• 한국미생물·생명공학회지
• /
• 제13권3호
• /
• pp.297-302
• /
• 1985

미생물중 urease생성능이 아주 강한 B. pasteurii의 Hind III partial digest 된 chromosomal DNA를 E. coli-B. subtilis bifunctional plasmid vector pGR 71으로 E. coli RR1 균주에 cloning 하므로써 그 urease gene을 expression시킬 수 있었다. 그러나 B. subtilis에서는 insertion DNA fragment의 deletion으로 expression되지 않았다. Cloning된 E.coli RR1 균주로부터 분리 정제한 urease gene함유 Plasmid(pGU66)의 restriction map을 작성하여 본 결과 7.1 Mdal의 insertion fragment가 삽입된 12.6Mdal의 plasmid에 Hind III, Bgl II, Xba I, Sal I등 몇 개의 cleavage site 위치를 찾을 수 있었다. Cloning된 E. coli의 urease는 periplasmic space에 많은 비율로 축적되며, 그 효소학적 성질은 donor인 B.pasteurii의 그것과 매우 유사하였다.

• 한국양식학회지
• /
• 제16권3호
• /
• pp.165-170
• /
• 2003

Isolation, cloning and sequencing of red seabream (Pagrus major) somatolactin (rsbSL) cDNA from pituitary gland revealed an open reading frame of 693 bp coding for a pre-growth hormone of 231 amino acids with a 22 amino acid putative signal peptide. Deduced amino acid sequence showed that there was one possible N-glycosylation site at Asn$^{145}$ and seven Cys residues (Cys$_{29}$ , Cys$^{39}$ , Cys$^{66}$ , Cys$^{89}$ , Cys$^{205}$ , Cys$^{222}$ , Cys$^{230}$ ). Except Cys$^{66}$ , others may be involved in disulfide bond formation. The rsbSL presented a 93% amino acid sequence identity with the SL of gilthead seabream (Sparus aurata) and contained the conserved hormone domain region. Expression of rsbSL in E. coli (BL2l) cells and gel analysis revealed a higher molecular weight for rsbSL than expected theoretically, implying posttranslational modifications.

• Animal cells and systems
• /
• 제8권2호
• /
• pp.105-109
• /
• 2004

Aminoalcoholphosphotransferase catalyzes the synthesis of phosphatidylcholine and phosphatidylethanolamine from diacylglycerol plus a CDP-aminoalcohol such as CDP-choline or CDP-ethanolamine. Previously we suggested the presence of possible isoforms of this enzyme from Chinese cabbage roots and now report the cDNA cloning and expression analysis of AAPT3 encoding a third isoform of aminoalcoholphosphotransferase (AAPT3). AAPT3 contains an open reading frame of 1,176 bp coding for a protein of 392 amino acids. It shares 96 and 95% identity with Chinese cabbage AAPT1 and AAPT2, respectively, at the deduced amino acid level. The results from reverse transcriptase-polymerase chain reaction analysis indicate that expression of AAPT3 is up-regulated by low temperature as well as AAPT1 and AAPT2.

• 한국어병학회지
• /
• 제8권1호
• /
• pp.31-36
• /
• 1995

HcNPV DNA genome 을 제한효소 EcoRI 으로 절단하여 그들의 일부 절편을 pUC8 vector 에 cloning 한 후 E. coli JM 83 세포에 형질 전환시켰다. 이 결과 24 개의 EcoRI 절편중 12 개의 절편이 cloning 되었다. 이들 제조합체중 4 개는 eNP-O, eNP-Q, eNP-R, eNP-S 라 명명하였다. 또한 이들 제조합체의 외래 유전자 발현을 SDS-PAGE 에 의해 단백질 패턴을 분석하였다. 그 결과 제조합체 eNP-O, eNP-Q, eNP-R 에서는 E. coli JM 83 숙주세포의 단백질 밴드와 비교하여 다른 분자량을 갖는 밴드가 나타났다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVII)
• /
• pp.991-991
• /
• 2005

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVII)
• /
• pp.997-997
• /
• 2005

• 한국미생물·생명공학회지
• /
• 제15권5호
• /
• pp.346-351
• /
• 1987

Clostridium thermocellum의 cellulase 유전자를 pBR322를 이용하여 Escherichia coli에 cloning하였다. 삽입된 Hind III 분해 DNA 단편의 크기는 약 1. 8kb였으며 이미 알려진 C. themocellum의 cellulase gene과는 다른 제한효소 분해위치를 가진 새로운 cellulase gene으로서 E, coli에서 CMCase와 FPase 활성을 나타내었다. 이 유전자는 생육의 전시기를 통해 표현되었고 고온성 cellulase의 구조적, 기능적 특성이 그대로 유지되었을 뿐만 아니라 상당량이 세포밖으로 분비되었다.

• Preventive Nutrition and Food Science
• /
• 제9권4호
• /
• pp.324-329
• /
• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• 미생물학회지
• /
• 제26권1호
• /
• pp.32-36
• /
• 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.

• 한국동물생명공학회지
• /
• 제36권4호
• /
• pp.314-322
• /
• 2021

Paired box protein, PAX7, is a key molecule for the specification, maintenance and skeletal muscle regeneration of muscle satellite cells. In this study, we identified and characterized the cDNA and amino acid sequences of PAX7 from black sea bream (Acanthopagrus schlegelii) via molecular cloning and sequence analysis. A. schlegelii PAX7 cDNA was comprised of 1,524 bp encoding 507 amino acids and multiple sequence alignment analysis of the translated amino acids showed that it contained three domains including paired DNA-binding domain, homeobox domain and OAR domain which were well conserved across various animal species investigated. Pairwise Sequence Alignment indicated that A. schlegelii PAX7 had the same amino acid sequences with that of yellowfin seabream (A. latus) and 99.8% identity and similarity with that of gilt-head bream (Sparus aurata). Molecular phylogenetic analysis confirmed that A. schlegelii PAX7 formed a monophyletic group with those of teleost and most closely related with those of the fish that belong to Sparidae family including A. latus and S. aurata. In the investigation of its tissue specific mRNA expression, the expression was specifically identified in skeletal muscle tissue and a weak expression was also shown in gonad tissue. The cultured cells derived from skeletal muscle tissues expressed PAX7 mRNA at early passage but the expression was not observed after several times of subculture.