• Title/Summary/Keyword: cDNA sequence

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Genetic Variations of Chicken MC1R Gene and Associations with Feather Color of Korean Native Chicken (KNC) 'Woorimatdag' (토종 '우리맛닭' 부계 및 실용계에서 MC1R 유전자 변이 및 모색과의 연관성 분석)

  • Park, Mi Na;Kim, Tae-Hun;Lee, Hyun-Jeong;Choi, Jin Ae;Heo, Kang-Nyeong;Kim, Chong-Dae;Choo, Hyo-Jun;Han, Jae-Yong;Lee, Taeheon;Lee, Jun-Heon;Lee, Kyung-Tai
    • Korean Journal of Poultry Science
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    • v.40 no.2
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    • pp.139-145
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    • 2013
  • There are several loci controlling the feather color of birds, of which one of the most studied is Extended black (E) encoding the melanocortin 1-receptor (MC1R). Mutations in this gene affect the relative distribution of eumelanin, phaeomelanin. The association of feather color and sequence polymorphism in the melanocortin 1-receptor (MC1R) gene was investigated using Korean native chicken H breed (H_PL) and 'Woorimatdag' commercial chickens (Woorimatdag_CC). In order to correlate gene mutation to Korean native chicken feather color, single nucleotide polymorphism (SNP) from MC1R gene sequence were investigated. A total of 307 birds from H_PL and Woorimatdag_CC were used. H_PL have black, black-brown feather color and Woorimatdag_CC have black with brown spots or brown with black spots. There are 6 SNPs in MC1R gene, locus T69C, C212T, A274G, G376A, G636A, T637C. 3 SNPs are nonsynonymous that change amino acid. But it is difficult to find correlation of feather color and polymorphisms. It will be needed to increase the population of Korean native chicken H breed and correlation analysis of genetic variation with feather colors.

Characterization of PR-10 gene derived from highly resistant '93-3-98' pear inoculated with scab (Venturia nashicola) (배 검은별무늬병(Venturia nashicola) 고도 저항성 '93-3-98' 유래 PR-10 유전자의 특성)

  • Chun, Jae An;Kim, Se Hee;Cho, Kang Hee;Kim, Dae Hyun;Choi, In Myong;Shin, Il Sheob
    • Journal of Plant Biotechnology
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    • v.42 no.1
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    • pp.25-33
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    • 2015
  • A PyrcpPR-10 gene with differentially expressed was isolated by using the suppression subtractive hybridization assay between '93-3-98' (highly resistant against scab caused by Venturia nashicola) and 'Sweat Skin'(highly susceptible) and analyzed the expression pattern according to organs and cultivars. The full length of PyrcpPR-10 was cloned as 743bp with 480bp's ORP, and was determined to encode a protein of 159 amino acid residues. On analyzing PyrcpPR-10 gene sequence compared with resistant and susceptible cultivars, 'Hwangsilri' (resistant), 'Gamcheonbae' (moderately resistant), 'Wonhwang' (moderately susceptible), 'Niitaka' (highly susceptible), and 'Sweat Skin' (highly susceptible) had identical gene sequence but 'Bartlett' (highly resistant) showed partly different sequences. The deduced amino acid sequence showed 64 ~ 98% homology and had the GXGGXG motif to known amino acid of other plants PR-10 by the BLAST X analysis. Among several organs or tissues, petal was showed highest expression level of PyrcpPR-10 gene followed by leaf, floral axis, bud, and bark. The expression level of PyrcpPR-10 gene was dramatically increased at 24 hr after inoculation in all cultivars and also up-regulated in accordance with resistant degree of cultivars. While resistant cultivars ('Bartlett', '93-3-98', and 'Hwangsilri') induced relatively high expression level of PyrcpPR-10 gene, susceptible cultivars ('Niitaka', and 'Sweat Skin') showed low expression level. PyrcpPR-10 gene is assumed that it is directly connected with defense mechanisms to pear scab.

