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H2AX Directly Interacts with BRCA1 and BARD1 via its NLS and BRCT Domain Respectively in vitro (H2AX의 BRCA1 NLS domain과 BARD1 BRCT domain 각각과의 in vitro 상호 결합)

  • Bae, Seung-Hee;Lee, Sun-Mi;Kim, Su-Mi;Choe, Tae-Boo;Kim, Cha-Soon;Seong, Ki-Moon;Jin, Young-Woo;An, Sung-Kwan
    • KSBB Journal
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    • v.24 no.4
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    • pp.403-409
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    • 2009
  • H2AX, a crucial component of chromatin, is implicated in DNA repair, cell cycle check point and tumor suppression. The aim of this study was to identify direct binding partners of H2AX to regulate cellular responses to above mechanisms. Literature reviews and bioinformatical tools were attempted intensively to find binding partners of H2AX, which resulted in identifying two potential proteins, breast cancer-1 (BRCA1) and BRCA1-associated RING domain 1 (BARD1). Although it has been reported in vivo that BRCA1 co-localizes with H2AX at the site of DNA damage, their biochemical mechanism for H2AX were however only known that the complex monoubiquitinates histone monomers, including unphosphorylated H2AX in vitro. Therefore, it is important to know whether the complex directly interacts with H2AX, and also which regions of these are specifically mediated for the interaction. Using in vitro GST pull-down assay, we present here that BRCA1 and BARD1 directly bind to H2AX. Moreover, through combinational approaches of domain analysis, fragment clonings and in vitro binding assay, we revealed molecular details of the BRCA1-H2AX and BARD1-H2AX complex. These data provide the potential evidence that each of the BRCA1 nuclear localization signal (NLS) and BARD1 BRCA1 C-terminal (BRCT) repeat domain is the novel mediator of H2AX recognition.

Host Preference of Ricania spp. (Hemiptera: Ricaniidae) at Different Developmental Stages (갈색날개매미충의 발육단계별 기주선호성)

  • Choi, Yong-Seok;Seo, Hwa-Young;Jo, Shin-Hyuk;Whang, In-Su;Lee, Young-Su;Park, Deog-Kee
    • Korean journal of applied entomology
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    • v.56 no.4
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    • pp.319-329
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    • 2017
  • We investigated the host preference of Ricania app. at different developmental stages to select the effective control methods. Ricania spp. at different developmental stages preferred different host plants. During the nymphal period, individuals moved to the bottom due to environmental factors such as wind and rain, rather than inhabiting trees, and fed on various plants ranging from 1 year-old herbaceous plants to shrubs. Especially, at Palbong Mt. in Gonju, Ricania spp. nymphs preferred Symplocos chinensis f. pilosa, Celastrus orbiculatus, Euonymus alatus, Kalopanax septemlobus, Aralia elata, Styrax japonicas, Rubus parvifolius, Ru. coreanus, Ru. crataegifolius, Quercus acutissima, Cornus officinalis, Lespedeza bicolor, Albizia julibrissin, L. maximowiczii, Rhododendron schlippenbachii and Rh. indicum. At Yongbong Mt. in Hongseong, Ricania spp. adults preferred herbaceous plants, such as Helianthus annuus, Solanum nigrum, and Oenothera biennis rather than trees during the pre-oviposition stage. Subsequently, as the oviposition season began, Ricania spp. adults were rarely found in H. annuus, S. nigrum, or O. biennis and appeared to migrate to the perennial trees and shrubs hosts to lay eggs. At Palbong Mt., the main laying hosts of Ricania spp. adults were Weigela subsessilis, Ailanthus altissima, Q. aliena, Boehmeria platanifolia, Diospyros lotus, Ziziphus jujubae var. inermis, St. japonicus, Prunus mume, P. serrulata var. spontanea, Spiraea prunifolia f. simpliciflora, Ru. crataegifolius, Salix koreensis, Ru. yedoense f. poukhanense, and Co. controversa. The degree of egg laying, number of egg masses and adult Ricania spp. were significantly higher in sunlit areas than in shaded areas, in addition, the lower the altitude, the more adults were present. This study contributes toward the development of traps that can attract and control Ricania spp. and reduce the density of Ricania spp. that disturb the ecosystem.

