• Asian-Australasian Journal of Animal Sciences
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• v.17 no.7
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• pp.897-902
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• 2004

PSMC5 subunit, which belongs to the 26S proteasomal subunit family, plays an important role in the antigen presentation mediated by MHC class I molecular. Full-length cDNA of porcine PSMC5 was isolated using the in silico cloning and rapid amplification of cDNA ends (RACE). Amino acid was deduced and the primary structure was analyzed. Results revealed that the porcine PSMC5 gene shares the high degree of sequence similarity with its mammalian counterparts at both the nucleotide level and the amino acid level. The RT-PCR was performed to detect the porcine PSMC5 expression pattern in seven tissues and the result showed that high express level was observed in spleen, lung, marrow and liver while the low express level was in muscle. The full-length genomic DNA sequence of porcine PSMC5 gene was amplified by PCR and the genomic structure revealed that this gene was comprised by 12 exons and 11 introns. Best alignment of the cDNA and genomic exon DNA sequence presents 4 mismatches and this information potentially bears further study in gene polymorphisms.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.332.1-332.1
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• 2002

Streptococcus vestibularis is a urease-producing oral bacterium. frequently isolated from vestibular mucosa of human oral cavity. Ureolysis by S. vestibularis and other ureolytic oral bacteria is believed to be crucially involved in oral microbial ecology and oral health. Genomic library of the S. vestibularis ATCC49124 was constructed in an E. coli plasmid vector and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for the urease activity was located on a 5.6 kb DNA fragment. (omitted)

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.619-624
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• 1992

Tn5 lac 삽입으로 채소입고병원균에 길항력이 약화된 T-67 및 고추역병균과 참깨역병균에 길항력이 약화된 T-81의 Tn5 lac 유전자 일부와 오른쪽 말단에 있는 길항관련 유전자의 flanking sequence가 cloning된 pAG67 및 pAG81 clone을 선발하였고, pAG67 및 pAG81 clone된 길항관련 유전자의 flanking sequence를 야생 길항균 Pseudomonas maltophilia B-14의 DNA를 probe로 사용하여 Southern hybridization으로 확인하였으며, 제한효소 지도를 작성하여 8Kb 및 4Kb 크기의 flanking sequence가 cloning되었음을 확인하였다. pAG6 및 pAG81의 flanking sequence를 EcoRi-BglII와 EcoRI-MpaI으로 분리하여 유전자 은행으로부터 길항관련 유전자가 cloning된 cosmid clone 7개주를 선발하였다.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.256-263
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• 2006

In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

• Journal of Plant Biology
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• v.37 no.3
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• pp.349-355
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• 1994

Viral RNA was extracted from a purified orchid strain of tobacco mosaic virus (TMV-O) from Cymbidium "Grace Kelly". Polyadenylated viral RNAs were primed with Not I-oligo (dT) primer-adapter. First-strand cDNAs were reversely transcribed by Moloney murine leukaemia virus reverse transcriptase (RNAse H-), and then second-strand cDNAs were synthesized by RNase H and DNA polymerase I. The resulting double-stranded cDNAs were ligated into pSPORT1 vector and transformed into competent E. coli strain JM109 cells. The size of cDNAs within the recombinant plasmids was ranging from 0.9 to 3.9 kb. Among the selected clones, pTMO-0205 and -0210 covered the 3' half and the 5' half of the viral genomic RNA, respectively, which were covering more than 99% of the viral genemo size based on sequencing analysis. Two cDNA fragments which were 3.1 kb BamHI and NotI fragement released from pTMO-0.205 and 3.3 kb SalI and BamHI fragment released from pTMO-0210 were ligated with T4 DNA ligase. The clone was almost entire length, lacking only 31 nucleotides from the 5' terminus based on the sequencing result. This method was shown to be efficiently applicable to other plant viral gnomic RNA for the construction of cDNA.n of cDNA.

