Biomedical subjects

Jae-Yean Kim

Publications and source records attributed to Jae-Yean Kim.

9 recordsLinked to original sources

PubMed2006

Engineering the lycopene synthetic pathway in E. coli by comparison of the carotenoid genes of Pantoea agglomerans and Pantoea ananatis.

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-D: -thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli.

Bacterial Proteins↗

PubMed2006

Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate.

To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichia coli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD(600) value of 15 for 72 h.

Carotenoids↗

PubMed2006

Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis.

Using the rice PEX14 cDNA as a bait in a yeast two-hybrid assay, two splice variants of the type I peroxisomal targeting signal (PTS1) receptor, OsPex5pL and OsPex5pS, were cloned from a pathogen-treated rice leaf cDNA library. The proteins were produced from a single gene by alternative splicing, which generated a full-length variant, OsPEX5L, and a variant that lacked exon 7, OsPEX5S. OsPex5pL contained 11 copies of the pentapeptide motif WXXXF/Y in its N-terminus, and seven tetratricopeptide repeats in its C-terminus. Expression of OsPEX5L and OsPEX5S predominantly occurred in leaf tissues, and was induced by various stresses, such as exposure to the pathogen Magnaporthe grisea, and treatment with fungal elicitor, methyl viologen, NaCl or hydrogen peroxide. The Arabidopsis T-DNA insertional pex5 mutant, Atpex5, which does not germinate in the absence of sucrose and was resistant to indole-3-butyric acid (IBA), was perfectly rescued by over-expression of OsPex5pL, but not by OsPex5pS. Using transient expression of OsPex5pL and OsPex5pS in the Atpex5 mutant, we show that OsPex5pL translocates both PTS1- and PTS2-containing proteins into the peroxisome by interacting with OsPex7p, whereas OsPex5pS is involved only in PTS1-dependent import in Arabidopsis.

Alternative Splicing↗

PubMed2005

A novel cell-to-cell trafficking assay indicates that the KNOX homeodomain is necessary and sufficient for intercellular protein and mRNA trafficking.

Cell-to-cell trafficking of regulatory proteins is a novel mechanism for communication during cell fate specification in plants. Although several developmental proteins traffic cell-to-cell, no signals that are both necessary and sufficient for this function in developmental proteins have been described. We developed a novel trafficking assay using trichome rescue in Arabidopsis. Fusion to KNOTTED1 (KN1) conferred gain-of-trafficking function to the cell-autonomous GLABROUS1 (GL1) protein. We show that the KNOX homeodomain (HD) is necessary and sufficient for intercellular trafficking, identifying a novel function for the HD as the minimal sequence required for trafficking of KN1 and its associated mRNA.

Arabidopsis↗

PubMed2005

Regulation of short-distance transport of RNA and protein.

The intercellular trafficking of proteins and RNAs has emerged as a novel mechanism of cell-cell communication in plant development. Plasmodesmata (PD), intercellular cytoplasmic channels, have a central role in cell-cell trafficking of regulatory proteins and RNAs. Recent studies have demonstrated that plants use either a selective or a non-selective PD trafficking pathway for regulatory proteins. Moreover, plants have developed strategies to regulate both selective and non-selective movement. Recent work has focused especially on integrating the recent understanding of the function and mechanisms of intercellular macromolecule movement through PD.

DNA, Plant↗

PubMed2003

Dispatch. Intercellular signaling: an elusive player steps forth.

Plasmodesmata play a central role in cell-to-cell communication in plants, allowing transport of regulatory proteins and mRNAs. A recent study has identified a specific protein that regulates the intercellular transport of macromolecules in plants, known as non-cell autonomous pathway protein 1.

Gene Expression Regulation↗

PubMed2003

Light-regulated translation mediates gated induction of the Arabidopsis clock protein LHY.

The transcription factor LHY and the related protein CCA1 perform overlapping functions in a regulatory feedback loop that is closely associated with the circadian oscillator of Arabidopsis: Overexpression of LHY abolished function of the circadian clock in constant light, but rhythmic expression of several circadian clock-regulated transcripts was observed under light- dark cycles. These oscillations correlated with high amplitude changes in LHY protein levels, caused by light-induced translation of the LHY transcript. Increases in LHY protein levels were also observed in light-grown wild-type plants, when light signals coincided with the circadian-regulated peak of LHY transcription at dawn. Unexpectedly, translational induction coincided with acute downregulation of LHY transcript levels. We suggest that the simultaneous translational induction and transcriptional repression of LHY expression play a role to narrow the peak of LHY protein synthesis at dawn and increase the robustness and accuracy of circadian oscillations. Strong phase shifting responses to light signals were observed in plants lacking function of LHY, CCA1 or both, suggesting that light-regulated expression of these proteins does not mediate entrainment of the clock to light-dark cycles.

Arabidopsis↗

PubMed2003

Developmental regulation and significance of KNOX protein trafficking in Arabidopsis.

Intercellular communication delivers critical information for position-dependent specification of cell fate. In plants, a novel mechanism for cell-to-cell communication involves the intercellular trafficking of regulatory proteins and mRNAs. The maize KNOTTED1 (KN1) gene acts non cell-autonomously in the maize leaf, and KN1 was the first plant protein shown to traffic cell-to-cell, presumably through plasmodesmata. We have compared the intercellular trafficking of green fluorescent protein (GFP) fusions of KN1 and Arabidopsis KN1-related homeobox proteins to that of the viral movement protein from turnip vein clearing tobamovirus. We show that there is specific developmental regulation of GFP approximately KN1 trafficking. GFP -- KN1 was able to traffic from the inner layers of the leaf to the epidermis, but not in the opposite direction, from epidermis to mesophyll. However, GFP or the GFP -- movement protein fusion moved readily out of the epidermis. GFP -- KN1 was however able to traffic out of the epidermal (L1) layer in the shoot apical meristem, indicating that KN1 movement out of the L1 was developmentally regulated. GFP -- KNAT1/BREVIPEDICELLUS and GFP -- SHOOTMERISTEMLESS fusions could also traffic from the L1 to the L2/L3 layers of the meristem. In a test for the functional significance of trafficking, we showed that L1-specific expression of KN1 or of KNAT1 was able to partially complement the strong shootmeristemless-11 (stm-11) mutant. However, a cell-autonomous GUS fusion to KN1 showed neither trafficking ability nor complementation of stm-11 when expressed in the L1. These results suggest that the activity of KN1 and related homeobox proteins is maintained following intercellular trafficking, and that trafficking may be required for their normal developmental function.

Arabidopsis↗

PubMed2002

MYB transcription factors in the Arabidopsis circadian clock.

The LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED (CCA1) genes encode closely related MYB transcription factors, which regulate circadian rhythms in Arabidopsis. LHY and CCA1 verify some of the properties of oscillator components, since (i) expression of their transcripts and protein exhibits circadian oscillations; (ii) their constitutive expression abolishes overt rhythmicity and (iii) they function as components of a negative transcriptional feedback loop. LHY and CCA1 have been proposed to function in conjunction with the pseudo response regulator TOC1, as components of the circadian oscillator. The regulation of their respective transcripts and protein levels in response to light signals suggests that these proteins may also mediate the regulation of circadian rhythms by light. This review discusses experimental evidence for these hypotheses.

Amino Acid Sequence↗