Construction of Retrovirus Vector System for the Regulation of Recombinant hTPO Gene Expression (재조합 hTPO 유전자의 발현 조절을 위한 Retrovirus Vector System의 구축)

  • Kwon, Mo-Sun;Koo, Bon-Chul;Kim, Do-Hyang;Kim, Te-Oan
    • Reproductive and Developmental Biology
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    • v.31 no.3
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    • pp.161-167
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    • 2007
  • In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

$V_H$ Gene Expression and its Regulation on Several Different B Cell Population by using in situ Hybridization technique

  • Jeong, Hyun-Do
    • Journal of fish pathology
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    • v.6 no.2
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    • pp.111-122
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    • 1993
  • The mechanism by which $V_H$ region gene segments is selected in B lymphocyte is not known. Moreover, evidence for both random and nonrandom expression of $V_H$ genes in matured B cells has been presented previously. In this report, the technique of in situ hybridization allowed us to analyze expressed $V_H$ gene families in normal B lymphocyte at the single cell level. The analysis of normal B cells in this study eliminated any posssible bias resulting from transformation protocols used previously and minimized limitation associated with sampling size. Therefore, an accurate measure of the functional and expressed $V_H$ gene repertoire in B lymphocyte could be made. One of the most important controls for the optimization of in situ hybridization is to establish probe concentration and washing stringency due to the degree of nucleotide sequence similarlity between different families which in some cases can be as high as 70%. When the radioactive $C{\mu}$ and $V_{H}J558$ RNA probes are tested on LPS-stimulated adult spleen cells, $2{\sim}4{\times}106cpm$/slide shows low background and reasonable frequency of specific positive cells. For the washing condition. 40~50% formamide at $54^{\circ}C$ is found to be optimum for the $C{\mu}$. $V_{H}S107$ and $V_{H}J558$ probes. The analyzed results clearly demonstrate that the level of each different $V_H$ gene family expression is dependent upon the complexity or size of that family. These findings are also extended to the level of $V_H$ gene family expression in separated bone marrow B cells depend upon the various stage of differentiation and conclude no preferential utilization of specific $V_H$ gene family. Thus, the utilization of VH gene segments in B lymphocyte of adult BALB/c mice is random and is not regulated or changed during the differentiation of B cells.

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Molecular Characterization of Cinnamate 4-Hydroxylase gene in Red Hot Pepper (Capsicum annuum L.) (고추에서 분리한 Cinnamate 4-Hydroxylase 유전자의 분자생물학적 특성)

  • Kim Kye-Won;Ha Sun-Hwa;Cho Kang-Jin;Kim Eun-Ju;Lee Min-Kyung;Yu Jae-Ju;Kim Jong-Guk;Lee Shin-Woo
    • Journal of Plant Biotechnology
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    • v.32 no.3
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    • pp.167-173
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    • 2005
  • Three different cDNAS for cinnamate 4-hydroxylase (C4H) which are involved in the second step of the general phenylpropanoid pathway were isolated and designated as pc4h1 (1,755 bp), pc4h2 (1,655 bp), and pc4h3 (1,316 bp), respectively. The nucleotide sequence analysis revealed that both pc4h1 and pc4h2 clones encode polypeptides of 505 amino acids frame but pc4h3 clone was truncated at the 5'-end of coding region. The alignment of the deduced amino acid sequences showed that PC4H1 and PC4H2 are highly homologous (95.8% identical) with each other and contain three conserved domains which are typical in cytochrome P450 monooxygenase: proline-rich region, threonine-containing binding pocket for the oxygen molecule, and heme binding region. In addition, result of the phylogenic tree analysis revealed that both pepper C4Hs belong to Class 1. pc4h2 transcription was strongly induced in wounded fruit (400%) and root (200%) relative to its very low basal level but not in leaf or stem tissue. In case of pc4h1, the basal level of transcription was higher than pc4h2 but induction by wounding was lower in fruit and root while leaf and stem tissues did not respond to wounding. The basal level of pc4h3 transcripts was not, if any, detectable and response to wounding was not observed.

