• Title/Summary/Keyword: DNA restriction

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Isolation and Characterization of a CDNA Encoding a Protein Homologous to the Mouse 70 kDa Heat Shock Protein (생쥐 섬 유아세포에서 70 kDa 고온충격 단백질의 CDNA 클로닝과 염기서열 분석)

  • 김창환;정선미최준호
    • The Korean Journal of Zoology
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    • v.35 no.2
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    • pp.203-210
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    • 1992
  • Hsp70, a 70 kDa protein, is the maior protein expressed when cells are heat-shocked. A cDNA library from mouse ID13 cells was screened with the human hsp70 gene as a probe, and a positive clone was obtained. The positive clone was subcloned into puc19 and the precise restriction was obtained. The CDNA was sequenced by the Sanger's dideoxv termination method. Single open reading frame that codes for a protein of 70 kDa was found. The DNA sequence of the cloned mouse DNA shows great homology (66-90%) with other mouse hsp70 genes and somewhat less homology (50",) with E. coli hsp70 gene (dnak). With the exception of one amino acid, the protein sequence deduced from the CDNA is identical to the mouse that shock cognate protein 70 (hsc70) that is constitutivelv expressed at normal temperature. The result suggests that the cloned CDNA encodes a hsc70 family rather than a heatinducible family.mily.

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Sequence Selectivity of DNA Alkylation by Adozelesin and Carzelesin

  • Yoon, Jung-Hoon;Lee, Chong-Soon
    • Archives of Pharmacal Research
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    • v.21 no.4
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    • pp.385-390
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    • 1998
  • Adozelesin and carzelesin are synthetic analogues of the extremely potent antitumor antibiotic CC-1065, which alkylates N3 of adenine in a consensus sequence $5^1$-(A/T)(A/T)$A^*$ ($A^*$ is the site of alkylation). We have investigated the DNA sequence selectivity of adozelesin and carzelesin by thermally ind ced DNA strand cleavage assay using radiolabeled restriction DNA fragments. An analysis of alkylation patterns shows that the consensus sequences for carzelesin and adozelesin have been found to be $5^1$-(A/T)(A/T)$A^*$ and $5^1$-(A/F)(G/C)(A/T)$A^*$. A new consensus sequence, $5^1$-(A/T)(A/T)$CA^*$, has been observed to display an additional alkylation site for adozelesin but not for carzelesin. These results indicate that the pattern of sequence selectivity induced by carzelesin is similar but not identical to those induced by adozelosin.

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Cloning of the 5'-end and Amplification of Full-Length cDNA of Genomic RNA of Lily symptomless virus

  • Park, Seon-Ah;Ryu, Ki-Hyun
    • The Plant Pathology Journal
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    • v.18 no.4
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    • pp.187-191
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    • 2002
  • This paper describes the cloning and sequence analysis of the 5'-terminal region and full-length cDNA production of genomic RNA of Lily symptomless virus (LSV), a Species Of the genus Carlavirus. A sing1e DNA band about 600 bp harboring the 5'-end of genomic RNA of the virus was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and was cloned for nucleotide sequence determination. Sequence analysis of selected RACE cDNA clones revealed that the LSV 5'non-translated region consists of 67 nucleotides long of AT rich stretch followed GC rich from the 5'-end. To produce full-length cDNA products for the viral genomic RNA, a set of LSV-specific primers could be designed based on the obtained sequence in this study and the known sequences of 3'-terminal region for the virus. Full-length cDNA copies of LSV, an 8.4 kb long, were directly amplified by the long-template RT-PCR technique from the purified viral genomic RNA samples. This full-length cDNA copies were analyzed by restriction mapping. The molecules produced in this study can be useful for the production of in vitro infectious cDNA clone, as well as, for the completion of genomic RNA sequence and genome structure for the virus.

