• Development and Reproduction
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• v.1 no.2
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• pp.189-202
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• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.

• BMB Reports
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• v.40 no.4
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• pp.511-516
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• 2007

A glutathione S-transferase (GST) related to the phi (F) class of enzymes only found in plants has been cloned from the Oryza sativa. The GST cDNA was cloned by PCR using oligonucleotide primers based on the OsGSTF5 (GenBank Accession No. $\underline{AF309382}$) sequences. The cDNA was composed of a 669-bp open reading frame encoding for 223 amino acids. The deduced peptide of this gene shared on overall identity of 75% with other known phi class GST sequences. On the other hands, the OsGSTF5 sequence showed only 34% identity with the sequence of the OsGSTF3 cloned by our previous study (Cho et al., 2005). This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF5 formed a homo-dimer composed of 28 kDa subunit and its pI value was approximately 7.8. The expressed OsGSTF5 displayed glutathione conjugation activity toward 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane and glutathione peroxidase activity toward cumene hydroperoxide. The OsGSTF5 also had high activities towards the herbicides alachlor, atrazine and metolachlor. The OsGSTF5 was highly sensitive to inhibition by S-hexylGSH, benastatin A and hematin. We propose from these results that the expressed OsGSTF5 is a phi class GST and appears to play a role in the conjugation of herbicide and GPOX activity.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.110-116
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• 1998

The secY gene of Streptomyces lividans TK24 was cloned by the PCR method with synthetic oligonucleotide primers designed on the basis of the conserved regions of Ll5-secY-adk operon from E. coli, B. subtilis, and M luteus. The deduced amino acid sequences of the SecY are highly homologous to those of other known SecY. It has 46%, 43%, 57%, 44%, 42%,56%, 90% similarity to Escherichia coli, Bacillus subtilis, Micrococcus luteus, Bacillus licheniformis Staphylococcus carnosus, Brevibacterium flavum, Streptomyces scabies, respectively and almost the same with Streptomyces coelicolor, The gene organization of Ll5- SecY-Adk is also similar to those of other bacteria. SecY and Adk are very likely translationally coupled that is overlapping stop codon of SecY and start codon of Adk with one base pair, which is common structure among high GC content strains of gram positive bacteria.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.472-477
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• 2017

Barnyardgrass (Echinochloa crus-galli) is one of the worst weeds in rice (Oryza sativa), but there are few reports about the genetic diversity and herbicide resistance of barnyardgrass in Vietnam. In this study, we used random amplified polymorphic DNA (RAPD) analysis and greenhouse testing to study the genetic diversity and quinclorac resistance levels of 15 Echinochloa crus-galli populations in the Mekong Delta, Vietnam, and the state of Arkansas, U.S. The quinclorac resistance of Echinochloa crus-galli populations in Vietnam was confirmed; 9 populations were resistant to quinclorac with R/S ratios ranging from 1.9 to 6.3. Six oligonucleotide primers produced a total of 55 repeatable bands of which 46 were polymorphic (83.3% average) among the 15 populations. Genetic distance was calculated, and cluster analysis separated the 15 populations into 2 main clusters with the genetic distances within the clusters ranging from 0.09 to 0.39. The two main clusters were divided into 7 subclusters, and the quinclorac resistant and susceptible populations were located randomly within each subcluster. Six out of 13 weed populations from Vietnam belonged to one cluster and a single Echinochloa species. The remaining 7 populations were identified as potentially different species in the Echinochloa genus. Nine Echinochloa populations from Vietnam were tested and identified as quinclorac resistant. The connection between quinclorac resistance levels and weed groups defined by RAPD analysis in the study is unclear; the quinclorac resistance of each resistant population could have evolved individually, regardless of differences in genetic diversity and location of the sampled populations.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.344-347
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• 2003