Etiological Properties and Coat Protein Gen Analysis of Potato Virus Y Occuring in Potatoes of Korea (우리나라 감자에 발생하는 PVY의 병원학적 특성 및 외피단백질 유전자 분석)

  • ;Richard M. Bostock
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 1995.06b
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    • pp.77-96
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    • 1995
  • To obtain basic informations for the improvement of seed potato production in Korea, some etiological properties of potato virus Y(PVY) distributed in the major seed potato production area(Daekwanryeong) were characterized, and the nucleotide and amino acid sequences of the coat protein gene of the PVY strains isolated were analyzed. PVY strains in Daekwonryeong, an alpine area, were identified to be two strains, PVYo and PVYN by symptoms of indicator plants, and their distribution in potato fields was similar. Major symptom on potato varieties by PVY was grouped as either mosaic alone or mosaic accompanied with veinal necrosis in the lower leaves. The symptom occurrence of the two symptoms was similar with Irish Cobbler, but Superior showed a higher rate of mosaic symptom than the other. The PVY strain which was isolated from potato cv. Superior showing typical mosaic symptoms produced symptoms of PVY-O on the indicator plants of Chenopodium amaranticolor, Nicotiana tabacum cv. Xanthi nc and Physalis floridana, but no symptom o Capsicum annum cv. Ace. Moreover, results from the enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies showed that the isolated PVY reacts strongly with PYV-O antibodies but does not react specifically with PVY-T antibodies. The purified virus particles were flexious with a size of 730$\times$11nm. On the basis of the above characteristics, the strain was identified to be a PVY-O and named as of PVY-K strain. The flight of vector aphids was observed in late May, however, the first occurrence of infected plants was in mid June with the bait plants surrounded with PVY-infected potato plants and early July with the bait plants surrounded with PVY-free potato plants. PVY infection rates by counting symptoms on bait plants (White Burley) were 1.1% with the field surrounded with PVY-free potato plants and 13.7% the fields surrounded with PVY-infected potato plants, showing the effect of infection pressure. The propagated PVY-K strain on tobacco(N. sylvestris) was purified, and the RNA of the virus was extracted by the method of phenol extraction. The size of PVY-K RNA was measured to be 9, 500 nucleotides on agarose gel electrophoresis. The double-stranded cDNAs of PVY-K coat protein(CP) gene derived by the method of polymerase chain reaction were transformed into the competent cells of E. coli JM 109, and 2 clones(pYK6 and pYK17) among 11 clones were confirmed to contain the full-length cDNA. Purified plasmids from pYK17 were cut with Sph I and Xba I were deleted with exonuclease III and were used for sequencing analysis. The PVY-K CP gene was comprised of 801 nucleotides when counted from the clevage site of CAG(Gln)-GCA(Ala) to the stop codon of TGA and encoded 267 amino acids. The molecular weight of the encoded polypeptides was calculated to be 34, 630 daltons. The base composition of the CP gene was 33.3% of adenine, 25.2% of guanine, 20.1% of cytosine and 21.4% of uracil. The polypeptide encoded by PVY-K CP gene was comprised of 22 alanines, 20 threonines, 19 glutamic acids and 18 glycines in order. The homology of nucleotide sequence of PVY-K CP gene with those of PVY-O(Japan), PVY-T(Japan), PVY-TH(Japan), PVYN(the Netherlands), and PVYN(France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. The amino acid sequence homology of the polypeptide encoded by PVY-K CP gene with those encoded by viruses was represented as 97.4%, 92.5%, 92.9%, 92.9%, and 98.5%, respectively.

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Genes of Wild Rice (Oryza grandiglumis) Induced by Wounding and Yeast Extract (상처와 효모추출물 처리조건에서 유발되는 야생벼 유전자 스크린)

  • Shin, Sang-Hyun;Im, Hyun-Hee;Lee, Jai-Heon;Kim, Doh-Hoon;Chung, Won-Bok;Kang, Kyung-Ho;Cho, Sung-Ki;Shin, Jeong-Sheop;Chung, Young-Soo
    • Journal of Life Science
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    • v.14 no.4
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    • pp.650-656
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    • 2004
  • Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.

