• Title/Summary/Keyword: molecular cloning

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Molecular cloning and restriction analysis of aspartokinase gene (HOM3) in the yeast, saccharomyces cerevisiae (아스파테이트족 아미노산 대사에 관여하는 효모유전자(HOM3)의 클로닝 및 구조분석)

  • 최승일;이호주
    • Korean Journal of Microbiology
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    • v.26 no.1
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    • pp.32-36
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    • 1988

  • The yeast gene HOM3 encodes aspartokinase, which catalyses the first step (aspartate to and from beta-aspartyl phosphate) of common pathway to threonine and methionine. The yeast HOM3 gene expression is known to be regulated by threonine and methionine specific control, and also by general control of amino acid biosynthesis. Isolation and characterization of the HOM3 gene are essential for the molecular genetic study on its regulation of expression. A recombinant plasmid pSC3 (15.5kb, vector YCp50) has been cloned into E. coli HB101 from yeast genomic library through their complementing activity of HOM3 mutation in a yeast recipient strain M34-24B. Organization of the plasmid was characterized by delineation of restriction cleavage sites in the insert fragment.

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Molecular Cloning and Expression of Human Dihydrolipoamide Dehydrogenase-Binding Protein in Excherichia coli

  • Lee, Jeong-Min;Ryou, Chong-Suk;Kwon, Moo-Sik
    • Journal of Microbiology and Biotechnology
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    • v.11 no.4
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    • pp.592-597
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    • 2001

  • The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

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Molecular Cloning and the Nucleotide Sequence of a Bacillus sp. KK-l $\beta$-Xylosidase Gene

  • Chun, Yong-Chin;Jung, Kyung-Hwa;Lee, Jae-Chan;Park, Seung-Hwan;Chung, Ho-Kwon;Yoon, Ki-Hong
    • Journal of Microbiology and Biotechnology
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    • v.8 no.1
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    • pp.28-33
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    • 1998

  • A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

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A Modified Mutation Detection Method for Large-scale Cloning of the Possible Single Nucleotide Polymorphism Sequences

  • Jiang, Ming-Chung;Jiang, Pao-Chu;Liao, Ching-Fong;Lee, Ching-Chiu
    • BMB Reports
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    • v.38 no.2
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    • pp.191-197
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    • 2005

  • Although the human genome has been nearly completely sequenced, the functions and the roles of the vast majority of the genes, and the influences of single nucleotide polymorphisms (SNPs) in these genes are not entirely known. A modified mutation detection method was developed for large-scale cloning of the possible SNPs between tumor and normal cells for facilitating the identification of genetic factors that associated with cancer formation and progression. The method involves hybridization of restriction enzyme-cut chromosomal DNA, cleavage and modification of the sites of differences by enzymes, and differential cloning of sequence variations with a designed vector. Experimental validations of the presence and location of sequence variations in the isolated clones by PCR and DNA sequencing support the capability of this method in identifying sequence differences between tumor cells and normal cells.

  • https://doi.org/10.5483/BMBRep.2005.38.2.191 인용 PDF