Biochemical Characteristics of the Granulosis Viruses DNA of Common Cabbage Worm, Pieris rapae and Pieris brassicae (배추흰나비 과립병바이러스 DNA의 생화학적 특성)

  • 류강선;진병래;강석권
    • Korean journal of applied entomology
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    • v.30 no.2
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    • pp.138-143
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    • 1991
  • This study was carried out to acquire some basic biochemical informations on the granulosis virus (GV) DNA of Pieris rapae and Pieris brassicae. The thermal denaturation temperature (Tm) and G+C content of the DNA of the viruses were $83.7^{\circ}C$ and 35.5% for P. rapae GV, $84.0^{\circ}C$ and 35.9% for P. brassicae GV, respectively. There were some differences in the DNA fragmentation patterns of the two GV's produced by digestion with restriction endonucleases such as EcoR I , BamH I and Hind m . The homololgy between the two DNAs was caculated to be 97.0%. The size of the genome was estimated to be 103 kbp for P. rapae GV and 108 kbp for P. brassicae GV.

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Amplified mitochondiral DNA identify four species of Tetranychus mites (Acarina: Tetranychidae) in Korea (미토콘드리아 DNA증폭을 이용한 한국의 잎응애속(Tetranychus;Acarina: Tetranychidae) 4종의 동정방법)

  • 이명렬;이문홍
    • Korean journal of applied entomology
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    • v.36 no.1
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    • pp.30-36
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    • 1997
  • Except for a cosmopolitan and major pest of apples, Tetranychus urticae Koch, Tetranychus mites in Korea such as T. viennensis Zaher, T. kanzawai Kishida, and T. truncams Ehara have been considered as quarantine pests by Canada and United States. Even though these mites are not feeders on apples, they are suspected to attach accidentally on apple h i t s in autumn as females enter the diapause. The characters used to identify Tetranychus mites have been confined to the shape of aedeagus in adult male. To develope a fast and accurate alternative identification protocol applied to hibernating female mites on apples, their mitochondrial DNA (mtDNA) were examined to find out any polymorphisms to discriminate each species from the other ones. Three pairs of primers for polymerase chain reaction (PCR) were used to amplify cytochrome oxidase subunit I (CO-I) coding region in mitochondrial DNA5 of four species of Tetranycus mites. The longest amplified product was estimated its size as about 680 bp. Digestion with restriction enzymes, AluI, Ddel, and Sau3A, showed length polymorphisms, which will he useful as diagnostic markers to identify Tetranychus mites. Schematic restriction maps in amplified region were shown for each species.

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Mitochondrial DNA Analysis of the Fleshy Prawn (Penaeus chinensis) for Stock Discrimination in the Yellow Sea (황해산 대하(Penaeus chinensis)의 계군분석을 위한 미토콘드리아 DNA 분석)

  • HWANG Gyu-Lin;LEE Yong-Chul;CHANG Chung-Soon
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.30 no.1
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    • pp.88-94
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    • 1997
  • The mitochondrial DNA (mtDNA) restriction fragment length polymorphi는 (RFLPs) of five populations were analyzed to delineate the stocks of Penaeus chinensis (Osbeck) in the Yellow Sea. Comparison of P. chinensis with P. japonicus to clarify the nucleotide divergence between two species was also carried out. Based on the fragment patterns, three composite haplotypes were analyzed in P. chinensis mtDNA as four naplotypes were in P. japonicus. Most individuals of each P. chinensis population are shared by one dominant haplotype. Another two haplotypes haying variations at the C/a I and hull sites were also distributed evenly in the Korean and Chinese populations. It is suggested that the gene exchange occurring between populations in the Yellow Sea is frequent. Average length of the mtDNA molecule was estimated to be about 16.44 kb in P. chinensis and 16.31 kb in P. japonicus, Sequence divergence (p) of mtDNA between two species estimated by using Upholt's (1977) fomula was $13.7\%$.

