• Journal of Microbiology and Biotechnology
• /
• 제2권4호
• /
• pp.237-242
• /
• 1992

In order to clone the gene coding for alkaline phosphatase in the yeast Kluyveromyces fragilis, a genomic library was constructed using the yeast-E. coli shuttle vector pHN114 as a cloning vector. From the genomic library, a clone carrying the gene was isolated and the plasmid was designated as pSKH101. A restriction enzyme map was made using this plasmid. Subcloning experiments and complementation studies showed that alkaline phosphatase was active only in the original 3.1 kb insert. Southern hybridization analysis confirmed that the cloned DNA fragment was derived from K. fragilis genomic DNA. Using a minicell experiment, the product of the cloned gene was identified as a protein with a molecular weight of 63 KDa. A 0.6 kb HindIII fragment, which showed promoter activity, was isolated using the E. coli promoter-probe vector pKO-1.

• 한국미생물·생명공학회지
• /
• 제26권5호
• /
• pp.387-392
• /
• 1998

B. lactofermentum의 lysine 생합성에 있어서 DDH경로 및 ddh gene이 지닌 중요성을 조사하기 위하여, site-specific mutagenesis technique를 통하여 B. lactofermentum의 ddh gene을 disruption함으로서 DDH 경로를 차단시켰다. B. lactofermentum ddh mutant는 wild type 및 AEC내성 균주보다 성장이 매우 저조하였으며 lysine 생산량에서도 급격한 저하를 가져왔다. 이와 같이 B. lactofermentum이 DAP 경로만을 가졌을 때 세포의 성장 및 lysine 생산량에 있어서 극적인 저하를 가져왔기 때문에 B. lactofermentum에서의 DDH 경로는 meso-DAP 및 lysine 생합성에 있어 필수적인 경로로 작용한다는 것을 확인하였다. 그러므로 C. glutamicum과 B. lactofermentum과 같은 corynebacteria가 lysine을 많이 생산하는 것은 DDH 경로가 부가적으로 존재하기 때문이며, 이러한 DDH 경로는 metabolic flux가 증가되면 중간 대사물을 lysine으로 변화시키는 중요한 경로로 작용할 것이라 사료된다.

• Reproductive and Developmental Biology
• /
• 제33권1호
• /
• pp.19-24
• /
• 2009

This study was conducted to investigate the specific expression genes in the cloned bovine tissues. Donor cells, cloned tissues were analysed by RAPD-RFLP method. The results were detected three genes (CH-U7B, CH-U7M and CH-U7P) in the cloned fetus. It was found a single copy genes by southern hybridization. Sequence analysis of CH-U7M gene was shown 99% homology to a previously reported EST from a cloned bovine fetus. The putative ORF was encode a protein of hydrophobicity index 0.03. Semi-quantitative RT-PCR by using the CH-LS001 specific primer was remarkably detected in the lung tissue of cloned fetus. Further investigation of these genes may provide one of the key information to explain the early death, abnormal fetus, large off-spring and the low pregnancy rate in the production of cloned bovine.

• Mycobiology
• /
• 제38권4호
• /
• pp.331-335
• /
• 2010

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/${\mu}g$ of DNA in $1{\times}10^7$ protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

• 한국가축번식학회지
• /
• 제17권1호
• /
• pp.43-48
• /
• 1993

These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1$\times$107 phagemids were screened with 32P labelled probe. Hybridization were carried on $65^{\circ}C$ for 16~20hours. And 1$\times$106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.

• 한국식물병리학회지
• /
• 제14권5호
• /
• pp.513-518
• /
• 1998

Beet curly top virus (BCTV) mutant has been constructed in vitro that contain G-to-T transversions at nucleotide 2727 within overlapping open reading frames (ORFs) L1 and L4. The mutations introduce termination codon in ORF L4 without affecting the amino acid encoded by ORF L1. When agroinoculated into Arabidopsis thaliana the mutant caused mild stunting and stem curling, but not the callus induction and hyperlasia on infected tissues of Sei-O ecotype. However, this mutant was not infectious on Col-O. Levels of single stranded DNA forms were similar in mutant and wild type BCTV infections. The DNA quantitation data showed that the DNA of BCTV-L4 mutant virus was accumulated in shoot tips, infection origin and roots with similar levels to those of wild type virus infected. Three tissues of asymptomatic ecotype Col-O also had as much as virus DNA from wild type virus infections. In both ecotypes infected with BCTV-Logan and BCTV-L4 mutant, root tissues contained more virus DNA than any other tissues by the Southern hybridization data. The results suggest that ORF L4 encodes a functional protein that is a major determinant of pathogenesis that might affect the hyperplastic response of the host to BCTV infection.

