Chul Wook Kim

Functional characterization of ObgC in ribosome biogenesis during chloroplast development

The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT.

RNA-Seq approach for genetic improvement of meat quality in pig and evolutionary insight into the substrate specificity of animal carbonyl reductases.

Changes in meat quality traits are strongly associated with alterations in postmortem metabolism which depend on genetic variations, especially nonsynonymous single nucleotide variations (nsSNVs) having critical effects on protein structure and function. To selectively identify metabolism-related nsSNVs, next-generation transcriptome sequencing (RNA-Seq) was carried out using RNAs from porcine liver, which contains a diverse range of metabolic enzymes. The multiplex SNV genotyping analysis showed that various metabolism-related genes had different nsSNV alleles. Moreover, many nsSNVs were significantly associated with multiple meat quality traits. Particularly, ch7:g.22112616A>G SNV was identified to create a single amino acid change (Thr/Ala) at the 145th residue of H1.3-like protein, very close to the putative 147th threonine phosphorylation site, suggesting that the nsSNV may affect multiple meat quality traits by affecting the epigenetic regulation of postmortem metabolism-related gene expression. Besides, one nonsynonymous variation, probably generated by gene duplication, led to a stop signal in porcine testicular carbonyl reductase (PTCR), resulting in a C-terminal (E281-A288) deletion. Molecular docking and energy minimization calculations indicated that the binding affinity of wild-type PTCR to 5α-DHT, a C21-steroid, was superior to that of C-terminal-deleted PTCR or human carbonyl reductase, which was very consistent with experimental data, reported previously. Furthermore, P284 was identified as an important residue mediating the specific interaction between PTCR and 5α-DHT, and phylogenetic analysis showed that P284 is an evolutionarily conserved residue among animal carbonyl reductases, which suggests that the C-terminal tails of these reductases may have evolved under evolutionary pressure to increase the substrate specificity for C21-steroids and facilitate metabolic adaptation. Altogether, our RNA-Seq revealed that selective nsSNVs were associated with meat quality traits that could be useful for successful marker-assisted selection in pigs and also represents a useful resource to enhance understanding of protein folding, substrate specificity, and the evolution of enzymes such as carbonyl reductase.

Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate RT-qPCR results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper. Combination analysis of these three programs showed that the stable and appropriate reference genes are PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. Because the four pig breeds had different suitable reference genes, the selection of appropriate reference genes is essential in RT-qPCR analyses. Taken together, our data could help to select reliable reference genes for the normalization of expression levels of various target genes in pigs.

Transfer of Intracellular HIV Nef to Endothelium Causes Endothelial Dysfunction

With effective antiretroviral therapy (ART), cardiovascular diseases (CVD) are emerging as a major cause of morbidity and death in the aging HIV-infected population. To address whether HIV-Nef, a viral protein produced in infected cells even when virus production is halted by ART, can lead to endothelial activation and dysfunction, we tested Nef protein transfer to and activity in endothelial cells. We demonstrated that Nef is essential for major endothelial cell activating effects of HIV-infected Jurkat cells when in direct contact with the endothelium. In addition, we found that Nef protein in endothelial cells is sufficient to cause apoptosis, ROS generation and release of monocyte attractant protein-1 (MCP-1). The Nef protein-dependent endothelial activating effects can be best explained by our observation that Nef protein rapidly transfers from either HIV-infected or Nef-transfected Jurkat cells to endothelial cells between these two cell types. These results are of in vivo relevance as we demonstrated that Nef protein induces GFP transfer from T cells to endothelium in CD4.Nef.GFP transgenic mice and Nef is present in chimeric SIV-infected macaques. Analyzing the signal transduction effects of Nef in endothelial cells, we found that Nef-induced apoptosis is mediated through ROS-dependent mechanisms, while MCP-1 production is NF-kB dependent. Together, these data indicate that inhibition of Nef-associated pathways may be promising new therapeutic targets for reducing the risk for cardiovascular disease in the HIV-infected population.

RNA-seq analysis and de novo transcriptome assembly of Jerusalem artichoke (Helianthus tuberosus Linne).

Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

Construction of a conditional lethal Salmonella mutant via genetic recombination using the ara system and asd gene

In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned into a plasmid carrying a p15A replication origin to create the recombinant plasmid pMMP55. The growth of Salmonella MMP10 harboring pMMP55 was dependent on the presence of arabinose. In the presence of arabinose, the Asd deficiency due to chromosomal deletion of asd in the Salmonella host was complemented by the asd gene transcribed and translated under the P(araBAD) promoter and araBAD Shine-Dalgarno (SD) sequence in pMMP55. Growth inhibition of the strain was demonstrated by arabinose depletion in M9 minimal medium, indicating that the strain were unable to grow in an arabinose-limited environment. In addition, the analysis of a 50% lethal dose (LD50) using mice revealed that the strain MMP10 exhibited attenuation by approximately 100-fold relative to that of the unmodified strain. In conclusion, these data suggest that the araC P(araBAD)::asd system developed in this study can be used to construct conditional lethal Salmonella mutants for application as safe, live-attenuated Salmonella vaccines.