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Development of PCR Technology for Identification of the Restriction Fragment Length Polymorphism(RFLP) of the Immunoglobulin Allotypes in Periodontal Patients (치주질환자의 면역글로블린 이종형에 따른 제한절편장 다변화 양상에 대한 PCR 기법의 개발)

  • Choi, Jeom-Il;Kim, Sung-Jo;Kim, In-Hoo
    • Journal of Periodontal and Implant Science
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    • v.29 no.2
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    • pp.349-355
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    • 1999
  • The present study has been performed to develop a PCR technology to identify human immunoglobulin(Ig) allotypes with restriction fragment length polymorphism(RFLP) using a probe. Genomic DNA were ampilified with PCR tecnology using primers from peripheral blood lymphocytes of 10 periodontal patiens, whose Ig allotypes have been pre-determined by serological tecnique using heagglutination technique. The result indicated that the RFLP patterns could successfully differentiate the Ig allotypes, which suggests that this technology can be developed as a tool useful for population genetics studies.

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Detection of Soybean Mosaic Virus Using RT-PCR

  • Kim, Yul-Ho;Kim, Ok-Sun;Lee, Bong-Choon;Roh, Jae-Hwan;Kim, Myoung-Ki;Im, Dae-Joon;Hur, Il-Bong;Lee, Sang-Chul
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.44 no.3
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    • pp.253-255
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    • 1999
  • Reverse transcription and polymerase chain reaction (RT-PCR) assay was used to detect SMV strains. A pair of oligonucleotide primers were designed to include the cylindrical inclusion (CI) coding region between 4,176 to 5,560 nt. Amplification from the total RNA extracted from infected plants with SMV yielded a 1,385 bp DNA fragment. RT-PCR was shown to be $10^3$ times more sensitive than the ELISA assay and it could detect a virus in $10^{-6}$ dilution. Restriction enzyme analysis of RT- PCR products using EcoR I showed that SMV isolates were classified into six groups according to the patterns of restriction fragments.

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Pseudomonas sp. W7로부터 Agarase 유전자의 Cloning 및 Escherichia coli에서의 발현

  • Ha, Jeong-Chul;Kim, Gu-Taek;Kim, Sung-Koo;Yu, Ju-Hyun;Kong, In-Soo
    • Microbiology and Biotechnology Letters
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    • v.23 no.6
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    • pp.665-670
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    • 1995
  • A marine bacterium which produces extracelluar agarase was isolated from sea water. Isolated strain was identified as Pseudomonas sp. by the morphological and biochemical properties (1). HindIII restriction fragment of 3.2 kb from Pseudomonas genomic DNA was cloned into pUC19 to obtain recombinant plasmid pJA1 which enables E. coli JM83 to produce agarase. Most of agarase produced in E. coli was secreted into the culture medium. The enzyme (pJA1) showed the highest agarase activity during the stationary phase (20 hrs) of E. coli. The optimum temperature and pH were 40$\circ$C and 7.8, respectively. Restriction gene map anlaysis revealed that it has different restriction pattern with three kind of agarase gene reported.

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Exprission of cellulomonas biazotea cellobiase gene in E. coli (Cellulomonas biazotea cellobiase gene의 대장균에의 형질발현)

  • 박영길;연창석;최영길
    • Korean Journal of Microbiology
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    • v.26 no.1
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    • pp.6-12
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    • 1988
  • Cellobiase ($\beta$-glucosidase) is an enzyme of the cellulase system in cellulolytic microor-ganisms. The chromosomal DNA fragment which include cellobiase gene of Cellulomonas biazotea was cloned in Eschericia coli via plasmid pBR 322 vector. Restriction enzyme Sal I was used to obtain adequate size of fragments from C. biazotea. chromosomal DNA. The transformant of E. coli HB101 with recombinant plasmid pBG101 showed cellobiase activity, which is not ordinary in E. coli HB101. The enzyme activity of the transformant was as of 20% lower than that of C. biazotea.

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