• Journal of Life Science
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• v.8 no.2
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• pp.126-130
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• 1998

A pair of designed primers (sequences from Gene Bank) amplified 16S fRNA gene of V. vulnificus within polymerase chain reaction (PCR) machine. This PCR product is about 1.3kb DNA fragment. Six enzymes (BamH I, Alu I, Sau3A I, Hind III, Sal I, Sma I) were used for restriction pattern analysis of amplified 16S rRNA gene of V. vulnificus ATCC 27562. Digested fragments are resolved by 3% agarose gel. BamH I did not show digested fragment so, there was no cutting site of BamH I in PCR product. Alu I produced three small fragments from 400 bp to 200 bp. Sau3A I produced three fragments larger than Alu I from 70 bp and 500 bp. One of fragments of Sal I was same with 500 bp of Hind III fragment and the other was 750 bp. Sma I showed two fragments of 800 bp and 470 bp. The profile of digested fragments of 16S rRNA of V.vulnificus ATCC 27562 will may be able to use standard profile for identification of V. vulnificus.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.355-360
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• 2013

Precise, rapid and simple methods for species identification in animals are among the most important techniques in the livestock industry and research fields including meat classification. In this study, polymerase chain reaction (PCR) based molecular identification using inter species polymorphisms were examined by PCR-restriction fragment length polymorphism (RFLP) analysis for mitochondrial DNA (mtDNA) cytochrome b (CYTB) gene sequences among four mammalian livestock animals (cattle, horse, goat and pig). The results from PCR-RFLP analysis using the AluI restriction enzyme were also provided for the species-specific band patterns among CYTB gene sequences in these four species. The AluI-digestion for CYTB genes provided interesting migration patterns differentially displayed according to each species. Cattle and horse had one AluI-recognition site at different nucleotide positions and their AluI-digested fragments showed different band patterns on the gels. Pig had two AluI-recognition sites within the amplified CYTB sequences and produced three bands on the gels. Goat had no AluI-recognition site and was located at the same position as the uncut PCR product. The results showed the species-specific band patterns on a single gel among the four livestock animal species by AluI-RFLP. In addition, the results from blind tests for the meat samples collected from providers without any records showed the identical information on the species recorded by observing their phenotypes before slaughter. The application of this PCR-RFLP method can be useful and provide rapid, simple, and clear information regarding species identification for various tissue samples originating from tested livestock species.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.225-230
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• 2009

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.8
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• pp.993-999
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• 2010

Serum lysozyme gene is one of the important genes influencing the immune system as its product can cause lysis of bacterial cell wall by cleaving the peptidoglycan layer. The present investigation on the serum lysozyme gene of Indian riverine buffalo was undertaken with the objectives to identify and characterize single nucleotide polymorphic patterns by PCR-SSCP method as well as to study the effect of different genotypes on serum lysozyme activity and somatic cell count. A total of 280 animals comprising four different famous bubaline breeds (Murrah, Mehsana, Surti and Bhadawari), spread over six different farms across the country were used for this study. A 276 bp (partial intron 2, complete exon 3 and partial intron 3) fragment of lysozyme gene was screened for polymorphism using the SSCP technique. Four genotypes namely AA, AB, BC and AC were observed, out of which BC genotype was found to be the most frequent. Among these three alleles, C allele (0.38) was most prevalent in these populations. Various SSCP allelic variants were cloned for sequencing and sequences were submitted to NCBI Genbank. From the alignment of the nucleotide sequences of various allelic variants, it was found that there were differences in 12 positions among the alleles, out of which maximum variation (at 8 places) was found in the intronic region. The allele A was closer to allele-C than allele-B. Allele B was phylogenetically equidistant from both of the other alleles. Mean lysozyme activity determined in serum samples of different animals of Murrah buffalo was $27.35{\pm}2.42\;{\mu}g$ per ml of serum, whereas the mean somatic cell count was $1.25{\pm}0.13{\times}10^5$ cells per ml of milk. The SSCP pattern-wise effects of various genotypes on lysozyme activity and SCC were analyzed. Although the mean values were apparently different in various genotypes, these differences were statistically non-significant. It can be concluded that the riverine buffaloes are sufficiently polymorphic with respect to serum lysozyme gene. The absence of AA genotype in Bhadawari breed of buffalo can be considered as a marker for breed characterization. The difference of four nucleotides in exon-3 indicates high selection pressure on the gene.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.102-102
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• 2001