A method using PCR was developed for the monitoring of genetically modified soybean (GMS) and GMS derived foods utilized in the market. We designed 3 pairs of specific oligonucleotide primers based on epsps and pat inserted in GMS and ferritin gene as internal standards. Template DNAs isolated from soybean and processed foods were used for multiplex PCR with 3 primer sets. PCR, used with specific primer sets for GMS detection, showed the amplified DNA fragments with GMS template DNA. In this study, GMS containing epsps was detected from soy processed foods manufactured before GM food labeling system, however, GMS containing epsps or pat was not detected from soy processed foods manufactured after GM food labeling system.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1359-1366
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• 2010

A search of the Streptomyces avermitilis genome reveals that its closest homologs are several O-methyltransferases. Among them, one gene (viz., saomt5) was cloned into the pET-15b expression vector by polymerase chain reaction using sequence-specific oligonucleotide primers. Biochemical characterization with the recombinant protein showed that SaOMT5 was S-adenosyl-L-methionine-dependent Omethyltransferase. Several compounds were tested as substrates of SaOMT5. As a result, SaOMT5 catalyzed O-methylation of flavonoids such as 6,7-dihydroxyflavone, 2',3'-dihydroxyflavone, 3',4'-dihydroxyflavone, quercetin, and 7,8-dihydroxyflavone, and phenolic compounds such as caffeic acid and caffeoyl Co-A. These reaction products were analyzed by TLC, HPLC, LC/MS, and NMR spectroscopy. In addition, SaOMT5 could convert phenolic compounds containing ortho-dihydroxy groups into O-methylated compounds, and 6,7-dihydroxyflavone was known to be the best substrate. SaOMT5 converted 6,7-dihydroxyflavone into 6-hydroxy-7-methoxyflavone and 7-hydroxy-6-methoxyflavone, and caffeic acid into ferulic acid and isoferulic acid, respectively. Moreover, SaOMT5 turned out to be a $Mg^{2+}$-dependent OMT, and the effect of $Mg^{2+}$ ion on its activity was five times greater than those of $Ca^{2+}$, $Fe^{2+}$, and $Cu^{2+}$ ions, EDTA, and metal-free medium.

• Fisheries and Aquatic Sciences
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• v.6 no.4
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• pp.180-186
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• 2003

The full-length cDNA encoding the pre-protein growth hormone (sfGH) from spotted flounder (Verasper variegatus) was amplified by the rapid amplification of cDNA ends (RACE) using degenerated oligonucleotide primers derived from conserved growth hormone sequences. It consists of 901 nucleotides in length, including the coding region of 609 nucleotides, 111 nucleotides of a 5' untranslated region, and 181 nucleotides of a 3' untranslated region. The conserved polyadenylation signal (AATAAA) lies 12 bases upstream from the poly (A) tail. The deduced amino acid sequence shows an open reading frame encoding a pre-protein of 203 amino acids and a putative signal peptide of 17 amino acids, suggesting that the mature hormone consists of 186 amino acids. The analyses of sfGH reveal some unique structural features. The repetitive sequences are located in the 5' untranslated region of sfGH cDNA and consist of tandem arrays of imperfect direct repeat monomers. Moreover, sfGH contains six Cys residues, as opposed to four or five in other GHs, and it is clearly distinguishable from olive flounder (Paralichthys olivaceus) GH, which lacks a region corresponding to residues 175-188 in alignment positions. It has important implications from an evolutionary standpoint, suggesting possible divergence among flatfishes.

• Parasites, Hosts and Diseases
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• v.40 no.1
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• pp.17-24
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• 2002

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healui OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healui cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healui (AhCPI) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, $Cys^{25},{\;}His^{159},{\;}and{\;}Asn^{175}$. Deduced amino acid sequence analysis indicates that AhCPI belong to ERFNIN subfamily of C 1 peptidases. By Northern blot analysis. no direct correlation was observed between AhCPI mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that AhCPI protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.

• Parasites, Hosts and Diseases
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• v.50 no.2
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• pp.103-111
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• 2012

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

• Parasites, Hosts and Diseases
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• v.51 no.2
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• pp.237-241
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• 2013

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from $10^{-1}$ to $10^{-5}ng/{\mu}l$ for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of $63^{\circ}C$ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha ($EF1{\alpha}$) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.