• 한국식물학회:학술대회논문집
• /
• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
• /
• pp.59-71
• /
• 1985

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.

• Journal of Microbiology
• /
• 제35권1호
• /
• pp.40-46
• /
• 1997

The pWHM220 cosmid with a 24-kb insert cloned from Streptomyces albus ATCC 21838 induces the biosynthesis of a polysther antibiotic similar to salinomycin in Streptomyces invidans. We have analyzed this region by DNA sequencing as well as Southern blot hybridization with type I and type II polyketide synthase (PKS) probes. Surprisingly, we found another set of type II SKS genes only 10-kb from the original PKS genes, salABCDE. The DNA sequence revealed two complete open reading frames (ORFs) named salB2 and salC2, and one partial ORF that does not resemble any known DNA or deduced protein sequence. The salC2 should code for chain length determining factor while the deduced amino acid sequence encoded by salB2 exhibits high similarity to .betha.-ketoacyl synthase from different PKS gene clusters. The highest identity was found for .betha.-keetoacyl synthases from S. argillaceus (MtmP. 59.1% identity), the mithramycin producer and from S. venezuelae ISP5230 (JadA, 52.3% identity), the jadomycin producer. The SalC2 protein clearly resembles its counterparts in order aromatic PKS gene clusters that are believed to influence the length of the polyketide chain. The highest identities observed were to that of S. argillaceus (MtmK, 62.3%) and S. venezuelae ISP 5230 (JadB, 55.1%) proteins, Moreover, the deduced amino acid sequences of the salB2 and salC2 products were 29.0% identical.

• Archives of Pharmacal Research
• /
• 제19권6호
• /
• pp.469-474
• /
• 1996

A rapid and sensitive method has been developed to detect hepatitis B virus DNA (HBV) by nested polymerase chain reaction (PCR) technique with primers specific for the surface and core regions in capillary thermal cycler within 80 min. The lower limit for detection by present PCR method is $10^{-5}$ pg of recombinant HBV DNA which is equivalent to that determined by one round of PCR amplification and Southern blot hybridization analysis. When boiled HBV positive serum was serially diluted 10-fold, HBV DNA was successfully determined in $1{\mu}l-10^{-3}$ of serum. HBV DNA was detected by present method in 69 clinical samples including HBsAg positives and negatives by enzyme-linked immunosorbent assay (ELISA). When serum samples were amplified by nested PCR using surface and core region primers, HBV DNAs were detected in 37 of 69 samples (53.6%) and 18 of 69 samples (26.1%), respectively. These results can inform the infectious state of HBsAg positive pateints. A simple and rapid nested PCR protocol by using boiled serum as DNA template has been described for the clinical utility to determine HBV DNA in human serum.

• Journal of Microbiology and Biotechnology
• /
• 제10권5호
• /
• pp.670-676
• /
• 2000

Several clones obtained from Serratia marcescens stimulated E. coli TP2139 (${\Delta}lac, \;{\Delta} crp$) cells to use maltose as a carbon source. The crp gene clone, pCKB12, was confirmed to stimulate the $\beta$-galactosidase activity, by Southern hybridization [31]. The nucleotide sequence of the crp region consisting of 1,979 bp was determined. The sequencing of the fragment led to the identification of two open reading frames: One of these, the crp gene, encoded 210 amino acid and the other encoded a truncated protein. The S. marcescens and E. coli crp genes showed a higher degree of divergence in their nucleotide sequence with 120 changes, however, the corresponding amino acid sequences showed only two amino acid differences. Yet, an analysis of the amino acid divergence revealed that the catabolite gene activator protein, the crp gene product, was the most conserved protein observed so far. Using a crp-lac protein fusion, it was demonstrated that S. marcescens CRP could repress its own expression, probably via a mechanism similar to that previously described for the E. coli crp gene.