• Journal of Mushroom
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• v.9 no.2
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• pp.53-58
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• 2011

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3'- and 5'-RACE PCR and RT-PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.690-693
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• 2000

Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA [5]. The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGe and Western blot. Proinsulin concentration was estimated to be around 200 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.467-472
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• 2018

Objective: Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Results: The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). Conclusion: ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

• The Korean Journal of Mycology
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• v.25 no.3 s.82
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• pp.167-175
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• 1997

The PCR fragments of two distinct chitin synthase genes, PdCHSl and PdCHS2, were cloned from Penicillium diversum KCTC 6786. The nucleotide sequences of PdCHSl and PdCHS2 contained uninterrupted open reading frames (ORFs) of 570 bp excluding the primer sequence. The similarity analysis of the deduced amino acid sequences using BLASTP indicated that the possible evolutionary relationship between P. diversum and ascomycetous fungi. Multialignment of the deduced amino acid sequences of PdCHSs using CLASTAL W revealed that the PdCHSs fell into two different classes: PdCHSl into Class I and PdCHS2 into Class II of chitin synthase defined by Bowen et al. (1992). By Southern blot analysis, it was shown that each of the two genes is present as a single copy in the genome of P. diversum KCTC 6786.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.113-113
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• 2017

Downy mildew (DM), caused by several species in the Peronosclerospora and Scleropthora genera, is a major maize (Zea mays L.) disease in tropical or subtropical regions. DM is an obligate parasite species in the higher plants and spreads by oospores, wind, and mycelium in seed surface, soil, and living hosts. Owing to its geographical distribution and destructive yield reduction, DM is one of the most severe maize diseases among the maize pathogens. Positional cloning in combination with phenotyping is a general approach to identify disease resistant gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combination strategy to improve the identification of disease resistant gene candidates. Downy mildew (DM) resistant maize was selected from five cultivars using the spreader row technique. Positional cloning and bioinformatics tools identified the DM resistant QTL marker (bnlg1702) and 47 protein coding genes annotations. Eventually, 5 DM resistant gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative RT-PCR without fine mapping of the bnlg1702 locus. Specifically, we provided DM resistant gene candidates with our new strategy, including field selection by the spreader row technique without population preparation, the DM resistance region identification by positional cloning using bioinformatics tools, and expression level profiling by quantitative RT-PCR without fine mapping. As whole genome information is available for other crops, we propose applying our novel protocol to other crops or for other diseases with suitable adjustment.

• Environmental Analysis Health and Toxicology
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• v.26
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• pp.5.1-5.11
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• 2011

Objectives: In order to identify the possibility of slender bitterling (SB) (Acheilognathus yamatsutae) being used as a test species for estrogenic endocrine disrupting chemicals (EEDCs), we carried out the cloning and sequence characterization of the estrogen receptor (ER). Methods: The ER from a slender bitterling was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (5'-RACE and 3'-RACE) and T-vector cloning. The expression of ER mRNA was also analyzed in six tissues (brain, liver, kidney, gill, gonad, and intestines) by real-time PCR. Results: We obtained an ER from the slender bitterling. The SB ER cDNA was 2189 base pairs (bp) in length and contained a 1707 bp open reading frame that encoded 568 amino acid residues. The SB ER amino acid sequence clustered in a monophyletic group with the $ER{\alpha}$ of other fish, and was more closely related to zebrafish $ER{\alpha}$(88% identity) than to the $ER{\alpha}$ of other fish. The SB ER cDNA was divided into A/B, C, D, E and F domains. The SB ER has conserved important sequences for ER functions, such as the DNA binding domain (D domain), which are consistent with those of other teleosts. Conclusions: The ER of the slender bitterling could provide basic information in toxicological studies of EEDCs in the slender bitterling.

• The Journal of the Korea Contents Association
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• v.21 no.4
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• pp.825-832
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• 2021

In this study, by selecting specific aptamers that selectively detection on P. gingivalis, the main cause of periodontal disease, and purifying and identifying protein molecules that bind to the selected aptamers, the mechanism of action of P. gingivalis was investigated. A DNA library having 39 random sequences was prepared, and aptamers with specificity for P. gingivalis were selected using the SELEX method, and the nucleotide sequence was analyzed by cloning using PCR2.1 cloning vector. 8 of aptamers with different nucleotide sequences were selected, and modified weston blot was performed using APG-3 among the selected aptamers to identify 11 proteins that act directly, and proteins were analyzed. As a result, a protein that selectively binds to P. gingivalis was isolated and identified. Therefore, aptamer selectively binds and attaches to proteins related to inhibition of sugar metabolism and cell activity of P. gingivalis, suggesting the possibility of a sensor for diagnosis of periodontal disease.

• Korean Journal of Veterinary Service
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• v.27 no.1
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• pp.103-109
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• 2004

Babesia equi ema-1 5' intergenic(IG) nucleotide was PCR amplified and analyzed for restriction sites in order to identify a promoter region in this IG nucleotide sequence. B equi ema-1 5' IG specific primers identified a 1268 bp PCR product. The sequence had restriction sites for 34 restriction enzymes when analyzed by a computer program. Among them, 26 enzymes had only one restriction site, but the others had more than one sites. When four restriction enzymes (Bgll , HindⅢ, Kpn1 and BamH1) were treated to digest the 1268 bp nucleotide, they had restriction sites as expected by the computer program. Information of restriction sites in the 1268 bp IG nucleotide will be applied to select restriction enzymes for cloning the IG nucleotide to a vector.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2005.05a
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• pp.197-197
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• 2005