• Title/Summary/Keyword: cDNA sequence

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Cloning of the 5'-end and Amplification of Full-Length cDNA of Genomic RNA of Lily symptomless virus

  • Park, Seon-Ah;Ryu, Ki-Hyun
    • The Plant Pathology Journal
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    • v.18 no.4
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    • pp.187-191
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    • 2002
  • This paper describes the cloning and sequence analysis of the 5'-terminal region and full-length cDNA production of genomic RNA of Lily symptomless virus (LSV), a Species Of the genus Carlavirus. A sing1e DNA band about 600 bp harboring the 5'-end of genomic RNA of the virus was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and was cloned for nucleotide sequence determination. Sequence analysis of selected RACE cDNA clones revealed that the LSV 5'non-translated region consists of 67 nucleotides long of AT rich stretch followed GC rich from the 5'-end. To produce full-length cDNA products for the viral genomic RNA, a set of LSV-specific primers could be designed based on the obtained sequence in this study and the known sequences of 3'-terminal region for the virus. Full-length cDNA copies of LSV, an 8.4 kb long, were directly amplified by the long-template RT-PCR technique from the purified viral genomic RNA samples. This full-length cDNA copies were analyzed by restriction mapping. The molecules produced in this study can be useful for the production of in vitro infectious cDNA clone, as well as, for the completion of genomic RNA sequence and genome structure for the virus.

Molecular Cloning of a cDNA Encoding a Ferritin Subunit from the Spider, Araneus ventricosus

  • Jin, Byung-Rea;Han, Ji-Hee;Kim, Seong-Ryul;Sohn, Hung-Dae
    • International Journal of Industrial Entomology and Biomaterials
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    • v.4 no.2
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    • pp.163-168
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    • 2002
  • We report for the first time the cDNA sequence encoding a ferritin subunit from the spiders Araneus ventricosus. The complete cDNA sequence of A. ventricosus ferritin subunit comprised 516 bp with 172 amino acid residues. The A. ventricosus ferritin subunit cDNA contained a conserved iron responsive element sequence in the 5 untranslated region. An alignment of the deduced protein sequence of the A. ventricosus ferritin subunit gene to that of other heavy chain ferritin molecules showed that A. ventricosus ferritin subunit is most similar to the great pond snail, Lymnaea stagnalis, ferritin with 70.2% of protein sequence identity.

Molecular Characterization of a Chinese cabbage cDNA, C-DH, Predominantly Induced by Water-Deficit Stress and Plant Hormone, ABA (수분부족 및 식물호르몬, ABA에 의하여 발현이 유도되는 배추의 C-DH cDNA에 대한 분자적 특성)

  • 정나은;이균오;홍창휘;정배교;박정동;이상열
    • Korean Journal Plant Pathology
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    • v.14 no.3
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    • pp.240-246
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    • 1998
  • A cDNA encoding desiccation-related protein was isolated from a flower bud cDNA library of Chinese cabbage (C-DH) and its nucleotide sequence was characterized. It contains 679 bp nucleotides with 501 bp open reading frame. The amino acid sequence of the putative protein showed the highest amino acid sequence homology (79 % identity) to dehydrin protein in Gossypium hirsutum. Also, the C-DH shares 48-52% amino acid sequence identity with the other typical dehydrin proteins in plant cells. When the amino acid sequence of their proteins were aligned, several peptide motifs were well conserved, of which function has to be solved. Particularly the C-DH contains 15 additional amino acids at its N-terminus. Genomic Southern blot analysis using the coding region of C-DH showed that the C-DH consists of a single copy gene in Chinese cabbage genome. The C-DH mRNA, whose transcript size is 0.7 kb, was expressed with a tissue-specific manner. It was highly expressed in seed, flower buds and low expression as detected in root, stem or leaf tissues of Chinese cabbage. And the transcript level of C-DH was significantly induced by the treatment of plant hormone, abscisic acid and water-deficit conditions.

