• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.837-842
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• 2013

A cryptic plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733 was isolated, characterized, and used for the construction of a cloning vector to engineer Leuconostoc species. pMBLR00 is a rolling circle replication plasmid, containing 3,370 base pairs. Sequence analysis revealed that pMBLR00 has 3 open reading frames: Cop (copy number control protein), Rep (replication protein), and Mob (mobilization protein). pMBLR00 replicates by rolling circle replication, which was confirmed by the presence of a conserved double-stranded origin and single-stranded DNA intermediates. An Escherichia coli-Leuconostoc shuttle vector, pMBLR02, was constructed and was able to replicate in Leuconostoc citreum 95. pMBLR02 could be a useful genetic tool for metabolic engineering and the genetic study of Leuconostoc species.

• 대한의생명과학회지
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• 제2권2호
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• pp.231-240
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• 1996

인체 혈액 응고 9인자는 간에서 생성되며 461개의 아미노산으로 구성된 당 단백질이다. 따라서 인체 혈액 응고 9인자 cDNA를 찾기 위해 태아의 간(fetal liver) cDNA library를 PCR(Polymerase Chain reaction) 방법으로 screening하였으며, 그 결과 ATG개시 코돈으로부터 TAA종료 코돈까지 포함하는 1.4 kb의 9인자 cDNA를 찾았다. 또한 클론된 9인자 cDNA를 박테리아에서 발현시키기 위해 박테리아 발현 벡터인 pGEX-2T 플라스미드에 클로닝하므로써 pGEX-F9 플라스미드를 제조하였다. pGEX-F9로 형질전환된 E. coli에서 PGEX-F9의 발현을 유도하면 73 kDa 크기의 GST-factor9 융합 단백질이 다량생성되며 , 이 단백질이 혈액 응고 9인자 단백질을 함유하는 융합 단잭질임을 혈액 응고 9인자 항체를 이용한 Western blot으로 입증하였다. E. coli에서 발현된 GST-factor 9 융합 단백질은 전체 단백질의 약 20%를 차지하며 GST agarose bead를 이용한 one step purificarion 방법을 통해 GST-factor9 융합 단백질을 쉽게 분리 할 수 있다.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.577-609
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• 1995

Bacteria have been regarded as one of the most important factors in pulpal and periapical diseases. Streptococci are frequently isolated facultative anaerobes in infected root canals. Recently molecular biological techniques have been rapidly progressed. This study was designed to apply the molecular biological tools to the identification and classification of streptococci in the endodontic microbiology. Streptococci isolated from infected root canals were identified with both Vitek Systems and API 20 STREP. Identification results were somewhat different in several strains of streptococci. Eighteen streptococci and enterococcal was difficult so to digest plasmid DNA using Hind III and EcoRI to differentiate strains by restriction enzyme analysis of plasmid DNA. 16S rDNA of chromosome was amplified by polymerase chain reaction(PCR) and then restricition fragment length polymorphism(RFLP) using several restriction enzymes was observed. The molecular mass of 16S rDNA of chromosomal DNA was approximately 1.4kb. There were three to five RFLP patterns using eight restriction enzymes. RFLP patterns digested with CfoI which recognizes four base sequences were identical in all stains. Hind III which recognizes six base sequences could not digest the 16S rDNA. Restriction enzymes which recognize five base sequences were suitable for RFLP pattern analysis. At least three different restriction enzymes were needed to compare each strains. 16S rDNA PCR-RFLP was simple and rapid to differentiate and classify strains and could be used in the epidemiological study of root canal infections.

• 미생물학회지
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• 제28권1호
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• pp.35-40
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• 1990

Thiostrepton 내성 유전자(tsr)를 포함하는 multi-copy 재조합 플라스미드 pJY7J2의 제한효소 절단지도를 작성하였다. pJY, 712는 Streptomyces에서 넓은 host range를 나타내었으며 cloning 목적에 사용할 수 있는 단일 BgtIl 제한효소 인식부위를 갖고 있었다. 플라스미드 pJY 712는 lethal zygosis(Ltz+) 현상을 보였다. pJY 712의 혁질전환빈도는 S. lividans에서 $5.0\times 10^{4}$ TFU였다. pJY 712의 Bell 제한효소 인식부위에 tyrosmase 유전자(mel)를 삽입하여 플라스미드 PJY713을 제조하였다. met 유전자를 포함한 재조합 플라스미드 pJY 714는 pJY 713의 일부분(1.9kb BgllI-BelI 단편)을 제거하여 제고하였다.

