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- NOTE] A Rapid PCR-Based Approach for Molecular Identification of Filamentous Fungi
- Yuanyuan Chen , Bernard A. Prior , Guiyang Shi , Zhengxiang Wang
- J. Microbiol. 2011;49(4):675-679. Published online September 2, 2011
- DOI: https://doi.org/10.1007/s12275-011-0525-3
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- 13 Scopus
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Abstract
- In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates.
- The Influence of the Nucleotide Sequences of Random Shine-Dalgarno and Spacer Region on Bovine Growth Hormone Gene Expression
- Soon-Young Paik , Kyung Soo Ra , Hoon Sik Cho , Kwang Bon Koo , Hyung Suk Baik , Myung Chul Lee , Jong Won Yun , Jang Won Choi
- J. Microbiol. 2006;44(1):64-71.
- DOI: https://doi.org/2335 [pii]
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Abstract
- To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region (SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 & bGH14) which have different 5''-coding regions and were induced in E. coli BL21(DE3). Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized and the distance between the SD region and the initiation codon were fixed to nine bases in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA, respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing only bGH14 cDNA produced considerable levels of bGH in the range of 6.9% to 8.5% of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary structure of the bGH14 gene. When the free energy was calculated from the transcription initiation site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization prediction, the constructs with values below ‒26.3 kcal/mole (toward minus direction) were not expressed. The constructs with the original sequence of bGH cDNA also did not show any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary structure may be a major factor regulating bGH expression in the translation initiation process. Accordingly, the first stem-loop among two secondary structures present in the 5''-end region of the bGH gene should be disrupted for the effective expression of bGH.
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