Analysis of Nutritional Compounds and Antioxidant Effect of Freeze-Dried powder of the Honey Bee (Apis mellifera L.) Drone (Pupal stage) (서양종 꿀벌(Apis mellifera L.) 수벌번데기 동결건조 분말의 영양학적 성분 및 항산화 효과)

  • Kim, Jung-Eun;Kim, Do-Ik;Koo, Hui-Yeon;Kim, Hyeon-Jin;Kim, Seong-Yeon;Lee, Yoo-Beom;Kim, Ji-Soo;Kim, Ho-Hyuk;Moon, Jae-Hak;Choi, Yong-Soo
    • Korean journal of applied entomology
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    • v.59 no.3
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    • pp.265-275
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    • 2020
  • In this study, we analyzed the nutritional ingredients of drone pupae (16th to 20th instar old) to evaluate the value of bee products and provide basic data for product diversification, and the extracts prepared using these pupae were tested for physiological activity. According to the analysis of the general ingredients of the freeze-dried powder of these bee pupae, the moisture, crude protein, crude fat, and crude ash was 1.69 ± 0.07%, 48.52 ± 0.20%, 23.41 ± 0.14%, and 4.05 ± 0.02%, respectively. Vitamin C and vitamin E were 14.92 ± 0.52 mg/100 g and 6.06 ± 0.11 mg α-TE/100 g, respectively. Regarding minerals, the highest content of K (1349.13 ± 34.57 mg/100 g) and P (1323.55 ± 43.85 mg/100 g) was observed and Ca and Fe were 55.43 ± 1.51 mg/100 g and 5.49 ± 0.19 mg/100 g, respectively. The fatty acids of the water extracted freeze-dried pupae powder accounted for approximately 59.62 of saturated fatty acids and 40.38 of unsaturated fatty acids, and high-quality fatty acids such as palmitic acid (C16:0) was 35.49 ± 0.08 and oleic acid (C18:1, n-9) was 35.91 ± 0.22 (g/100 g total fatty acids). The total amino acid content was 38.99 ± 2.63 g/100 g and the free amino acid was a total of 5129.04 mg/100 g, of which 1257.68 mg/100 g was proline and 759.12 mg/100 g glutamic acid. The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the drone pupae extract showed values of 0.8 for distilled water extract, 3.2 for 50% EtOH extract, 6.4 for 70% EtOH extract, and approximately 90% for 32 ㎍/mL for 100% EtOH extract. These results suggest that the main compound contributing to the antioxidant activity is a polar compound, and it is highly likely to be a low-molecular protein or a free amino acid. In conclusion, the honey bee drone pupa is excellent as a food resource and can be utilized as a new functional material for food and functional food.

Microarray Analysis of Gene Expression in Rat Glioma after Ethanol Treatment (에탄올 처리에 의한 흰쥐 신경아교종(Glioma) 세포에서의 유전자 발현 - DNA 칩을 이용한 분석 -)

  • Lee, So Hee;Oh, Dong-Yul;Han, Jin-Hee;Choi, Ihn-Geun;Jeon, Yang-Whan;Lee, Joon-Noh;Lee, Tae Kyung;Jeong, Jong-Hyun;Jung, Kyung Hwa;Chai, Young-Gyu
    • Korean Journal of Biological Psychiatry
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    • v.14 no.2
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    • pp.115-121
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    • 2007
  • Objetives : Identification of target genes for ethanol in neurons is important for understanding its molecular and cellular mechanism of action and the neuropathological changes seen in alcoholics. The purpose of this study is to identify of altered gene expression after acute treatmet of ethanol in rat gliom cells. Methods : We used high density cDNA microarray chip to measure the expression patterns of multiple genes in cultured rat glioma cells. DNA microarrays allow for the simultaneous measurement of the expression of several hundreds of genes. Results : After comparing hybridized signals between control and ethanol treated groups, we found that treatment with ethanol increased the expression of 15 genes and decreased the expression of 12 genes. Upregulated genes included Orthodenticle(Drosophila) homolog 1, procollagen type II, adenosine A2a receptor, GATA bindning protein 2. Downregulated genes included diacylglycerol kinase beta, PRKC, Protein phosphatase 1, clathrin-associated protein 17, nucleoporin p58, proteasome. Conclusion : The gene changes noted were those related to the regulation of transcription, signal transduction, second messenger systems. modulation of ischemic brain injury, and neurodengeneration. Although some of the genes were previously known to be ethanol responsive, we have for the most part identified novel genes involved in the brain response to ethanol.