• Journal of Microbiology
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• v.40 no.3
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• pp.245-247
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• 2002

A genomic DNA library of the fungus Trametes versicolor was constructed in a yeast integration vector which contains the URA3 gene of the budding yeast Saccharomyces cerevisiae and the gene responsible for hygromycin B resistance, and fragments acting as autonomously replicating sequences (ARSes) in the budding yeast were identified from the genomic DNA library. Sixteen recombinant plasmids from the library transformed the budding yeast Saccharomyces cerevisiae to Ura+ at high frequencies. They were maintained stably under selective conditions, but were gradually lost from yeast cells at different rates under nonselective conditions, indicating that they contain eukaryotic origins of DNA replication and exist as extrachromosomal plasmids. Base sequences of four ARS DNAs among the 16 cloned fragments revealed that all or the four contain at least one 11 bp ［(A/T)TTTA(T/C)(A/G)TTT(A/T)］consensus sequence of the budding yeast ARS.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.21 no.1
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• pp.53-66
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• 1995

The gene for ${\gamma}$-casein, a milk protein, is a member of a family of casein gene which is expressed in mammary glands of the animal during the late gestation and lactation periods binder the influence of various hormones. In order to elucidate tile mechanisms b)'which hormones regulate the coordinate induction of milk protein genes, the mouse ${\gamma}$-casein gene was isolated and characterized. The ${\gamma}$-casein gene was screened from a mouse genomic library constructed in bacteriophage EMBL3 with the ${\gamma}$-casein CDNA used as probe and one clone was obtained. The ${\gamma}$-casein CDNA as probe was partially sequenced and contained ATG start codon and 5'-noncoding region. The cloned genomic DNA was digested with Sal I restriction enzyme, by which the insert DNA can be isolated from EMBL3 vector. Three DNA bands were observed and the size of DNAs was approximately 28kb, 14kb and 9Kb, respectively Accordingly the size of the insert DNA was calculated with approximately 23Kb. The result of Southern blot analysis, however, showed that the cloned genomic DNA was not hybridized with the synthetic oligonucleotides (40 mer) of cDNA 5'-end region, but it was hybridized with the y -casein CDNA. This means that tile cloned y -casein gene may not contain its promoter region. The ${\gamma}$ -casein genomic DNA containing the promoter region has been screening from mouse genomic library with oligonucleotides of CDNA 5'-end region as probe, and twenty-nine clones was obtained.

• 대한생식의학회:학술대회논문집
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• 2002.05a
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• pp.11-17
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• 2002

Animal clones derived from somatic cells have been successfully produced in a variety of mammalian species such as sheep, cattle, mice, goats, pigs, cat and rabbits. However, there are still many unsolved problems in the present cloning technology. Somatic cell nuclear transfer has shown several developmental aberrancies including high rate of abortion in early gestation and increased perinatal death. These developmental failures of cloned embryos may arise from abnormal reprogramming of donor genome and/or incomplete cloning procedure. We have found that overall genomic methylation status of cloned bovine embryos is quite different from that of normal embryos in various genomic regions, suggesting that the developmental failures of cloned embryos may be due to incomplete reprogramming of donor genomic DNA. Many of the advances in understanding the molecular events for reprogramming of donor genome will more clarify the developmental defects of cloned embryos.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.50-55
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• 1992

A genomic library of a mesophilic cellulolytic anaerobe, Clostridium sp. KCTC 8440 DNA was constructed in Escherichia coli using plasmid pUC9. Clones of E. coli exhibiting carboxymethyl cellulose-hydrolyzing activity (CMCase) were isolated and divided into seven types based on the restriction enzyme patterns of recombinant plasmids. E. coli strains carrying type A genes showed activity on carboxymethyl cellulose about 7-8 times greater than clones carrying genes of other types. Restriction maps of the cloned DNA fragments were determined, and homologies between them were investigated. The results suggest that Clostridium sp. KCTC 8440 has seven distinct CMCase genes.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.369-374
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• 1996

The ${\beta}$-lactamase inhibitory protein (BLIP) produced by Streptomyces exfoliatus SMF19 was purified(33 kDa) and the N-terminal amino acid sequence was determined as NH2-ATSVVAWGGNND. Genomic DNA library of S. exfoliatus SMF19 was constructed in pWE15 and recombinants harbouring the corresponding gene were selected by colony hybridization to the mixture of 36-mer oligonucleotide designed from the N-terminal amino acid sequence. The corresponding gene (bliX) was isolated on a 4-kb ApaI fragment of S. exfoliatus SMF19 chromosomal DNA and then sequenced. The bliX consisting of 1, 119bp encoded a mature protein with a deduced amino acid sequence of 342 residues and also encoded a 40-amino-acid signal sequence. No significant sequence similarity to bliX was found by pairwise comparison using various protein and nucleotide sequences.