• Journal of Microbiology
• /
• 제38권4호
• /
• pp.230-238
• /
• 2000

Class III chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class III chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5'flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• 미생물학회지
• /
• 제38권4호
• /
• pp.230-230
• /
• 2002

Class Ⅲ chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class Ⅲ chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5′flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Genomics & Informatics
• /
• 제7권4호
• /
• pp.181-186
• /
• 2009

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans-splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

• Journal of Microbiology
• /
• 제41권3호
• /
• pp.239-247
• /
• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

• Archives of Pharmacal Research
• /
• 제22권6호
• /
• pp.542-548
• /
• 1999

The UL47 gene (b 60390-b 60388) located in the unique long region of the human cytomegalovirus (HCMV) AD169 strain genome was analyzed RNA mapping. Northern blot analysis showed that the UL47 gene was expressed at late times after infection (72 h postinfection). The 9.7-kb transcript was expressed in the infected cells but not in phosphonoformate-treated cells at 72 hpi, indicating that the UL47 gene was only expressed at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer extension and RNase protection analysis were performed. Primer extension analysis revealed that the transcription initiation site of UL47 was located in 27 bp downstream (b 60323) of the TATA box motif. The sizes of UL47 ORF (approximately 2.9-kb) and UL48 ORF (approximately 6.7-kb) deduced from computer sequence analysis suggest that the expressed 9.7-kb transcript of UL47 uses the 3'-end polyadenylation signal of Ul48. The result of RNase protection determined that the 3'-end of UL47 RNA utilized the 3'-end polyadenylation signal of UL48, which is located in HCMV genome b 70082.

• Journal of Microbiology and Biotechnology
• /
• 제1권4호
• /
• pp.256-260
• /
• 1991

In B. subtilis, $\alpha$-amylase synthesis is regulated by amyR located directly on the upstream of amyE. Three different amyR alleles have been reported, amyR1, amyR2 and amyR3. Strains bearing the gra-10 mutation which confers derepression for catabolite repression has GlongrightarrowA transition mutation at ＋5 of amyR1. S1 nuclease mapping demonstrated that transcription initiated at 8 bases downstream from the -10 region of putative E$\sigma^{A}$ promoter P1 in amyR1 and gra-10. In amyR2, the major transcription initiatd at the same place and the minor, 10 bases downstream from -10 of P2. The transcript from P2 contributed approximately 15-20% of total amyE mRNA. S1 nuclease protection experiment indicated that amyE mRNA levels corresponded to the rate of synthesis assumed by specific activities of $\alpha$-amylase in culture supernatants, suggesting that $\alpha$-amylase synthesis is regulated at the level of transcription.n.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권11호
• /
• pp.1399-1404
• /
• 2010

We have created a Bovine Genome Database, an integrated genomic resource for Bos taurus, by merging bovine data from various databases and our own data. We produced 55,213 Korean cattle (Hanwoo) ESTs from cDNA libraries from three tissues. We concentrated on genomic information based on Hanwoo transcripts and provided user-friendly search interfaces within the Bovine Genome Database. The genome browser supported alignment results for the various types of data: Hanwoo EST, consensus sequence, human gene, and predicted bovine genes. The database also provides transcript data information, gene annotation, genomic location, sequence and tissue distribution. Users can also explore bovine disease genes based on comparative mapping of homologous genes and can conduct searches centered on genes within user-selected quantitative trait loci (QTL) regions. The Bovine Genome Database can be accessed at http://bgd.nabc.go.kr.

• Journal of Microbiology
• /
• 제38권4호
• /
• pp.239-244
• /
• 2000

The cats gene encodes the major catalase in Sreptomyces coelicolor, whose production increases upon H$_2$O$_2$treatment. Besides the previously identified primary promoter (catApl), a minor promoter (catAp2) was newly assigned by S1 nuclease mapping. The catAp2 transcript was observed transiently upon entry into the stationary phase in liquid culture and upon differentiation on solid plates, whereas the level of catApl transcription did not chance significantly during this growth transition. ThecatApl promoter was transcribed by the major vegetative RNA polymerase holoenzyme containing $\sigma$$\^$HrdB/, whereas the catAp2 was transcribed in vitro by the holoenzyme containing $\sigma$$\^$R/ that is activated under oxidative conditions. The cia-element regulating the H$_2$O$_2$-inducibility of catApl was identified within the 23 bp inverted repeat sequence located between -65 and -43 of the catApl promoter. We roamed this sequence HRE (H$_2$O$_2$-responsive Element). The distal half of the inverted repeat was more crucial for H$_2$O$_2$-dependent induction of the catApl transcript than the proximal half. HRE most likely serves as a binding site for the H$_2$O$_2$-responsive repressor CatR.

• 대한소아치과학회지
• /
• 제33권2호
• /
• pp.304-310
• /
• 2006

본 연구는 치주질환 주요 병인균주 중의 하나인 P. intermedia를 대상으로 하여, 이 균주에서의 hemin 결합 단백질 유전자를 분리하고 염기서열을 결정하기 위하여 수행되었다. 본 연구에서는 약 5,000개의 recombinant colony들을 스크리닝하여 hemin 결합 단백질 유전자를 포함하고 있는 것으로 여겨지는 1개의 클론(pHem1)을 확인하였다. Restriction enzyme mapping 결과 pHem1의 insert DNA의 크기는 약 2.5kb이었으며 P. intermedia chromosomal DNA 내에는 hemin 결합 단백질 유전자가 single copy로 존재하였고, transcript의 크기는 약 1.8kb이었다. 분리한 pHem1 유전자는 1개의 ORF를 가지고 있으며, ORF의 크기는 2,550bp로 약 850개의 아미노산 폴리펩타이드로 구성되어 있다. 또한, pHem1 유전자는 이미 밟혀진 다른 유전자들과 상동성을 보이지 않았다. 본 연구는 P. intermedia에서의 porphyrin생리 및 hemin 획득기전을 분자생물학적으로 규명하는데 있어 중요한 의의가 있으리라 사료된다. 향후 pHem1 유전자의 특성 분석 등이 수행되어야 할 것으로 여겨진다.

• 한국생명과학회:학술대회논문집
• /
• 한국생명과학회 2000년도 제28회 학술심포지엄
• /
• pp.3-8
• /
• 2000

The complete genome sequence of a Gram-positive bacterium .Bacillus subtilis has recently been reported and it is now clear that more than 50% of its ORFs have no known function (1). To study the global gene expression in B. subtilis at single gene resolution, we have tested the glass DNA microarrays in a step-wise fashion. As a preliminary experiment, we have created arrays of PCR products for 14 ORF whose transcription patterns have been well established through transcriptional mapping analysis. We measured changes in mRNA transcript levels between early exponential and stationary phase by hybridizing fluorescently labeled cDNA (with Cy3-UTP and Cy5-UTP) onto the array. We then compared the microarray data to confirm that the transcription patterns of these genes are well consistent with the known Northern analysis data. Since the preliminary test has been successful, we scaled up the experiments to ${\sim}$94% of the 4,100 annotated ORFs for the complete genome sequence of B. subtilis. Using this whole genomic microarray, we searched genes that are catabolite-repressive and those that are under the control of ${\sigma}^{Y}$, one of the functionally unknown ECF sigma factors. From these results, we here report that we have established DNA microarray techniques that are applicable for the whole genome of B. subtilis.