- Quantitative Analysis of Expressed Genes in Aspergillus Oryzae by Sequencing 3'-directed cDNA Clones
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Hwang, Hyun Ah , Lee, Dong Whan , Kim, Jong Hwa , Lee, Tae Kyoo , Yang, Moon Sik , Chae, Keon Sang
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J. Microbiol. 1998;36(2):111-117.
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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.
- Quantitative analysis of gene expression pattern in aspergillus nidulans mycelia by sequencing of 3'-directed cDNA clones
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Park, Yoon Dong , Lee, Dong Whan , Lee, Seog Jae , Kim, Jong Hwa , Chae, Keon Sang
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J. Microbiol. 1996;34(1):23-29.
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Since sequencing of randomly selected cDNA clones has been known to be a powerful approach to obtain information on gene expression pattern in specific cells or tissues, we have analyzed a 3'-directed cDNA library of vegetative mycelia of A. nidulans by single-pass sequencing of hundreds of randomly selected clones. Sequencing of 292 cDNA clones yielded 209 gene signatures (GSs) probably representing highly or lesser expressed genes in the vegetative mycelia. Among the 209 GSs, 25 (79 cDNA clones) appeared more than once and 184 only once. One GS appeared at a highest frequency of 6 times, 2 GSs5 times, 4 GSs 4 times, a GSs 3 times and 16 GSs twice. About 6.6% GSs comprizing of 13 GSs showed alternative polyadenylation. Among 23 redundant GSs, three were common in both mycelia and sexual organs, and 22 were probably mycelia-specific. Out of 209 GSs, 36 were identified in GenBank showing of 70% or greater similaritis. Only six GSs were for A. nidulans genes, and 13 GSs were of DNA or genes encoding cytoplasmic or organellar proteins. This pattern is similar to those in the human HepG2 cell line and in human colonic mucosa, although very few genes for nuclear proteins and for protein synthesis were in A. nidulans.
- Cloning and mulecular characterization of a nprX gene of bacillus subtilis NS15-4 encoding a neutral protease
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Lee, Seung Hwan , Yoon, Ki Hong , Nam, Hee Sop , Oh, Tae Kwang , Lee, Seog Jae , Chae, Keon Sang
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J. Microbiol. 1996;34(1):68-73.
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An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (0RF) possibly encoding 521 amino acid polypeptide. The ORF used all codons expected two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a major transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6. 7. 8. 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. subtilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences form those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.
- Heat Inducible Expression of the CDC70 Gene under the Control of Heat Shock Element in Saccharomyces cerevisiae
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Lee, Seok Jae , Jahng, Kwang Yeop , Lee Young Hoon , Chae, Keon Sang
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J. Microbiol. 1995;33(3):196-200.
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In order to express the CDC70 gene of Saccharomyces cerevisiae by heat shock, we have designed heat inducibe hybrid promoters using the Drosophila melanogaster heat shock elements (HSEs). A 220 bp-long upstream fragment of the D. melanogaster hsp70 gene comprised of four HSEs was placed upstream of the putative proximal TATA box of the CDC70 gene. Hybrid promoters containing different fusion joints were tested for their ability to drive the CDC70 gene expression by heat shock. The results showed that the HSEs of D. melanogaster conferred the heat-induced CDC70 gene expression, but the heat inducibility was much lower than that in D. melanogaster.
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