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Molecular Phylogenetic Study of the Endangered Land Snail Satsuma myomphala Based on Metallothionein Gene. (Metallothionein 유전자를 기초로 한 멸종위기 육상 달팽이 Satsuma myomphala (거제외줄달팽이) 의 분자계통학적 연구)

  • Sang, Min Kyu;Kang, Se Won;Hwang, Hee-Ju;Chung, Jong Min;Song, Dae Kwon;Min, Hye Rin;Park, Jie Eun;Ha, Hee Cheol;Lee, Hyun Jun;Hong, Chan Eui;Ahn, Young Mo;Park, So Young;Park, Young-Su;Park, Hong Seog;Han, Yeon Soo;Lee, Jun Sang;Lee, Yong Seok
    • The Korean Journal of Malacology
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    • v.32 no.4
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    • pp.263-268
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    • 2016
  • Metallothionein (MT) family of metal-binding proteins are involved in maintaining homeostasis and heavy metal poisoning. Recently, MT has been considered as a biomarker that can identify a particular species, very similar to the use of cytochrome oxidase I (COI) gene. Satsuma myomphala species of land snails have been reported from North-East Asia, including South Korea and Japan. In particular, the land snail species have been known from only a limited area of Geoje Island, Gyeongsangnam-do province of South Korea. Genetic studies of S. myomphala has been limited with only 6 nucleotide, 2 protein registered on the NCBI server. For elucidating the genetic information of S. myomphala, we conducted RNA sequencing analysis using Illumina HiSeq 2500 next-generation platform. We screened the MT gene from the RNA-Seq database to confirm the molecular phylogenetic relationship. After sequencing, the de novo analysis and clustering generated 103,774 unigenes. After annotation against PANM database using BLAST program, we obtained MT sequence of 74 amino acid residues containing the coding region of 222 bp. Based on this sequence, we found about 53 sequences using the BLAST program in NCBI nr database. Using ClustalX alignment, Maximum-Likehood Tree of MEGA program, we confirmed the molecular phylogenetic relationships that showed similarity with mollusks such as Helix pomatia and H. aspersa, Megathura crenulata.

Anti-aging Effects of the Extracts from Leaf. Stem, Fruit and Seed of Yew (Taxus cuspidata Sieb) by Solvent Extraction Method (용매추출법에 의한 주목의 잎, 줄기, 과실 추출물의 항 노화 효과)

  • Kim, In-Young;Jung, Sung-Won;Ryoo, Hee-Chang;Zhoh, Choon-Koo
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.2
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    • pp.211-219
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    • 2004
  • Yew (Taxus cuspidata Sieb.) chose that grow as medicine, food, decorative plant in Korea's Kyong-Gi province surroundings. Extracts of yew extracted leaf of 250g and stems of 300g with 1,3-butylene glycol (l,3-BG), propylene glycol (PG) and water. As results, external appearance of leaf extract of yew was slightly brown clear extract. The pH was 5.3${\pm}$0.5, and specific gravity was 1.012${\pm}$0.05, and refractive index was l.375${\pm}$0.05. Also, appearance of stem's extract was slightly brown clear extract, and the pH was 5.4${\pm}$0.5, and specific gravity was 1.016${\pm}$0.05, and refractive index was 1.358${\pm}$0.05. Oil of yew separated from seeds, and extracted polysaccharide high purity from fruits. As a result, specific gravity of oil was 0.987, and obtained 40.0% of yield. Total polyphenols amount of yew extract is detected 0.563% in leaves, 0.325% in stems, whereas total tannins amount contained 0.054% and 0.037% each in leaves and stems. As effect in cosmetics, the anti-oxidative effect by DPPH method is 75.0% in leaves, and stems was 64.0%. Collagen synthesis rate was shown high activity by 54.16% in stem's extract, 33.18% in leaves' extract. Also, PPE-inhibitory activities were 13.7% and 23.5% each in leaves and stems. Anti-inflammatory effect of yew seed oil displayed superior effect of 41% than control. Polysaccharide's molecular weight that is gotten from fruits was 5${\times}$10$^4$-3${\times}$10$\^$5/ dalton, and got 20.0${\pm}$5% of yield.

