Biomedical subjects

Byung-Dong Kim

Publications and source records attributed to Byung-Dong Kim.

14 recordsLinked to original sources

PubMed2006

Exploitation of pepper EST-SSRs and an SSR-based linkage map.

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR-ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum 'TF68' and C. chinense 'Habanero'. Dinucleotide SSRs and EST-SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST-SSRs were mapped onto the previously generated pepper linkage map, using 107 F(2) individuals from an interspecific cross of TF68 x Habanero. One-hundred and thirtynine EST-SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST-SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.

Capsicum↗

PubMed2006

Molecular cloning of a pepper gene that is homologous to SELF-PRUNING.

"Determinate" and "indeterminate" inflorescences in plants are controlled by a single recessive gene, for example, SELF-PRUNING (SP) in Solanum lycopersicum, TERMINAL FLOWER1 in Arabidopsis, CENTRORADI-ALIS in Antirrhinum, and CENTRORADIALIS-like gene in tobacco. Pepper (Capsicum annuum L.) is an indeterminate species in which shoots grow indefinitely. In this study, we cloned and characterized the pepper SP-like gene (CaSP). RT-PCR revealed that the CaSP transcript accumulates to higher levels in floral buds than in other organs. Comparison of genomic DNA and cDNA sequences from indeterminate and determinate pepper plants revealed the insertion of a single base in the first exon of CaSP in the determinate pepper plants. CaSP is annotated in linkage group 8 (chromosome 6) of the SNU2 pepper genetic map and showed similar synteny to SP in tomato. Transgenic tobacco plants overexpressing CaSP displayed late-flowering phenotypes similar to the phenotypes caused by overexpression of CaSP orthologs in other plants. Collectively, these results suggest that pepper CaSP is an ortholog of SP in tomato.

Amino Acid Sequence↗

PubMed2006

Oral immunogenicity of potato-derived HBsAg middle protein in BALB/c mice.

The antibodies to preS2 synthetic peptides have been probed to neutralize hepatitis B virus (HBV), and also the addition of preS2 sequence could enhance the antibody response compared with a conventional vaccine in the non- and low responders. Previously, we generated transgenic potatoes expressing middle protein, which contains additional 55 amino acid preS2 region at the N-terminus of the S protein, of HBV to determine the feasibility of developing a plant-delivered HBV vaccine. In this study, we monitored the immune response after induction of immunoglobulin by boosting and assessed the efficacy of the mucosal immune response with regard to generate IgA antibodies. The HBsAg middle protein expressed in our transgenic potatoes was well immunized at low antigenic quantities in mice and the induced anti-S or anti-preS2 antibodies were sustained for the whole period without decrease. Orally delivery of plant-derived HBsAg middle protein to mice resulted in fecal anti-S or anti-preS2 as well as serum IgG. In addition, we used antibodies induced from the immunized mice with the potato-derived rHBsAg in competition assay as competitors to confirm the binding ability of preS2 antibodies to surface antigen of hepatitis virus. Anti-preS2 antibodies induced from immunized mice with transgenic potatoes effectively competed with anti-preS2 murine antibody H8 as expected. From these results, the inclusion of preS2 antigen to HBV plant vaccine may provide additional protective immunity in the HBV prevention.

Animals↗

PubMed2006

Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.).

Cytoplasmic male sterility (CMS) in plants, which is due to failure to produce functional pollen, is a maternally inherited trait. Specific nuclear genes that sup-press CMS, termed fertility restorer (Rf) genes, have been identified in several plants. In this study, Rf-linked molecular markers in pepper (Capsicum annuum L.) were detected by bulked segregant analysis of eight amplified fragment length polymorphisms (AFLPs). Only AFRF8 was successfully converted to a cleaved amplified polymorphic sequence (CAPS) marker. This was named AFRF8CAPS and genotype determination using it agreed with that obtained with the original AFRF8. A linkage map with a total size of 54.1 cM was constructed with AFRF8CAPS and the seven AFLP markers using the Kosambi function. The AFRF8CAPS marker was shown to be closest to Rf with a genetic distance of 1.8 cM. These markers will be useful for fast and reliable detection of restorer lines during F(1) hybrid seed production and breeding programs in pepper.

