from a standpoint of environmental safety, but also limit market potential. Thus, development of improved Bacillus thuringiensis strains having broad host spectrum will contribute to increase its use. For the construction of Bacillus thuringiensis strain having broad host spectrum, we cloned cry1C gene encoding a toxin protein highly toxic against Spodoptera exigua from a B. thuringiensis isolate and constructed two recombinant plasmids, pUBClC and plC60. The plasmid PUBC1C has a replication origin of the natural plasmid pBC16 from B. cereus which is closely related species to B. thuringiensis, and the pBC16 was known to be replicated by rolling-circle mechanism. The plasmid pIC60 has a replication origin of a resident 60 MDa plasmid from B. thuringiensis subsp. kurstaki HD263, and it is believed that the pIC60 is replicated in a theta mode. The two plasmids were introduced into B. thuringiensis subsp. kurstaki cryB strain, and the transformed strains produced well-shaped bipyramidal crystals. We confirmed the expression of the cry1C gene by SDS-PAGE, and Western blotting. By investigating the segregational stability, it was found that the plasmid pIC60 is more stable than the pUBC1C.

• Animal cells and systems
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• v.1 no.2
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• pp.363-370
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• 1997

RNase MRP is a ribonucleoprotein complex with a site-specific endonuclease activity. Its original substrate for cleavage is the small mitochondrial RNA near the mitochondrial DNA replication origin, thus it was proposed to generate the primer for mtDNA replication. Recently, it has been shown to have another substrate in the nucleus, such as pre-S.8S ribosomal RNA in nucleolus. The gene for the RNA component of RNase MRP (MRP RNA) was found to be encoded by the nucleus genome, suggesting an interesting intracellular trafficking of MRP RNA to both mitochondria and nucleolus after transcription in nucleus. In this study, genomic DNA encoding MRP RNA was microinjected into the nucleus of Xenopus oocytes, to analyze promoter regions involved in the transcription. It showed that the proximal sequence element and TATA box are important for basal level transcription; octamer motif and Sp1 binding sites are for elevated level transcription. Most of Xenopus MRP RNA was exported out to the cytoplasm following transcription in the nucleus. Utilizing various hybrid constructs, export of MRP RNA was found to be regulated by the promoter and the 5' half of the coding region of the gene. Interestingly, the transcription in nucleus seems to be coupled to the export of MRP RNA to cytoplasm. Intracellular transport of injected MRP RNA can be easily visualized by whole-mount in situ hybridization following microinjection; it also shows possible intra-nuclear sites for transcription and export of MRP RNA.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.529.2-529
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• 1986

pSp-Si (1.6kbp) was originally found in pediococcus halophilus to be a cryptic multicopy-plasmid. Hoping that the plasmid can also replicate in Bacillus subtilis, protoplast transformation of strain 207-25 (recE) was performed using pSP-Sl onto which was added the marker of tmrB8 (on 4.9 kbp EcoRI fragment ) or tmrB+ (on 0.9 kbp xbaI fragment) gene. Though the tmrB8 gene can expres tunicamycin-resistance at the single copy state, and the tmrB+ gene exerts the resistance only at the multicopy state, we could not confirm the replication of pSP-Sl (tmrB8) or pSP-Sl(tmrB+) in B. subtilis. During the experiment, however, we unexpectedly found that the circularized 0.9 kbp xgaI fragment (tmrB+) itself, which had no replication origin, could transform strain 207-25 to tunicamycin-resistant by protoplast transformation. Southern hybridization analyses with tmrB+ and other probes revealed the integration of the fragment at a single copy state into a position other than the homologous tmrB gene. This recE independent integration of another tmrB+ gene into the chromosome may contribute to the tunicamycinresistance in the transformants.