• 대한의생명과학회지
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• 제9권2호
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• pp.67-74
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• 2003

Viral and non-viral vectors have been used in the delivery of genetic materials into animal cells and tissues, with each approach having pros and cons. Non-viral vectors have many useful merits such as easy preparation, low immunity and size tolerance of a transgene when compared to those of viral vectors. Delivery specificity may be achieved by complex formation between receptor ligands and a non-viral vector. In the present study, non-viral vector systems are investigated in an effort to find a practical delivery means for gene therapy, Receptor-ligand interaction between transferrin-receptor and transferrin was utilized for efficient gene transfer into cancer cells. A plasmid vector, pcDNA3 (LacZ) was ligated with a small duplexed oligo fragment in which a Biotin- VN$^{TM}$ phosphoramidite was placed in the middle of the oligo. The plasmid vector labeled by biotin was then conjugated with biotin-labeled transferrin via streptavidin. This trimeric conjugates were delivered to a hepatoma cell line, HepG2. The delivery efficiency of the trimeric conjugate was 2-fold higher than that of cationic liposomes used for transfection of a plasmid vector. These results demonstrate that a plasmid vector can be efficiently transferred into cells by forming a trimeric complex of plasmid vector-linker-ligand.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권3호
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• pp.225-229
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• 2005

To investigate impediments to plasmid transformation in Brevibacterium flavum BF4 and B. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties. B. lactofermentum was more sensitive to growth inhibition by glycine than B. flavum. Release of cellular proteins during sonication was more rapid for B. lactofermentum than for B. flavum. Plasmid DNA (pCSL 17) isolated from B. flavum transformed recipient $McrBC^+$ strains of Escherichia coli with lower efficiency than $McrBC^-$. McrBC digestion of this DNA confirmed that B. flavum contain methylated cytidines in the target sequence of McrBc sequences but B. lactofermentum contained a different methylation pattern. DNA derived from the B. lactofermentum transformed recipient $EcoKR^+$ strains of E. coli with lower efficiency than $EcoKR^-$, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.

• KSBB Journal
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• 제10권3호
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• pp.264-270
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• 1995

Trigonopsis variabilis는 버l타락탐 항생제인 cephalosporin C (ceph C)를 ${\alpha}$-keto-adipyl-7 a aminocephalosporanic acid(AKA-7 ACA)로 생변 환하는 강력한 D-amino acid oxidase 효소를 갖고 있다. 본 연구는 이 D-AAO 효소의 유전자를 추출하기 위하여 polymerase chain reaction (PCR)을 사용하였다. PCR 단편을 콜로닝하기 위하여 Taq D DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal와 T4 kinase 의 효소반응을 이용하여 4가지의 방법을 샤용한 결 과, blunt - end 의 PCR fragment 를 phosphory­l lation하고 blunt -end의 pUC18 plasmid를 dephos phorylation 한 후 ligation 한 것 이 가장 좋은 클로 닝 효율을 보였다. Ceph C에 대한 D-AAO 효소의 활성은 재조합 E. coli의 세포추출액과 permea bilized cells에서 모두 확인할 수 있었다.

• 한국식물병리학회지
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• 제11권1호
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• pp.73-79
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• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.

• 한국양식학회지
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• 제13권1호
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• pp.21-27
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• 2000

The successful gene transfer and transient expression was demonstrated in olive flounder embryos using lipofected sperm. Olive flounder sperm interacted with foregn plasmid DNA encapsidated by positively charged liposome. The maximum plasmid copy number that associated with the sperm was 5 copies/sperm based on the examination of DNA blot assay. The foreign DNA was transferred into fertilized eggs without any adverse effect on fertilization and survival of embryos (P>0.05) and retained in embryos until at least 42 hours with successful expression. The maximal expression was detected in 18 hours after fertilization at 18$^{\cird}C$ and gradually decreased with development of embryo. Most of DNA transferred into embryos persisted extrachromosomally without significant sign of integration or replication.

• 미생물학회지
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• 제27권1호
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• pp.21-26
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• 1989

한국산 Klebsiella pnelµnoniae KNFl의 chromosomal DNA를 Hind III로 부분소화하고 pBR 322도 같은 효소로 완전소화했다. 소화된 pBR 322 의 5'- 말단연산기를 제거하여 두 DNA 표품을 ligation 한 후 E. coli K060 으로 transformation 하 였다. 이러한 transformants중 N-free 한천배지에서 자라는 단일 colony을을 얻었으며, 이들은 모두 ampicillin에 대해 저 항성을 가지고 있었고 tetracycline에 대해 저항성이 없었으며 cunng 되였을 때는 질소고정능을 잃었다. 이런 사실로부터 이 transformants가 K. pneumoniae의 질소고정능 유전자을 포함하는 recombinant plasmid를 가지고 있음을 확인할 수 있었다. 이들 transformants의 nitrogenase 역가는 K. pneumoniae KNF 1보다 더 높았다.