Capsicum↗

PubMed2006

A differentially expressed proteomic analysis in placental tissues in relation to pungency during the pepper fruit development.

Using proteomic analysis including 2-DE, image analysis, and protein identification with LC-MS/MS, an investigation aimed at a better understanding of the differentially expressed proteins and/or gene products was carried out with total cell extracts from placental tissues in nonpungent (Capsicum annuum cv. Saeng-Ryeog #213) and pungent peppers (C. annuum cv. Saeng-Ryeog #211). Mobilization of the most abundant proteins, which were on the gels of pH ranges of 4-7, 4.5-5.5, 5.5-6.7, and 6-9, and showed very similar profiles in the two tissues, revealing approximately 2600 protein spots consisting of 1200 on pH 4-7, 600 on 4.5-5.5, 550 on 5.5-6.7, 250 on 6-9. Of these, 37 protein spots, which appeared in only pungent tissues but not in nonpungent tissues or markedly increased in their staining intensities on the gels from pungent tissue, were selected, excised, in-gel trypsin digested, and analyzed by LC-ESI-MS/MS. Peptide MS/MS data were searched against publicly available protein and EST databases, and 22 proteins were identified. Based on this result, we tested and compared the differential expression during fruit development on the 2-DE gels with total cell extracts from placental tissues of pungent and nonpungent peppers at an interval of 10 days from 10 to 40 days after flowering. In addition, this differential protein expression was further confirmed for some subsets of candidates by Northern-blot analysis with RNA samples from placental tissues harvested from each pepper fruit at the same sampling intervals. In this study, the physiological implications, revealed from the experimental data in the levels of proteome and transcripts, are discussed in the context of a complex biosynthesis network of capsaicinoids in pepper cells responsive to pungency.

Amino Acid Sequence↗

PubMed2006

A functional screen to characterize the secretomes of eukaryotic pathogens and their hosts in planta.

Complex suites of proteins that are secreted by plants and phytopathogens into the plant apoplast play crucial roles in surveillance, assault, defense, and counter-defense. High-throughput genome-scale strategies are being developed to better understand the nature of these "secretomes" and the identity of pathogen-derived effector proteins that subvert plant defenses and promote pathogenicity. Although combined bioinformatic and experimental approaches recently have provided comprehensive coverage of secreted proteins from bacterial phytopathogens, far less is known about the secretomes and batteries of effectors of eukaryotic phytopathogens; notably fungi and oomycetes. The yeast secretion trap (YST) represents a potentially valuable technique to simultaneously target pathogen and host secretomes in infected plant material. A YST screen, using a new vector system, was applied to study the interaction between tomato (Solanum lycopersicum) and the oomycete Phytophthora infestans, revealing sets of genes encoding secreted proteins from both pathogen and host. Most of those from the oomycete had no identifiable function and were detectable in planta only during pathogenesis, underlining the value of YST as a tool to identify new candidate effectors and pathogenicity factors. In addition, the majority of the P. infestans proteins had homologs in the genomes of the related oomycetes R. sojae and P. ramorum.

Algal Proteins↗

PubMed2005

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.).

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.

3' Untranslated Regions↗

PubMed2005

The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.).

The mitochondrial atp6 gene in male fertile (N) and CMS (S) pepper has previously been compared and was found to be present in two copies (Kim et al. in J Kor Soc Hort Sci 42:121-127 2001). In the current study, these atp6 copies were amplified by an inverse PCR technique, and the coding region as well as the 5' and 3' flanking regions were sequenced. The atp6 copies in CMS pepper were detected as one intact gene and one pseudogene, truncated at the 3' coding region. When the atp6 genes in pepper were compared to other plant species, pepper, potato, and petunia all possessed a sequence of 12 identical amino acids at the 3' extended region, which was considered a hallmark of the Solanaceae family. Northern blot analysis showed differences in mRNA band patterns between CMS and restorer lines, indicating that atp6 gene is one of the candidates for CMS in pepper.

Amino Acid Sequence↗

PubMed2005

Use of SSR markers to complement tests of distinctiveness, uniformity, and stability (DUS) of pepper (Capsicum annuum L.) varieties.

