• Title/Summary/Keyword: Cloning and sequencing

Search Result 300, Processing Time 0.033 seconds

Complementary DNA Cloning and Sequencing of the Coat Protein Gene of Potato Virus Y-Ordinary Korean Strain (감자바이러스 Y의 OK계통에 대한 외피단백질 유전자 cDNA 클로닝 및 염기서열 분석)

  • 정승룡;최장경;길전행이;이부영
    • Korean Journal Plant Pathology
    • /
    • v.11 no.1
    • /
    • pp.73-79
    • /
    • 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.

  • PDF

Cloning, Sequence Analysis, and Characterization of the astA Gene Encoding an Arylsulfate Sulfotransferase from Citrobacter freundii

  • Kang, Jin-Wook;Jeoung, Yeon-Joo;Kwon, Ae-Ran;Yun, Hee-Jeong;Kim, Dong-Hyun;Choi, Eung-Chil
    • Archives of Pharmacal Research
    • /
    • v.24 no.4
    • /
    • pp.316-322
    • /
    • 2001
  • Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy of Citrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homologyl they were designated as astA and dsbA, respectively. The amino acid sequence of astA deduced from C. freundii was highly similar to that of the Salmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, and Campylobacter jejuni, encoded by the astA genes. However, the ASST activity assay revealed different acceptor specificities. Using p-nitrophenyl sulfate (PNS) as a donor substrate, $\alpha$-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine and tyrosine.

  • PDF

Reverse Transcription and Amplification of Halobacterial gvp Genes with Polymerase Chain Reaction Method (Polymerase Chain Reaction 방법에 의한 Halobacteria gvp 유전자의 역전사 및 증폭)

  • 윤병수;이상섭
    • Korean Journal of Microbiology
    • /
    • v.30 no.6
    • /
    • pp.456-459
    • /
    • 1992
  • The genes of Halohacteria. gvpD and gvpE. take part in formation of gas vesicle. These mRNA cause a lot of experimental prohlems due to its eharacteristic instahility in the analysis of transcripts. This study allowed easy cloning and sequencing of RNA hy substituting a stable complementary DNA for the mRNA of the genes for an analysis. The weak 111 RNA was reverse transcribed to DNA using reverse transcriptase. and was amplified using PCR method. The transcripts confirmed in this ~,tudy have not heen round in the northern hybridization covering almost all ranges of ORF of the gene. gvpD.

  • PDF

A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

  • Choi, Sangdun;Chang, Mi Sook;Stuecker, Tara;Chung, Christine;Newcombe, David A.;Venkateswaran, Kasthuri
    • Genomics & Informatics
    • /
    • v.10 no.4
    • /
    • pp.249-255
    • /
    • 2012
  • In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular- weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

Isolation and Identification of Urease-Positive Photobacterium sp. Strain HA-2 from Sea Water (해수에서 urease 양성 Photobacterium sp. Strain HA-2의 분리 및 동정)

  • Kim, Kang-Jin;No, A-Reum;Park, Kwon-Sam
    • Korean Journal of Fisheries and Aquatic Sciences
    • /
    • v.42 no.6
    • /
    • pp.531-536
    • /
    • 2009
  • A urease-positive bacterium isolated from sea water was identified as Photobacterium sp. by morphological, biochemical, and 16s rRNA gene analyses and named Photobacterium sp. strain HA-2. 2.0-fold increase enzyme activity was observed in LB medium containing 3% NaCl and 0.1% urea or not and the enzyme activity was 16.0-fold lower compared to urease-positive Vibrio parahaemolyticus AQ4673 strain when grown in the LB medium containing 3% NaCl with 0.1% urea. The cloning and sequencing of Photobacterium sp. strain HA-2 urease gene cluster is currently being analyzed in our laboratory.