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Isolation of a cDNA Encoding a Chloroplast Triosephosphate Isomerase from Strawberry

  • Kim, In-Jung;Lee, Byung-Hyun;Jinki Jo;Chung, Won-Il
    • Journal of Plant Biotechnology
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    • v.2 no.3
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    • pp.115-121
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    • 2000
  • A cDNA clone encoding chloroplast triosephosphate isomerase (TPI-cp) was isolated from strawberry fruit cDNA library. Sequence analyses indicated that the cDNA contains an open reading frame of 314 amino acids (33.5 kDa) composed of a transit peptide (59 amino acids) in amino terminal region and mature protein (255 amino acids). The existence of transit peptide in the deduced amino acid sequence implies that it encodes a chloroplast isoform. The protein sequence is more similar to other plant chloroplast isoforms than cytosolic isoforms. RNA blot analysis indicated that its expression is ubiquitous in examined five tissues, flowers, leaves, petioles, roots and fruits, and shows differential pattern according to fruit ripening. Genomic DNA blot analysis showed that TPI-cp is encoded by multiple genes in strawberry. Through sequence comparison and phylogenetic tree construction, TPI-cp is distinctively grouped into dicot and chloroplast isoforms.

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cDNA Cloning and Nucleotide Sequence Determination for VP7 Coding RNA Segment of Human Rotavirus Isolated in Korea (한국에서 분리된 사람 로타바이러스의 VP7 코딩 RNA 분절의 cDNA 합성과 염기서열 결정)

  • Kim, Young Bong;Kim, Kyung Hee;Yang Jai Myung
    • Korean Journal of Microbiology
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    • v.30 no.5
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    • pp.397-402
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    • 1992
  • The cDNA of RNA segment coding for VP7 of human rotavirus isolated from patient's stool at Seoul area was synthesized, amplified by polymerase chain reaction, field in with Klenow fragment of DNA polymerase I and cloned into pUC19. The cDNA sequence was determined and compared with that of VP7 coding RNA segments of group A rotaviruses isolates in foreign country. Over 90% sequence homology was found with serotyppe I sepcific WA1 and RE9 strains. Comparative analysis of the deduced amino acid sequences within the two variable regions (amino acid residue 87 through 101 and 208 through 221) with WA1 and RE9 strains also showed high degree of sequence similarity with each other.

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Isolation and Characterization of cDNA Encoding Pyridoxal Kinase from Ovine Liver

  • Lee, Hyun-Shik;Choi, Soo-Young;Kwon, Oh-Shin
    • BMB Reports
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    • v.32 no.5
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    • pp.502-505
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    • 1999
  • cDNA fragments of ovine liver pyridoxal kinase were amplified by PCR using degenerate oligonucleotide primers derived from partial amino acids sequences of the enzyme. Using PCR products as probes, several overlapping cDNA clones were isolated independently from an ovine liver and a human brain cDNA library. The largest cDNA clone for each was selected for sequence analysis. The ovine liver cDNA encodes a polypeptide of 297 amino acid residues with Mr of 32,925, whereas the human clone is comprised of an open reading frame encoding 312 amino acid residues with Mr of 35,102. The deduced sequence of the human brain enzyme is completely identical to that of human testes cDNA recently reported (Hanna et al., 1997). The ovine enzymes have approximately 77% sequence identity with the human enzyme although the two sequences are completely different in the N-terminus comprising 32 residues. This result suggests that pyridoxal kinase is highly homologous in mammalian species.

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Cloning and Nucleotide Sequencing of a Partial Glutamate Decarboxylase Gene from Arabidopsis thaliana cDNA Library (애기장대 cDNA library로부터 Glutamate Decarboxylase 유전자의 부분 클로닝 및 서열분석)

