Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) have a important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Recently, hnRNP A2/B1 can recognize m6A modifications on pre-mRNA or pre-miRNA and affect alternative splicing and miRNA processing in HeLa Cells. However, roles of hnRNP A2/B1 in various cells and tissues, especially in elary embryo development, are unclear. Here, we investigated the temporal and spatial expression patterns of hnRNPA2B13 during mammalian early embryo development. In mouse, hnRNPA2B1 was localized at the nucleus after 1-cell stage, however, hnRNPA2B1 was expressed after 2-cell stage in pig. Then, knockdown of hnRNP A2/B1 induced by RNA interference (RNAi) was used to analyze the effect of hnRNP A2/B1 in preimplantation develop in pigs. Knockdown of hnRNP A2/B1 delayed embryo development. Interestingly, ICM marker OCT4 and Sox2 was significantly decreased in blastocyst stage. mRNA expression show that transcription factors which is Pou5f1, Sox2, Nanog, Cdx2 and AP2γwas decreased the transcription levels without the changing of junction protein, ZO-1, occludin, and CXADR. Outgrowth results indicated that knock-down of hnRNPA2B1 embryos cannot format the colony. Knock-down of Methyltransferase like 3(METTL3) embryos mislocalized the hnRNPA2/B1 at the nucleus. In summary, the expression patterns of hnRNPA2/B1 differ between mouse and porcine embryos, and these differences may reflect species-specific functions during preimplantation embryo development. Our results suggested that hnRNPA2/B1 is necessary for newly synthesis of mRNA related with transcription factor, and early embryo development by the RNA epigenetic modification.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.

Cadherin gene, which is a receptor of the Bacillus thuringiensis toxins, was predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, Spodoptera exigua. The S. exigua cadherin gene (SeCad1) encodes 9 cadherin repeats and a tranmembrane domain. The SeCad1 gene was expressed in all developmental stage specifically in gut tissue by RT-PCR analysis. Expression of SeCad1 gene was suppressed by both injection and feeding of its specific dsRNASeCad1 in 5th instar larval stage. The suppression of SeCad1 expression did not significantly influence on pupal and adult development of S. exigua. However, the larval treated with dsRNASeCad1 (100 ng/larva) significantly reduced susceptibility to B. thuringiensis ssp. aizawai (3 × 106 CFU/larva). By contrast, the dsRNASeCad1-treated larvae did not show any change in susceptibility to B. thuringiensis ssp. krustaki (4 × 107 CFU/larva). These results suggest that SeCad1 is a specific receptor of Cry1A toxin from B. thuringiensis in S. exigua, but not Cry1C toxin.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

There has been a substantial controversy on the phylogenetic relationships among butterfly families and several competing phylogenetic hypothesis have been suggested. Among them the relationships of (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae) has been further widely accepted. In this study, we sequenced EF1-α, COI, and 16S rRNA from 62 species belonging to four true butterfly families, Papilionoidea. Phylogenetic analyses using BI, ML, and MP showed that the traditionally recognizable families were strongly supported as monophyletic groups, with the exception of Nymphalidae, wherein the singly included species of Danainae was placed as basal lineage of the Nymphalidae + Lycaenidae group. Phylogenetic relationships among families supported the sister group relationship of Nymphalidae and Lycaenidae strongly by all analyses and placed Papilionidae as the most basal lineage of the Papilionoidea. On the other hand, the relationships of Nymphalidae and Lycaenidae group to Pieridae were either unresolved, revealing trichotomy, or the relationships of (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae) as previously supported by several morphological and molecular works supported. Detailed within-family relationships among some genera also are shown in the presentation.

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The L-type amino acid transporter 1 (LAT1) is over-expressed to support cell growth in malignant tumors. The double stranded RNA-mediated RNA interference (RNAi) analysis can be in a wide variety of eukaryotes to induce the sequence-specific inhibition of gene expression. In this study, we examined the effect of LAT1 short interfering RNA (siRNA) on cell growth using siRNA of LAT1 in the KB human oral squamous cell carcinoma. In the RT-PCR analysis and western blot analysis, the siRNA of LAT1 inhibited expressions of LAT1 mRNA and protein. The uptake of [14C]L-leucine was inhibited by siRNA of LAT1. In the MTT assay, the siRNA of LAT1 inhibited the growth of the KB cells in the time-dependent manner, indicating that the growth inhibition of KB cell by the siRNA of LAT1 is induced by the blocking of neutral amino acid transport mediated by LAT1. These results suggest that the transport of neutral amino acids including several essential amino acids into the KB human oral squamous cell carcinoma is mediated mainly by LAT1. Further, the LAT1 would be a new target for the inhibition of cancer cell growth.

The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0). Cmr1 restricts the systemic spread of CMV-Fny, whereas this gene cannot block the spread of CMV-P1 to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection. To identify the key amino acids determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with CMV-GFP clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993) substitutions in CMV-P1 were required for complete loss of virulence in ‘Bukang’.

An isolate of barley yellow mosaic virus(BaYMV-HN) obtained from Haenam, Korea was compared with two BaYMV strains. BaYMV-Ⅱ-1 from Japan and BaYMV-G from Germany. The sequence of the 3'-terminal 3817nucleotides[excluding the poly (A) tail] of RNA 1 of BaYMV-HN was determined to start within a long open reading frame coding for a part of the NIa-VPg polymerase(26 amino acids). NIa-Pro polymerase (343 amino acids), NIb polymerase(528 amino acids) and the entire capsid protein(297 amino acids), which is followed by a noncoding region(NCR) of 235 nucelotides. In the partial ORFs, BaYMV-HN shows higher sequence homology with BaYMV-Ⅱ-1(99.5%) than BaYMV-G(92.7%). The 3' non-coding regions of BaYMV-HN(235nt) shows higher nucleotide sequence homology with BaYMV-G(235nt)(99.6%) than BaYMV-Ⅱ-1(231nt)(97.0%). The 3' NIa-Pro protein sequence of BaYMV-HN shows higher amino acid sequence homology with BaYMV-Ⅱ-1(95.0%) than BaYMV-G(93.6%), but, NIb protein sequence of BaYMV-HN shows same all amino acid sequence. The capsid protein sequence of BaYMV-HN(297aa) shows same with BaYMV-Ⅱ-1, and shows higher nucleotide sequence homology with BaYMV-UK (from United Kingdom)(97.3%) than BaYMV-G(96.9%) and G2(96.9%). Difference of capsid protein amino acid were 0-9 between the Japan, United Kingdom and Germany and were 2-6 between all Korean isolates. Many of the amino acid differences are located in the N-terminal regions of the capsid proteins from 1 to 74 amino acid positions.