• 한국가축번식학회지
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• 제20권4호
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권2호
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• pp.211-215
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• 2004

Genetic diversity within the natural populations of Daba ecorace of Antheraea mylitta Drury was studied using individual silkworms collected from the South Singhbhum district of Jharkhand state of India with 21 inter simple sequence repeat (ISSR) primers. A total of 148 bands were produced, of which 79% was polymorphic. The pair wise genetic distance among the individuals varied from 0.186 to 0.329. The dendrogram grouped the individuals into 3 major clusters. Nei's heterozygosity analysis revealed 0.265 ${\times}$ 0.18 variability within the population. The high genetic variability present within the natural population of Daba ecorace of A. mylitta is indicative of their adaptational strategy in nature and have much importance for in situ conservation as well as utilization in breeding programs.

• Asian-Australasian Journal of Animal Sciences
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• 제17권8호
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• pp.1053-1061
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• 2004

Of the 20 arbitrarily chosen primers, six oligonucleotides decamer primers were used on the basis of the number of the polymorphisms generated in catfish (Silurus asotus) from Yesan and bullhead (Pseudobagrus fulvidraco) from Dangjin in Korea. Six primers were used generating a total of 602 scorable bands in catfish and 195 in bullhead population, respectively, ranging in size of DNA fragments from less than approximately 100 to larger than 2,000 base pairs (bp). Six primers yielded 199 polymorphic fragments (33.1%) in catfish and 47 (24%) in bullhead, respectively. In the present study, a total of 328 common fragments (an average of 54.7 per primer) were observed in catfish population, whereas 84 (an average of 14.0 per primer) in bullhead. The total number of specific fragments in catfish and bullhead population were 76 and 64, respectively. In catfish population, random decamer, OPA-17 (GACCGCTTGT) generated the highest number of fragments (a total of 141) in comparison with other primers used, with an average of 11.8. The common bands in the molecular weight of 300 bp generated by random primer OPA-06 (GGTCCCTGAC) were present in every individuals in bullhead population. The major polymorphic bands in the molecular weight of 100 bp generated by OPA-17 were identified in lane 14, 15, 17, 18, 19 20 and 21, which were identifying species in bullhead population. The average bandsharing values (BS values) of all of the samples within catfish population ranged from 0.575 to 0.945, whereas 0.063-1.000 within bullhead population. The bandsharing value (index of similarity between individuals) between individual No. 5 and No. 9 showed the highest level within catfish population, whereas the bandsharing value between individual No. 1 and No. 2 showed the lowest level. The single linkage cluster analysis resulted from four primers, indicating four genetic groupings composed of group 1 (C1-C10, all of the catfish samples), group 2 (B11, B12, B13, B14, B16, B17, B18, B19), group 3 (B15) and group 4 (B20 and B21). The dendrogram reveals close relationships between individual identities within two species populations and individuals derived from the same ancestor, respectively. However, genetic distances between two species populations ranged from 0.124 to 0.333. The shortest genetic distance (0.042) displaying significant molecular differences was between individual No. 6 and No. 9 catfish population. The shortest genetic distance (0.033) displaying significant molecular differences also was between individual No. 18 and No. 19 in bullhead population. Reversely, the genetic distance of individual No. 20/21 among individuals in bullhead population was highest (0.333). This result showed that bullhead No. 20 and 21 were distinct from other individuals within bullhead population.