Taurine (2-ethaneaminosulfonic acid) is one of the major intracellular ${\beta}$ -amino acids in mammals and is required for a number of biological processes including membrane stabilization, osmoregulation, antioxidation, detoxification, modulation of calcium flux and neurornodulation. The taurine transporter (TAUT) which contains 12 hydrophobic membrane-spanning domains has been cloned from dog kidney, rat brain, mouse brain, human thyroid, placenta and retina. In this study, The TAUT cDNA from the human intestinal epithelial cell, HT-29 was cloned and sequenced. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify partial cDNA encoding human intestinal TAUT. The coding region of the PCR product was 732 bp long. The primers were designed to encode highly conserved amino acid sequences near the transmembrane domains III (IPYFIFLF) and Ⅵ (KYKYNSYR) both in human and mouse. The TAUT cDNA amplified was ligated into the pGEX 4T-1 expression vector. The resulting sequence of human intestinal TAUT cDNA (Accession number of NCBI Genebank is AF346763) was identical to the sequences of the TAUTs previously determined in the human placenta and retina except 3 base pairs from that of the reported human thyroid. TAUT specific antibodies were generated to use them as biological tools in the studies of the biological role of TAUT. Peptides of 149-162 amino acid residue (14 amino acids) of the TAUT were synthesized. The synthetic peptide used in this study was LFQSFQKELPWAHC. This region was chosen not only to avoid putative glycosylation sites but also to exclude regions of known homology with GABA transporters in the extracellular hydrophilic domains. The synthetic peptide, TAUT-1 was conjugated with carrier protein, kehole lympet hemocyanin (KLH) to use as an antigen. When used for immunization on a rabbit to produce polyclonal antiserum, the conjugates elicited high -titered specific anti-TAUT-1 antibodies, which reacted well with the ovalbumin (OVA) conjugated peptides in ELISA. The KLH-conjugated peptide was also used as immunizing antigen in BALB/c mice to produce TAUT specific monoclonal antibodies. From the culture supernatant of the hybridoma, the specificity of anti-TAUT-1 monoclonal antibodies was confirmed by ELISA. Further applications of more tools in TAUT expression analysis will be performed such as western blotting and flow cytometry.

• Journal of Korean Society of Forest Science
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• v.87 no.3
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• pp.334-340
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• 1998

This research was conducted to identify plant regulatory regions by gene tagging method. A promoterless GUS coding sequence was introduced to Populus nigra ${\times}$ P. maximowiczii via Agrobacterium strains(LBA4404/EHA101), and putative transgenic poplars were selected by culturing on medium containing G418($60mg/{\ell}$) and by GUS assay. Among them one positive plant was to amplify the native sequences flanking to the introduced GUS gene in plant genome by inverse PCR method and from this 730 by DNA product was obtained. After subcloning and sequencing, it has 88% homology to the Eucalyptus gunnii CAD(cinnamyl alcohol dehydrogenase) gene. The GUS gene fused with the putative promoter reinserted into poplar leaves by particle bombardment method to test the funtional promoter activity. Upon staining with X-gluc, many blue spots appeared on the leaf segments bombarded by the chimeric gene 2-3 days, thus the isolated DNA fragment contain some possible coding region as well as a putative regulatory sequences of poplar CAD gene.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.5 no.2
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• pp.157-168
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• 2000

Sea urchin S. intermedius occurring in the Korean east coast is a cold water species that belongs to the family Strongylocentrotidae of Echinoidea. Although it is known that there are nine species in the family, species identification criteria, phylogenetic relationships, time and process of evolution of the family members have not been uncovered clearly. In the present study, I tried to find some clues to such problems for S. intermedius by means of DNA sequences. For this, cytochrome c oxidase subunit I (COI), one of the mitochondrial genes that evolve fast and follow maternal inheritance was analyzed. DNA was extracted from the female gonad of S. intermedius, a segment of COI gene amplified by polymerase chain reaction (PCR), and finally a total of 1077 base pair sequence of COI obtained by cloning and sequencing the PCR product. The sequence was compared with homologous genes of other sea urchins and echinoderm species. Phylogenetic trees of the COI gene segment revealed that S. intenedius is a sister species of S. purpuratus which lives along the east coast of the Paciflc. With reference to the fossil records of sea urchins and genetic distances in the molecular phylogenies, it is estimated that the two species were separated about 0.89 million years ago when the earth temperature fluctuated significantly. The current disjunct distribution patterns of the two species and the climate change of the earth at the time of separation suggest that speciation might have occurred by vicariance. The COI gene sequence obtained here now can be used as a molecular character which discerns S. intermedius from the other sea urchin species of Strongylocentrotidae.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.318-326
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• 2002