Endogenous Phenoloxidase Purified from an Earthworm, Lumbricus rubellus (붉은 지렁이(Lumbricus rubellus) 체내로부터 정제한 Phenoloxidase)

  • 백승렬;조은정;유경희;김유삼;서정진;장정순
    • The Korean Journal of Zoology
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    • v.39 no.1
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    • pp.36-46
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    • 1996
  • An endogenous phenoloxidase (EPO) from earthworm, Lumbricus rubellus, has been purified and characterized. The purified EPO using ammonium sulfate fractionation, Blue-2, Phenyl-, and Q-sepharose chromatography steps was revealed in SDS-PAGE as a single protein banri with Mr. of 59 kl)a. A native strudure of the enzyme was examined with an in situ staining of a nondenatudng-PAGE using DL-dopa as a substrate. The result showed that a single band due to the EPO activity was located siighdy above a standard polypeptide with Mr. of 210 kl)a. These fads indicate that the EPO is an oligomeric enzyme. The presence of a monophenolase activity of the purified EPO, which hydroxylates tyrosine to dopa, was confirmed by observing dopachrome accumulation at 475 nm at PH 8.0 with a typical lag phase during 60 mm. of meausrement. A series of inhibition study has been performed for the enzyme with several divalent cation chelators such as phenyithiourea (Flu), 1, lO-phenanthroline, EDTA, and EGTA. Among them, only V'flj inhibited the enzyme with 1C0.5 of 65 MM, which indicated that copper was critical for the catalysis of EPO. The enzyme was maximally active at 35'C and pH 8.0 when L-dopa to dopachrome conversion was spectrophotometricaily monitored at 475 nm. The apparent Km values of P0 for L-opa were obtained as 1.86 mM and 13.8 mM at pH 6.5 and 8.0, respectively. The catalytic efficiencies at both pH were almost identical [(kat/Km)pH8.0/(kcat/Km)pH6.5 = O.92] while the Vmax at p11 8.0 was 6.6-fold higher than that at pH 6.5. This fact may indicate that pH affeds the catalysis at substrate and/or enzyme-substrate complex level rather than the enzyme itself. Taken together, the EPO was an oligomeric enzyme which did not require proteolysis for its activation. These results also indicated that the enzyme can exist, at least, in part as a latent form In vivo, which might be distinct from the prophenoloxidase activating system. Therefore, it is pertinent to consider that there must be certain regulatory molecules or phenomena in L. rubellus which make the 1,0 in a latent form in vivo before the foreign invasions.

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Identification and Characterization of Wild Yeasts Isolated from Korean Domestic Grape Varieties (국산 포도로부터 분리한 야생효모의 동정 및 특성)

  • Choi, Sang-Hoon;Hong, Young-Ah;Choi, Yoon-Jung;Park, Heui-Dong
    • Food Science and Preservation
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    • v.18 no.4
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    • pp.604-611
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    • 2011
  • Several wild yeasts were isolated from Korean grape varieties before and during spontaneous fermentation. Among them, four strains were isolated based on the alcohol content and flavor production in wine after fermentation of apple juice. In this study, the four yeast strains were identified and characterized. PCR-restriction fragment length polymorphism analysis of ITS I-5.8S-ITS II region with restriction endonuclease Hae III and Hinf I resulted in that all the strains showed a typical pattern of Saccharomyces cerevisiae. Pulse field gel electrophoresis showed three different chromosome patterns with a same band between strains SS89 and SS812. When ITS I-5.8S-ITS II sequences of the four strains were compared with one another, they were similar to those of Saccharomyces cerevisiae CBS 4054 type strain. Identity of the sequences was higher than 97% with those of the type strain. Phylogenetic analysis showed based on the sequences showed they were genetically closed to the type strain. The four identified strains were tested in a medium containing 200 ppm potassium metabisulfite, and the MM10 and WW108 inhibition rates resulted at up to 24 h. The four strains were tested at an incubation temperature of $30^{\circ}C$. The 30% sugar concentration in the medium (w/v) showed the highest growth in 36 h, especially in the case of SS89, which was close to growth 40. The four strains were tested in an 8% ethanol medium (v/v). Alcohol tolerance was initially kept in the incubation process. The strains began to adapt, however, to the exceeded resistance. The four strains showed the lowest inhibition rate at 24 h.