• Asian-Australasian Journal of Animal Sciences
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• 제18권4호
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• pp.458-462
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• 2005

The study was aimed at detecting polymorphism of the fifth intron in lipoprotein lipase (LPL) gene and analyzing association between the polymorphism and productive traits. A pair of primers was designed for amplifying the fifth intron. Sequence analysis indicated that a G1171C substitution existed in Large White breed. The mutation was detected by PCR-AfaI-RFLP. Polymorphism analysis in a pig resource family showed that there existed significant effects on carcass and meat quality traits. Thoraxwaist fat thickness of BB genotype was significantly higher (14.2%, p<0.05) than that of AA on carcass traits, while BB genotype was significantly lower (3.6% p<0.01, 4.1% p<0.01; 2.3% p<0.01, 1.9% p<0.01; 1.8% p<0.01, 1.4% p<0.05) than AA and AB genotype in pH of m. Longissimus Dorsi (LD), m. Biceps Femoris (BF), m. Semipinali Capitis (SC). The allelic frequencies were also significantly different between indigenous Chinese breeds and exotic breeds. Data analyzed revealed that the mutation locus affected production traits mostly by additive effects. Based on these results, it is necessary to do more studies on LPL gene before making the LPL locus into the application of marker-assisted selection (MAS) programs.

• 한국균학회지
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• 제37권2호
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• pp.173-180
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• 2009

This study was carried out to obtain basic data on mycelial growth characteristics for an artificial cultivation of Volvariella volvacea. Twelve URP primers were used to assess genetic relationship of V. volvacea and its result was divided into two groups. But there was nothing different in morphological characteristics in V. volvacea. Among five kinds of mushroom media, MCM medium was selected as the favorable culture medium. The optimal range of temperature and pH for mycelial growth on V. volvacea were 35oC and pH 6~8, respectively. Carbon sources had not an effect on 10 strains of V. volvacea, and nitrogen source for the optimum mycelial growth was yeast extract. Also, we selected GMVV 79004 as a superior strain of 10 strains V. volvacea based on the mycelial growth and yield.

• Journal of Microbiology
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• 제40권3호
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• pp.211-218
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• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권5호
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• pp.373-384
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• 2001

Cellular proliferation is an intricately regulated process mediated by the coordinated interactions of critical growth control genes. Two of these factors in mammalian cells are the p53 and mdm-2 genes. A protein product of the mem-2 oncogene has been recently shown to associate with the protein encoded by the tumor suppressor gene p53. The p53 tumor suppressor protein is stabilized in response to DNA damage and other stress signals and causes the cell to undergo growth arrest or apoptosis, thus preventing the establishment of mutations in future cellular generations. Mutation or loss of p53 is a very common event in tumor progression. It occurs in about 50% of all tumors analysed including of colon, lung, breast and liver. The cellular mdm-2 gene, which has potential transforming activity that can be activated by overexpression, is amplified in a significant percentage of human sarcoma and in other mammalian tumors. Proteins encoded by the mdm-2 gene are able to bind to the p53 protein and, when overexpressed, can inhibit p53's transcriptional activation function, thus mdm-2 can act as a negative regulator of p53 function. Experimental study was performed to observe the relationship between p53 gene mutation and mdm-2 protein expression and apply the results to the clinical activity. 36 golden syrian hamster each weighing $60{\sim}80g$ were used and painted with 0.5% DMBA by 3 times weekly on the right buccal cheek(experimental side) for 6, 8, 10, 12, 14 and 16 weeks. Left buccal cheek(control side) was treated with mineral oil as the same manner to the right side. The hamsters were sacrificed on the 6, 8, 10, 12, 14 & 16 weeks. Normal and tumor tissues from paraffin block were examined for histology and immunohistochemistry observation, and were completely dissected by microdissection and DNA from both tissue were isolated by proteins K/phenol/chloroform extraction. Segments of the hamster p53 exons 5, 6, 7 and 8 were amplified by PCR using the oligonucleotide primers, and then confirmational change was observed by SSCP respectively. The results were as follows : 1. Dysplasia at 6 weeks, carcinoma in situ at 8 weeks and invasive carcinoma from 10 weeks could be observed in experimental groups. 2. p53 mutations were detected in 10 of the 36(28%) and the exons 6(6 of the 10 : 60%) was the most hot spot area among the highy conserved region(exons 5, 6, 7 & 8). 3. Immunohistochemical study confirmed 22 of the 36(61%) of p53 expression involving 10 of p53 mutations. 4. mdm-2 expression of was showed in 3 of the 36(8%) involving 1 of the 22 of p53 expression and 2 of the 14 of p53 non-expression. From the above results, mutation of p53 gene or expression of p53 protein may have the influence of the DMBA induced carcinoma of hamster buccal pouch but the expression of mdm-2 protein may not have relationship with tumorigenesis.