Genomic Analysis of Satellite RNA of Cucumber mosaic virus-Paf Related with Mild Symptoms (Cucumber mosaic virus Paf 계통의 약독 병징과 관련된 satellite RNA의 유전자 해석)

  • Sung, Mi-Young;Jung, Min-Young;Lee, Sang-Yong;Ryu, Ki-Hyun;Choi, Jang-Kyung
    • Research in Plant Disease
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    • v.10 no.4
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    • pp.241-247
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    • 2004
  • Recently, we reported a satellite RNA (Paf-satRNA) which is encapsidated in a pepper isolate of Cucumber mosaic virus (CMV-Paf) regulated symptom attenuation of the helper virus. To characterize mild symptom domain of Paf-satRNA, a series of chimeric cDNAs of satRNAs were created by using full-length cDNA clones of Paf-satRNA and a Pep-satRNA, chlorosis-inducing satRNA in pepper plants, and analyzed for determinants of symptom attenuation. When compared the nucleotide sequences, the 3' and 5' terminal sequences of the two wild-type (wt) satRNAs contained relatively conserved sequences which are the typical to CMV satRNA. Ten bases insertions were found in PepY-satRNA, and two variable regions, 81st to 113th and 183rd to 265th from the 5'-end, were located in the middle parts of the satRNAs. To delineate the attenuated symptom-related domain for the Paf-satRNA, in vitro transcripts RNAs transcribed from the wt cDNAs and constructed chimeric cDNAs were combined with genomic RNAs, RNA1, RNA2 and RNA3, of CMV-Fny and inoculated onto Nicotiana benthamiana plants. These transcripts were fully infectious onto the N. benthamiana and infectivity was confirmed by the RT-PCR. Chimeric Paf(H/N)-satRNA and PepY(N/A)-satRNA as well as Paf-satRNA induced very mild mosaic or symptomless infection on N. benthamiana. By contrast, typical mosaic symptom and stunting of infected plants were induced when PepY-satRNA, PepY(H/N)-satRNA and Paf(N/A)-satRNA were infected to N. benthamiana. Paf-satRNA coinfected with CMV-Fny RNAs induced very mild to sympomless on pepper plants whereas PepY-satRNA-infected pepper expressed typical chlorosis mosaic symptom. Two kinds of chimeric mutants, Paf(H/N)-satRNA and PepY(N/A)-satRNA, induced mild mosaic or symptomless infection onto pepper plants, while PepY(H/N)-satRNA and Paf(N/A)-satRNA showed typical chlorosis and mosaic symptom with stunting. This results suggest that mild symptom-related domain for the Paf-satRNA was located on HpaI-NarI region.

Characterization of a Monoclonal Antibody Specific to Human Siah-1 Interacting Protein (인체 SIP 단백질에 특이적인 단일클론 항체의 특성)

  • Yoon, Sun Young;Joo, Jong Hyuck;Kim, Joo Heon;Kang, Ho Bum;Kim, Jin Sook;Lee, Younghee;Kwon, Do Hwan;Kim, Chang Nam;Choe, In Seong;Kim, Jae Wha
    • IMMUNE NETWORK
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    • v.4 no.1
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    • pp.23-30
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    • 2004
  • Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.

Analysis of Mitochondrial Gene Sequence in Etoxazole Resistant Two-Spotted Spider Mite, Tetranychus urticae (Etoxazole 저항성 점박이응애의 미토콘드리아 유전자 서열 분석)

  • Park, Sang-Eun;Koo, Hyun-Na;Yoon, Chang-Mann;Choi, Jang-Jeon;Kim, Gil-Hah
    • The Korean Journal of Pesticide Science
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    • v.16 no.1
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    • pp.54-61
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    • 2012
  • The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important pest species devastating many horticultural and ornamental crops and fruit trees. Difficulty in managing this mite is largely attributed to its ability to develop resistance to many important acaricides. Development of 3,700-folds resistance to etoxazole was found in the population of T. urticae collected from rose greenhouses in Buyeo, Chungnam Province in August 2000. This population has been selected for eleven years with etoxazole (over 500 times), and increased over 5,000,000-folds in resistance as compared with susceptible strain. Also, etoxazole-resistant strain was shown to be maternally inherited. The objective of this study was to determine whether resistance of T. urticae to etoxazole was linked with point mutations in the mitochondrial gene. DNA sequencing of cytochrome c oxidase subunit I (COX1), COX2, COX3, cytochrome b (CYTB), NADH dehydrogenase subunit 1 (ND1), ND2, ND3, ND4, ND5, and ND6 were analyzed by comparing two etoxazole-susceptible and etoxazole-resistant strains. As a result, differences were not detected between the nucleotide sequences of two strains within a mitochondrial gene.