The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases such as life-threatening septicemia. To better understand this organism's strategies to survive osmotic stress, a mutant that was more sensitive to high osmolarity was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, putAP genes encoding a proline dehydrogenase and a proline permease were identified and cloned from V. vulnificus. The amino acid sequences deduced from nucleotide sequences of putAP from V. vulnificus were 38 to $59\%$ similar to those of PutA and PutP reported from other Enterobacteriaceae. Functions of putAP genes were assessed by the construction of mutants, whose putAP genes were inactivated by allelic exchanges. When proline as the sole carbon or nitrogen source was used, the putA mutant was not able to grow to the substantial level, revealing the proline dehydrogenase is the only enzyme for metabolic conversion of proline into other amino acids. Although the growth rate of the putP mutant on proline as the sole carbon or nitrogen source was significantly reduced, the mutant still grew. This indicated that at least one more proline permease is produced by V. vulnificus. The putP mutant decreased approximately $2-log_10$ CFU/ml after a hyperosmotic challenge, while the parent strain decreased approximately $l-log_10$ CFU/ml. This result suggests that the gene product of putP contributes to the osmotic tolerance of V. vulnificus.

• The Korea Journal of Herbology
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• v.36 no.1
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• pp.9-17
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• 2021

Objectives : Tetrapanacis Medulla and Akebiae Caulis are one of the most frequently adulterated herbal medicines because of their confusability of terms in the ancient writings and the similarity of morphological features of dried herbal products. The major adulterant is Aristolochia manshuriensis (Guanmutong) which has a serious safety concern with its toxicity. To ensure the safety and quality of the two herbal medicines, it is necessary to discriminate the toxic adulterant from authentic species. The aim of this study is to develop SCAR markers and to establish the multiplex-SCAR assay for discrimination of four plant species related to Tetrapanacis Medulla and Akebiae Caulis. Methods : ITS regions of fifteen samples of four species (Tetrapanax papyrifer, Fatsia japonica, Aristolochia manshuriensis, and Akebia quinata) collected from different sites were amplified and sequenced. Fifteen obtained ITS sequences were aligned and analysed for the detection of species-specific sequence variations. The SCAR markers were designed based on the sequence alignments and then, multiplex-SCAR assay enhancing rapidity was optimized. Results : ITS sequences clearly distinguished the four species at the species level. The developed SCAR markers and multiplex-SCAR assay were successfully discriminated four species and detected the adulteration of commercial product samples by comparison of the amplified DNA fragment sizes. Conclusions : These SCAR markers and multiplex-SCAR assay are a rapid, simple, and reliable method to identify the authentic Tetrapanacis Medulla and Akebiae Caulis from adulterants. These genetic tools will be useful to ensure the safety and to standardize the quality of the two herbal medicines.

• Korean Journal of Ecology and Environment
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• v.37 no.1 s.106
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• pp.122-129
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• 2004

In an effort to develop the biomarker for monitoring the contamination of xenoestrogen in the freshwater environment of Korea, reverse transcription-polymerasechain reaction (RT-PCR) analysis of vitellogenin (VTG) gene expression was optimized in Hearisarsus Iaseo, Based on the homology of the VTG cDNA sequences between the common carp and zebra fish, a set of PCR primers for VTG mRNA amplification for H; labo was designed. VTG mRNA level in livers from female and male fishes was analyzed by RT-PCR following single injection of 17 beta estradiol($E_2$ 10 mg $kg^{-1}$ B.W.). As an internal control, beta actin mRNA was amplified. One us of total liver RNA was subjected to RT-PCR. In female the amount of PCR productof VfC gradually increased in the range from 16 to 34 cycles of amplification. On the contrary, in control male, PCR product first detected at 32 cycles of amplification and linearly increased up to 40 cycles of amplification. In $E_2$ injected male liver, the VTC mRNA level was similar to that in the female. Taken together, this result suggests that liver of male H. labo expresses minute amount of VTG mRNA which are2-l6 equivalent of female and that induction of VTG mRNA occurs in male liver after estrogen treatment. In conclusion, the optimized protocol for RT-PCR analysis of VTG mRNA expression in liver of male H. labo will provide the environmental monitoring method for the xenoestrogen contamination in the rivers in Korea.