• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.789-793
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• 1999

The effect of acetic acid formation deficiency on recombinant E. coli fermentation was investigated using a mutant strain deficient in acetic acid formation. A mutant strain which does not grow under anaerobic conditions was isolated. The acetic acid production in this strain was negligible in aerobic batch fermentation. The cloned-gene expression in the mutant strain was higher than the wild-type strain. Fed-batch fermentations with controlled specific growth rates were carried out in order to compare the cloned-gene expression between the wild-type and the mutant strains. The expression decreased along with the specific growth rate in both strains. The cloned-gene expression in the mutant strain was 60% higher than in the wild-type strain at the same specific growth rate.

• BMB Reports
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• 제44권8호
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• pp.535-540
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• 2011

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.

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

• Reproductive and Developmental Biology
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• 제37권1호
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• pp.1-8
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• 2013

In order to investigate genetic stability and gene expression profile after cloning procedure, two groups of cloned pigs were used for swine leukocyte antigen (SLA) gene nucleotide alteration and microarray analyses. Each group was consist of cloned pigs derived from same cell line (n=3 and 4, respectively). Six SLA loci were analyzed for cDNA sequences and protein translations. In total, 16 SLA alleles were identified and there were no evidence of SLA nucleotide alteration. All SLA sequences and protein translations were identical among the each pig in the same group. On the other hand, microarray assay was performed for profiling gene expression of the cloned pigs. In total, 43,603 genes were analyzed and 2,150~4,300 reliably hybridized spots on the each chip were selected for further analysis. Even though the cloned pigs in the same group had identical genetic background, 18.6~47.3% of analyzed genes were differentially expressed in between each cloned pigs. Furthermore, on gene clustering analysis, some cloned pigs showed abnormal physiological phenotypes such as inflammation, cancer or cardiomyopathy. We assumed that individual environmental adaption, sociality and rank in the pen might have induced these different phenotypes. In conclusion, the results of the present study indicate that SLA locus genes appear to be stable following SCNT. However, gene expressions and phenotypes between cloned pigs derived from the same cell line were not identical even under the same rearing conditions.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.21-27
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• 1998

The xylA gene of Bacillus stearothermophilus encoding the major ${\beta}$-xylosidase was previously cloned and sequenced. In the present study we examined the regulation of the cloned xylA gene expression in Bauillus subtilis MW15 carrying the xylA::aprA fusion plasmids. The induction of the fused xylA gene expression remained uninfluenced by any of the carbon sources tested but the gene expression was repressed about 2-3 fold in the presence of glucose. Two CRE-like sequences (CRE-1: nucleotides ＋ 124 to ＋136 and CRE-2: ＋247 to ＋259) were recognized within the reading frame region of the xylA gene. The deletion experiments showed that the CRE-2 sequence had a role in catabolite repression (CR) as a true CRE of the xylA gene, but the CRE-1 had no effect on CR of the xylA gene expression. Surprisingly, the deletion of the CRE- 1 sequence reduced about 2~3 fold of the expression of the xylA fused gene. The repression ratios of the xylA gene expression were estimated to be about 0.4 from the assay of subtilisin activity, and about 0.3 at the level of transcription by determining the amounts of xylA transcripts in B. subtilis. While, the level of CR of the xylA gene was assessed to be about l0-fold in previous work when the relative amounts of the xylA transcripts were measured in B. stearothermophilus.

• 한국미생물·생명공학회지
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• 제28권1호
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• pp.21-25
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• 2000

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in Escherichia. coli TP2139 ( lac, crp). One of the cloned genes, pCKB17, was further analyzed. In order to find whether the increased expression of the gent was under the direction of maltose metabolism, we constructed several recombinant subclones. We have found that the clone, pCKB17AV, codes maltose metabolism stimulation(mms) gene. E. coli transformed with the cloned gene showed increase in the activity of maltose utilzation, The recombinant proteins expressed by multicopy and induction with IPTG, one polypeptide of 29-kDa, was confirmed by SDS-PAGE. The overexpression of maltose-binding proter protein in the presence of mms gene was confirmed by Western blot analysis. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• 한국양식학회지
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• 제16권2호
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• pp.124-127
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• 2003

Our previous studies of both microarray analysis in channel catfish muscle gene expression of 2 different ages and channel catfish muscle expressed sequence tag profiles demonstrated parvalbumin beta is one of the highly expressed muscle transcriptome. We have cloned and sequenced complementary DNA encoding the channel catfish parvalbumin which encode 109 amino acids. The deduced amino acid sequences of the catfish parvalbumin are highly conserved with those cloned from other teleosts. The availability of the catfish parvalbumin provides the opportunity of studying fish epitopes.

• Journal of Life Science
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• 제9권1호
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• pp.54-57
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• 1999

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in E. coli TP2139 (${\Delta}$lac, ${\Delta}$crp). One of the cloned genes, pCKB13, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several recombinant subclones. We have confirmed that the clone, pCKB13 codes Coenzyme A transferase gene by partial nucleotide sequencing in the terminal region. The enzyme activity of Coenzyme A transferase increased after introduction of the multicopy of the cloned gene in E. coli. The recombinant proteins expressed by multicopy and induction with IPTG, two polypeptide of 26-and 28-kDa, were confirmed by SDS-PAGE. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

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

• Reproductive and Developmental Biology
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• 제31권4호
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• pp.227-233
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• 2007

This study was conducted to examine the mRNA expression of apoptosis-related and imprinted genes and methylation pattern of the differentially methylated region (DMR) of H19 gene in day 35 of SCNT pig fetuses. The day 35 of natural mating (control) or cloned (clone) pig fetuses were recovered from uterus. Endometrium from dam and liver from fetus were obtained, respectively. mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. The Bcl-2 mRNA expression in clone was significantly lower than that of control (p<0.05). The mRNA expression of H19 gene in both endometrium and liver was significantly higher in clone than that of control, respectively (p<0.05). The level of IGF-2 mRNA in liver of clone was significantly lower than that of control (p<0.05), whereas the mRNA expression of IGF2-R gene in liver of clone was significantly higher than that of control (p<0.05). The DMR of H19 was lower methylation pattern in clone than that of control. These results suggest that the aberrant mRNA expression of apoptosis-related and imprinted genes and the lower DMR methylation pattern of imprinted gene may be closely related to the inadequate fetal development of cloned fetus.