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Chunpoong is one of the most valuable cultivars of Panax ginseng C. A. MEYER, and is widely grown in Korea and China. Insertion/deletion (InDel) markers and single nucleotide polymorphism (SNP) markers are useful tools for marker-assisted selections. The SNP marker for determinate Chunpoong was previously developed from the nad7 gene of mtDNA by Wang et al. (2009) but was effective only on a limited range of cultivars. In this study, we studied the reasons for this limited application and developed new useful markers for application in Chunpoong-breeding programs. The new markers of InDel and SNP were designed in the major latex-like protein (MLP-like) gene which was highly expressed in 4-year-old Chunpoong expressed sequence tags (ESTs). To validate the marker in polymerase chain reaction (PCR), we used an InDel marker for identification of Chunpoong in the 70 Panax samples based on a double-blind test, and the success rate was 100%. For rapid and reliable assay of Chunpoong in numerous samples, we utilized an EvaGreen dye and melting curve method on real-time PCR. Furthermore, we designed an SNP marker that depended on the InDel region for more efficient detection of Chunpoong in real-time PCR. Compared with PCR-based assays, our Chunpoong SNP marker and real-time PCR assay offers a significant savings of time and labor in the analysis of large numbers of Chunpoong samples.

The expressed sequence tags (ESTs) referenced in this report are the first transcriptomes in a leaf from a half-shade ginseng plant. A cDNA library was constructed from samples of the leaves of 4-year-old Panax ginseng plants, which were cultured in a field. The 2,896 P. ginseng cDNA clones represent 1,576 unique sequences, consisting of 1,167 singletons and 409 contig sequences. BLAST comparisons of the cDNAs in GenBank's non-redundant databases revealed that 2,579 of the 2,896 cDNAs (89.1%) exhibited a high degree of sequence homology to genes from other organisms. The majority of the identified transcripts were found to be genes related with energy, metabolism, subcellular localization, and protein synthesis and transport. The chlorophyll a/b-binding protein ESTs in the ginseng leaf samples manifested a substantially higher level of expression than was observed in other plant leaves. The ESTs involved in ginsenoside biosynthesis were also identified and discussed.

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.

Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a posttranslational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.

Expressed sequence tags (ESTs) provide valuable tools that can be used to predict the genes involved in primary and secondary metabolite synthesis. To the best of our knowledge, ESTs have not yet been developed for Codonopsis. lanceolata, and therefore, the EST referenced in this report is the first transcript for C. lanceolata. A cDNA library was constructed using the roots of C. lanceolata plants that were grown in a field. The selected 881 cDNA clones were sequenced and processed with an EST pipeline, resulting in 636 unique sequences, including 517 singletons and 119 contig sequences. Using bioinformatics tools, 81% of the EST sequence was putatively annotated. Data for unique transcripts were mined from biological databases and functionally classified using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes Orthology, KEGG pathway maps, and protein family. The GO-based analyses were examined in terms of biotic and abiotic stress response, transport, cellular component organization, biogenesis, and secondary metabolic processes. The KEGG-based analyses of most transcripts were sorted by carbohydrate metabolism, energy metabolism, and biosynthesis of secondary metabolites. Five randomly-selected putative genes were used for an expression study using various stresses such as salt, H(2)O(2), salicylic acid, and methyl jasmonic acid. Mined data were organized in "The Codonopsis EST Database" (www.bioherbs.khu.ac.kr/Codonopsis).

Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.

Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. RRN9 encodes one of the UAF subunits. We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II). This change is reversible and does not involve a simple mutation. The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis. Thus, S. cerevisiae has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state.

Like most eukaryotic rDNA promoters, the promoter for rDNA in Saccharomyces cerevisiae consists of two elements: a core element, which is essential, and an upstream element, which is not essential but is required for a high level of transcription. We have demonstrated that stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF (upstream activation factor) which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. The three genes were originally defined by mutants that show specific reduction in the transcription of rDNA. These genes were cloned and characterized. Epitope tagging of RRN5 (or RRN9), combined with immunoaffinity purification was used to purify UAF, which complemented all three (rrn5, rrn9, and rrn10) mutant extracts. Using rrn10 mutant extracts, a large stimulation by UAF was demonstrated for template containing both the core element and the upstream element but not for a template lacking the upstream element. In the absence of UAF, the mutant extracts showed the same weak transcriptional activity regardless of the presence or absence of the upstream element. We have also demonstrated that UAF alone makes a stable complex with the rDNA template, committing that template to transcription. Conversely, no such template commitment was observed with rrn10 extracts without UAF. By using a series of deletion templates, we have found that the region necessary for the stable binding of UAF corresponds roughly to the upstream element defined previously based on its ability to stimulate rDNA transcription. Differences between the yeast UAF and the previously studied metazoan UBF are discussed.

Loss of function of any one of three UPF genes prevents the accelerated decay of nonsense mRNAs in Saccharomyces cerevisiae. We report the identification and DNA sequence of UPF3, which is present in one nonessential copy on chromosome VII. Upf3 contains three putative nuclear localization signal sequences, suggesting that it may be located in a different compartment than the cytoplasmic Upf1 protein. Epitope-tagged Upf3 (FLAG-Upf3) does not cofractionate with polyribosomes or 80S ribosomal particles. Double disruptions of UPF1 and UPF3 affect nonsense mRNA decay in a manner indistinguishable from single disruptions. These results suggest that the Upf proteins perform related functions in a common pathway.