Biomedical subjects

Gyoo Yeol Jung

Publications and source records attributed to Gyoo Yeol Jung.

5 recordsLinked to original sources

PubMed2006

A precise mRNA quantification method using CE-based SSCP.

Even though mRNA quantification provides significant information for biological analysis, current methods such as Northern blot analysis and real-time PCR are known to be laborious and lacking in precision. In this study, we demonstrate a new precise mRNA quantification method using CE based on SSCP (CE-SSCP) coupled with reverse transcription. mRNA samples could be simply analyzed for the quantification directly with reverse transcript obtained from a single reaction. This helps to avoid considerable errors generated by a series of the tedious manual steps. Also, unlike real-time PCR, reverse transcripts can be directly quantified by CE-SSCP in this method without further data estimation. Reproducibility and accuracy of CE-SSCP for mRNA quantification was examined using enhanced green fluorescent protein (eGFP) mRNA transcribed in vitro. Specific reverse transcription primer was determined for the accurate quantification of eGFP mRNA from total RNA obtained from the recombinant Escherichia coli. Using elongation factor Tu mRNA as an internal standard, it was shown that sample-to-sample variation could be minimized. Expression kinetics at both mRNA level and protein level was studied and the potential of CE-SSCP in expression analysis was demonstrated by comparison with the eGFP activity assay.

Electrophoresis, Capillary↗

PubMed2004

A functional protein chip for pathway optimization and in vitro metabolic engineering.

Pathway optimization is difficult to achieve owing to complex, nonlinear, and largely unknown interactions of enzymes, regulators, and metabolites. We report a pathway reconstruction using RNA display-derived messenger RNA-enzyme fusion molecules. These chimeras are immobilized by hybridization of their messenger RNA end with homologous capture DNA spotted on a substrate surface. Enzymes thus immobilized retain activity proportional to the amount of capture DNA, allowing modulation of the relative activity of pathway enzymes. Entire pathways can thus be reconstructed and optimized in vitro from genomic information. We provide concept validation with the sequential reactions catalyzed by luciferase and nucleoside diphosphate kinase and further illustrate this method with the optimization of the five-step pathway for trehalose synthesis.

Catalysis↗

PubMed2004

Psychrophilicity of Bacillus psychrosaccharolyticus: a proteomic study.

Psychrophilicity of Gram-positive bacterium, Bacillus psychrosaccharolyticus was investigated in a proteomic approach. One hundred and thirty-one protein spots were analyzed by electrospray ionization-quadrupole-time of flight-tandem mass spectrometry and identified using an unpublished translated contig database as well as a nonredundant Gram-positive bacteria protein database from NCBI because of the lack of a genome sequence of this organism. Results focused on proteomic behavior of cold-response show that global up-regulation of metabolic functions and protective mechanism by stress responses might play a major role in psychrophilicity of B. psychrosaccharolyticus.

Bacillus↗

PubMed2003

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression.

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.

Aspartate Kinase↗

PubMed2002

Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum.

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.

Aspartic Acid↗