Abacavir, didanosine and tenofovir do not induce inflammatory, apoptotic or oxidative stress genes in coronary endothelial cells.

BACKGROUND The use of abacavir and didanosine in HAART has been associated with an increased risk of myocardial infarction in HIV-infected patients. The aim of this study was to address the development of endothelial dysfunction in cultivated coronary artery endothelial cells (HCAECs) in response to abacavir, didanosine and tenofovir. We examined the impact of these drugs on the expression levels of the proinflammatory, oxidative stress and apoptosis regulating genes in HCAECs. METHODS We tested gene and protein expression changes in HCAECs in response to abacavir, didanosine and tenofovir using quantitative real-time reverse transciptase PCR, FACS and ELISA. The assessed genes/proteins included the proinflammatory molecules VCAM-1, ICAM-1, MCP-1, RANTES and IL-6. In addition, we assessed the gene expression of the intracellular reactive oxygen producing NADPH oxidase subunit gp91(PHOX) and the apoptosis regulating molecules Bcl-2 and BAD. RESULTS Exposure of HCAECs to abacavir, didanosine and tenofovir resulted in no statistically significant changes in any of the tested genes/proteins at any time point or at any concentration. CONCLUSIONS We found no evidence that abacavir, didanosine or tenofovir had direct in vitro effects on coronary endothelial cell gene transcription and protein expression of the selected mediators. If abacavir or didanosine increase cardiovascular risk, it is likely not through the direct endothelial activation pathways tested in these experiments. However, further studies are needed to completely exclude the toxicity of abacavir or didanosine on endothelial cells.

Identification of Differentially Expressed Genes Associated with Litter Size in Berkshire Pig Placenta

Improvement in litter size has become of great interest in the pig industry because fecundity is directly related to sow reproductive life. Improved reproduction has thus been achieved by elucidating the molecular functions of genes associated with fecundity. In the present study, we identified differentially expressed genes (DEGs) via transcriptomic analysis using RNA-sequencing (RNA-Seq) in Berkshire pig placentas from larger (LLG, mean litter size >12) and smaller (SLG, mean litter size < 6.5) litter size groups. In total 588 DEGs were identified (p < 0.05, > 1.5-fold change), of which 98 were upregulated, while 490 were downregulated in the LLG compared with the SLG. Gene Ontology (GO) enrichment was also performed. We concluded that 129 of the 588 DEGs were closely related to litter size according to reproduction related genes selected based on previous reports, as 110 genes were downregulated and 19 upregulated in the LLG compared with the SLG. RT-qPCR utilizing specific primers targeting the early growth response 2 (EGR2), pheromaxein c subunit (PHEROC) and endothelial lipase (LIPG) genes showed high accordance with RNA-Seq results. Furthermore, we investigated the upstream regulators of these three genes in the placenta. We found that WNT9B, a Wnt signaling pathway molecule, and IL-6, known inducers of EGR2 and LIPG, respectively, were significantly increased in LLG compared with SLG. We believe that the induction of IL-6 and LIPG may play an important role in increasing nutrition supply through the placenta from the sow to the piglet during gestation. These results provide novel molecular insights into pig reproduction.

Single-Nucleotide Polymorphisms in Pig EPHX1 Gene are Associated with Pork Quality Traits

Epoxide hydrolase 1 (EPHX1) plays an important role in both the activation and detoxification of exogenous chemicals. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the highest level of EPHX1 expression occurred in Berkshire liver, which is an organ that plays a key role in detoxification. We examined EPHX1 SNPs to analyze effect on increased expression of EPHX1 gene in Berkshire liver by total of 192 pigs of a pure Berkshire line (males = 97; females = 95). As a result, two nonsynonymous SNPs (nsSNPs) of EPHX1 were found from c.685T>G and c.776C > T, and located in 5th and 6th exons, respectively, which constitute the A/b hydrolase 1 domain of epoxide hydrolase. The nsSNP c.685T > G was significant differences in meat color, protein content, collagen content, and pH24 hr. Especially, T and G alleles of the nsSNP c.685T > G were significantly associated with CIE a*/CIE b* and protein content/pH24 hr, respectively. The nsSNP c.776C > T was significant differences in drip loss and protein content. Among meat quality traits to associate with SNPs, the protein content was only significantly associated with sex. Therefore, it is suggested that nsSNP c.685T > G in EPHX1 gene is a potential to apply as appropriate DNA markers for improvement of porcine economic traits.

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H+-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.