• Genes and Genomics
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• 제40권11호
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• pp.1127-1136
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• 2018

The Greater Sudbury Region has been known as one of the most ecologically disturbed areas in Canada for the past century. Plant adaptation to environmental stressors often results in modifications in gene expression at the transcriptional level. The main objective of the present study was to compare the expression of genes associated with nickel resistance in Acer rubrum and Populus tremuloides growing in areas contaminated and uncontaminated with metals. Primers targeting Nramps4, Nas 3, At2G, MRP4 and alpha-tubulin genes were used to amplify cDNA of both species. The expression of the At2G gene, was $2{\times}$ and $9{\times}$ higher in P. tremuloides than in A. rubrum for St. Charles (uncontaminated site) and Kelly Lake (metal contaminated site), respectively. There was a much smaller difference between the two species for the Nramps 4 gene as its expression was $2.5{\times}$ and $3{\times}$ higher in P. tremuloides compared to A. rubrum from St. Charles and Kelly Lake, respectively. The same trend was observed for the MRP4 gene whose expression was $2{\times}$ and $14{\times}$ higher in P. tremuloides than in A. rubrum from St. Charles and Kelly Lake, respectively. For the Nas 3 gene, the expression was similar in both sites. This gene was upregulated $11{\times}$ and $10{\times}$ in P. tremuloides compared to A. rubrum in samples from St. Charles and Kelly Lake, respectively. In general, no significant difference was observed between the metal contaminated and uncontaminated sites for gene expression. In depth analysis revealed that AT2G and MRP4 genes were significantly down regulated in A. rubrum from the metal contaminated sites compared to those from uncontaminated areas, but environmental factors driving this differential gene expression couldn't be established.

• Journal of Microbiology
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• 제45권2호
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• 대한임상검사과학회지
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• 제49권4호
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• pp.413-419
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• 2017

폐렴의 한(일개) 환자의 임상검체에서 연속적으로 분리된 Imipenem 내성세균 4균주를 분리하였다. 분리균을 동정하기 위해 Vitek II system의 GN card를 이용하였으며 16S rRNA유전자 염기서열을 기초로 계통학적 분석을 실시하였다. 분리균은 P. aeruginosa (2 strains), P. monteilii (1) 및 P. putida (1) 으로 동정되었다. 분리균들의 항생제에 대한 내성시험은 Vitek II system AST-N225 card를 이용해서 imipenem의 최소억제 농도가 모두 $${\geq_-}8{\mu}g/mL$$을 확인한 후 실험에 사용하였다. ${\beta}-Lactamase$ 유전자의 특이 시발체를 이용하여 증폭한 PCR 산물로 imipenem 내성 유전자형을 결정하였는데 분리된 4균주 모두에서 MBL 유전자를 확인하였으며 2균주의 P. aeruginosa는 MBL유전자중 VIM형과 SHV형 유전자를 그리고 또다른 균주는 VIM형과 OXA group II형 유전자를 동시에 보유하고 있었다. 항생제 감수성 결과에서는 amikacin이 다른 항생제보다 감수성을 보였을 뿐 대체적으로 내성율이 높았다. 균주들간의 역학적 연관성 분석을 위해 ERIC-PCR을 이용한 DNA 지문 분석결과, 분리된 2 균주의 P. aeruginosa는 유사한 균주일 것으로 추정하였으나 DNA band 유형의 상동성은 서로 다른 유형임을 알아 볼 수 있었다. 특이하게 한 환자에게서 imipenem 내성세균이 4균주가 검출 된 것은 이례적이며 동종의 DNA band 유형도 서로 상이하였다.