This study was carried out to assess the potential of SSR markers for variety identification by comparing SSR markers and morphological traits in tests of distinctiveness, uniformity, and stability (DUS) of pepper (Capsicum annuum L.) varieties. Twenty-seven SSR markers were polymorphic in 66 pepper varieties, revealing a total of 89 alleles. Average polymorphism information content (PIC) value was 0.529, ranging from 0.03 to 0.877. Cluster analysis of the band patterns separated the varieties into three groups corresponding to varietal types. Morphological trait-based clustering showed some degree of similarity to dendrogram topologies based on the SSR index. However, no significance correlation was found between the SSR and morphological data. SSR markers could be used to complement a DUS test of a candidate variety and to select complimentary varieties by pre-screening existing varieties in the context of protecting new varieties of pepper.

Capsicum↗

PubMed2005

A coupled yeast signal sequence trap and transient plant expression strategy to identify genes encoding secreted proteins from peach pistils.

Many developmental processes and induced plant responses have been identified that are directly or indirectly influenced by wall-localized, or apoplastic, molecular interactions and signalling pathways. The yeast-based signal sequence trap (YSST) is a potentially valuable experimental tool to characterize the proteome of the wall and apoplast, or 'secretome', although few studies have been performed with plants and to date this strategy has not been coupled with a subsequent analysis to confirm extracellular localization of candidate proteins in planta. This current report describes the use of the YSST, together with transient expression of a selection of identified proteins as fusions with the reporter GFP, focusing on the complex extracellular interactions between peach (Prunus persica) pollen and pistil tissues. The coupled YSST and GFP localization assay was also used to confirm the extracellular localization of a recently identified pistil-specific basic RNase protein (PA1), as has been observed with S-RNases that are involved in self-incompatibility. This pilot YSST screen of pollinated and unpollinated pistil cDNAs revealed a diverse set of predicted cell wall-localized or plasma membrane-bound proteins, several of which have not previously been described. Transient GFP-fusion assays and RNA gel blot analyses were used to confirm their subcellular localization and to provide further insights into their expression or regulation, respectively. These results demonstrated that the YSST strategy represents an effective means either to confirm the extracellular localization of a specific candidate secreted protein, as demonstrated here with PA1, or to conduct a screen for new extracellular proteins.

Flowers↗

PubMed2005

Non-pungent Capsicum contains a deletion in the capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers.

The capsaicinoid synthetase (CS) gene cosegregated perfectly with the C locus, which controls the presence of pungency, in 121 F2 individuals from a cross between 'ECW123R' and 'CM334', both of Capsicum annuum. We concluded that CS and C are tightly linked. Sequence analysis of the genes of four pungent and four non-pungent pepper lines showed that the non-pungent peppers had a 2,529 bp-deletion in the 5' upstream region of CS. We have developed molecular markers of the C locus to detect pungency at the seedling stage. Based on the deleted sequence, we developed five SCAR markers, two of them being codominant. These SCAR markers will be useful for easy, accurate, and early detection of non-pungent individuals in breeding programs.

Breeding↗

PubMed2005

Digging deeper into the plant cell wall proteome.

The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function.

Cell Wall↗

PubMed2005

FESD: a Functional Element SNPs Database in human.

We have created the Functional Element SNPs Database (FESD) that categorizes functional elements in human genic regions and provides a set of single nucleotide polymorphisms (SNPs) located within each area. In the FESD, the human genic regions were divided into 10 different functional elements, such as promoter regions, CpG islands, 5'-untranslated regions (5'-UTRs), translation start sites, splice sites, coding exons, introns, translation stop sites, polyadenylation signals and 3'-UTRs, and subsequently, all the known SNPs were assigned to each functional element at their respective position. With the FESD web interface, users can select a set of SNPs in the specific functional elements and get their flanking sequences for genotyping experiments, which will help in finding mutations that contribute to the common and polygenic diseases. A web interface for the FESD is freely available at http://combio.kribb.re.kr/ksnp/resd/.

Databases, Nucleic Acid↗

PubMed2005

The Pun1 gene for pungency in pepper encodes a putative acyltransferase.

Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

Acyltransferases↗