  • 오석흥;최원규;최동성
    • KSBB Journal
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    • v.16 no.1
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    • pp.36-40
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    • 2001
  • In order to study the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in plants, we cloned and sequenced a partial glutamate decarboxylase (GAD) cDNA from the Arabidopsis thaliana cDNA library, using primers targeted at highly conserved sequences of the petunia GAD gene. The cDNA fragment was inserted into TA cloning vector with T7 promoter and the recombinant plasmid obtained was used to transform E. coli. The plasmid DNA purified from the transformed E. coli was digested with EcoRI and the presence of the insert was confirmed. Nucleotide sequence analysis showed that the fragment is a partial Arabidopsis thaliana GAD gene and that the sequence showed 98% and 78% identity to the region of the putative Arabidopsis thaliana GAD sequences deposited in GenBank, Accession nos: U46665 and U10034, respectively. The amino acid sequence deduced from the partial Arabidopsis thaliana GAD gene showed 99% and 91% identities to the GAD sequences deduced from the genes of the U46665 and U10034, respectively. The partial cDNA sequence determined may facilitate the study of the molecular mechanism of GABA metabolism in plants.

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(CA/GT)n Simple Sequence Repeat DNA Polymorphism in Chlamydomonas reinhardtii (녹조류 Chlamydomonas reinhardtii의 (CA/GT)n Simple Sequence Repeat DNA 다형현상)

  • ;;Marvin W. FAWLEY
    • Korean Journal of Plant Tissue Culture
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    • v.24 no.2
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    • pp.113-117
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    • 1997
  • Simple sequence repeats (SSR) are widely dispersed throughout eukaryotic genomes, highly polymorphic, and easily typed using polymerase chain reaction (PCR). The objective of this study was to determine the polymorphism of different Chlamydomonas reinhartdtii strains and to determine the mode of inheritance of the SSR locus in Chlamydomonas. A genomic DNA library of C. reinhardtii was constructed and screened with a radiolabeled $(AC)_{11}$ probe for the selection of (CA/GT)n repeat clone. Selected clone was seqeuenced, and PCR primer set flanking (CA/GT)n sequence was constructed. PCR was used to specifically amplify the SSR locus from multiple isolates of C. reinhardtii. The locus was polymorphic in some of the C. reinhardtii isolates. However, the locus was amplified only 4 of 6 isolates of C. reinhardtii, not in other 2 isolates of C. reinhardtii, suggesting that this locus is not extensively conserved. A simple Mendelian inheritance pattern was found, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that this simple sequence repeat DNA polymorphism will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.

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Cloning and Sequence Analysis of Spinach (Spinacia oleracea L. cv Ace) Nitrate Reductase cDNA (시금치 nitrate reductase cDNA 클로닝 및 염기서열 분석)

  • Park, Nu-Ri;Chung, Jong-Bae;Park, Sang-Gyu
    • Applied Biological Chemistry
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    • v.45 no.3
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    • pp.129-133
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    • 2002
  • Suppression of nitrate accumulation in spinach and lettuce through foliar application of chitosan formula containing micronutrients is related with the increase of the nitrate reductase (NR) activity. If NR in spinach were highly expressed to increase the assimilatory activity, nitrate content could be reduced. For this, NR cDNA was cloned from the isolated mRNAs of spinach using reverse transcriptase-PCR. Nucleotide sequence of cloned spinach NR cDNA showed highly deduced amino acid sequence identity ($71{\sim}82%$) with other known plant NR genes. Only two nucleotide-base differences were observed in the cloned NR cDNA compared with that of the published spinach NR cDNA.

Isolation of cDNA Encoding Double-Stranded RNA Binding Protein (RBFII) (이중선RNA결합담백질(RBFII)의 cDNA분리)

  • 박희성
    • Journal of Life Science
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    • v.7 no.3
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    • pp.167-171
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    • 1997
  • As an initial effort to elucidate RNA: protein binding in a way to regulate translation initiation and phosphorylation, a cDNA encoding a double-stranded RNA binding factor (RBFII)was isolated from Hela ZAPII cDNA library by affinity screening using [$\alpha$$^{-32}$P] UMP-labeled HIV Rev-responsive element(RRE) RNA. The nucleotide sequence of RBF (or TRBP) cDNA except the 5’end. At the 5’end, This common ORF was fused in-frame to N-terminal residues of Lac-Z through a unique 138 nt sequence encoding 46residues in the case of RBFII and a 63 nt sequence encoding 21 residuces in the case of RBFI. The context of ATG appearing first in the sequences suggests that both these cDNA inserts are incomplete at the 5’end.

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