Journal of Microbiology

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Generation of Infectious Transcripts from Korean Strain and Mild Mottle Strain of Potato Virus X: Sun Hee Choi , Ki Hyun Ryu; J. Microbiol. 2008;46(5):502-507. Published online October 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0078-2

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Full-length cDNAs of two different strains of Potato virus X (PVX-Kr and PVX-Mo) have been directly amplified by long template reverse transcription polymerase chain reaction (RT-PCR) using the 5’-end primer containing a SP6 or T7 RNA promoter sequence and the virus-specific 3’-end primer, and then constructed in plasmid vectors. Capped in vitro transcripts from cloned full-length cDNAs as well as those RTPCR amplicons proved to be infectious systemically on tobacco plants. Symptom expression on tobacco plants from PVX-Mo transcripts was faster and severer than that from PVX-Kr. In replication stability test of transcripts derived from PVX clones, progeny viruses showed stable replication according to sequencing through passages. This highly infectious transcript system from the full-length cDNA clones for PVX can be useful for recombinant molecules for functional analysis of viral proteins in plant-virus interaction study as well as for expression of foreign protein in planta.

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Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco
Eseul Baek, Ju-Yeon Yoon, Peter Palukaitis
Virology.2017; 510: 29. CrossRef
Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study
Fater Youssef, Armelle Marais, Chantal Faure, Pascal Gentit, Thierry Candresse
Virology Journal.2011;[Epub] CrossRef

Cloning and sequence determination of α-tubulin, β-tubline and Flagellar Calmodulin cDNAs of Naegleria gruberi: Choi, Youn Jeong , Park, Hye Lee , Lee, Joo Hun; J. Microbiol. 1995;33(1):40-45.

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Abstract PDF: Five cDNAs encoding two α-tubulins(α13 and α15), two β-tubulins(β5 and β5), and one flagellar calmodulin (Cal-1) were cloned from naegleria gruberi NB-1 and their nt sequences were determined. The α13(EMBL number X81049) and β1(EMBL number X81050) contained a complete open reading frame for α-tubulin and β-tubulin, respectively. The other three clones (α15, β5 and Cal-1) had a part of coding region and a 3’ untranslated region of the respective genes. The α13, β1 and Cal-1 had no homologous sequences in the coding regions and in the 3’ untranslated sequences. However, the α13 and β1 shared an eight nucleotide (AATACAAA) sequence in front of the respective initiation codons. The AATACAAA sequence was also found in N. gruberi strain NEG α-tubulin cDNA clone(αpT1) at the same position. Comparison of the α13 to the αpT1 revealed another stretch of identical sequence, which is 30 nts long, in the 5’ untranlated region.

Quantitative Analysis of Expressed Genes in Aspergillus Oryzae by Sequencing 3'-directed cDNA Clones: Hwang, Hyun Ah , Lee, Dong Whan , Kim, Jong Hwa , Lee, Tae Kyoo , Yang, Moon Sik , Chae, Keon Sang; J. Microbiol. 1998;36(2):111-117.

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Abstract PDF: Sequence analysis of randomly selected 3'-directed cKNA clones has been known to be one of the most powerful methods of examining the genes highly expressed in a tissue or cell type. We constructed a 3'-directed cDNA libraty from Aspergillus oryzae mycelia, and sequenced 345 randomly selected 3'-directed cDNA clones. Determined nucleotide sequences, not shorter than 30nt, were compared with one other to generate gene signatures (GSs) and were then compared with GenBank entries to analyze sequence similarity to known genes. A GS for the most highly expressed gene appeared six times, one GS five times, five GSs four times, five GSs three times and 22 GSs twice. In total, 324 clones yielded 268 GSs consisting of 34 redundant GSs appeaning at least twice and 234 solitary ones. Forty-three GSs showed similarities ranging from 60% to 99% with known sequences from Genbank. A considerable number of A. oryzae GSs mateched those obtained from the sexual structures of A. nidulans suggests that A. oryzae may not be phylogentically distant from A. nidulans and that A. oryzae may have a sexual life cycle from the ancient period.

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