Studies on the Raw Starch Saccharifying Enzyme from the Aspergillus niger and Its Mutants (Aspergillus niger 및 그 변이주(變異株)의 생전분당화효소(生澱粉糖化酵素)에 관(關)한 연구(硏究))

  • Sohn, Cheon Bae;Park, Yoon Joong
    • Korean Journal of Agricultural Science
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    • v.10 no.1
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    • pp.166-185
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    • 1983
  • Aspergillus niger IFO 8541 (NRRL 3112) was investigated through a series of UV rays and N-Methyl-N'-Nitro-N-Nitrosoguanidine (NTG) treatments to induce mutants that produce highly active raw starch saccharifying enzyme, and two mutants with strong enzymatic productivity were obtained. The mutants obtained were investigated for their fungal characters, condition of enzyme production, and other activities. Furthermore, the raw starch saccharifying enzyme was purified and the characteristics of purified enzyme were studied. The results obtained were summarized as follows; 1. The color of conidial head of UV-46 mutant obtained from UV rays treatment was changed to tan type and the gelatinated starch saccharifying enzyme productivity and the raw starch saccharifying enzyme productivity increased up to twice and 1.8 times compared to the productivities of original Aspergillus niger IFO 8541 cultured on the wheat bran, respectively. 2. The conidial head color of NG-41 mutant obtained from NTG treatment became lighter than that of parent strain. The gelatinated starch saccharifying enzyme productivity and raw starch saccharifying enzyme productivity increased about 1.8 times, and twice over the Aspergillus niger IFO 8541 parent strain cultured on wheat bran, respectively. The productivity of ${\alpha}$-amylase increased about 3 times more than the parent strain. 3. Two peaks of glucoanlylase and a peak of ${\alpha}$-amylase were obtained when enzyme solution of mutants and parent strain were passed through DEAE-Sephadex A-50 column chromatography. Glucoamylase I showed only gelatinated starch saccharifying enzyme activity. However, glucoamylase II (raw starch saccharifying enzyme) showed both raw starch saccharifying enzyme activity and gelatinated starch saccharifying enzyme activity. 4. Mutant, UV-46 was strengthened in glucoamylase II productivity and mutant NG-41 was strengthened in ${\alpha}$-amylase productivity. 5. Glucoamylase II of mutants and parent strain were appeared to have the same enzymatic properties. 6. Glucoamylase II of mutants and parent strain were recognized as simple enzyme through electrophoresis. 7. The glucoamylase II crystallized showed rhombic board type. 8. The molecular weight, isoelectric point, optimum pH, and optimum temperature of the glucoamylase II crystallized were estimated as 76,000, 3.4, 3.5 and $60^{\circ}C$, respectively.

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Analysis of Genetic Polymorphisms and Similarity Using Random Amplified Polymorphic DNAs in Cattle (RAPD기법을 이용한 축우의 유전적 다형성과 유사도 분석)

  • Lee, S.H.;Seo, K.W.;Kwon, I.;Sung, C.K.;Kim, S.K.;Sang, B.C.
    • Korean Journal of Agricultural Science
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    • v.26 no.2
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    • pp.39-48
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    • 1999
  • This study was carried out to investigate the difference and genetic similarity at the level of molecular genetics. Genomic DNA was extracted from blood of Holstein, Korean cattle, Charolais, and hybrid between Korean cattle and charolais and RAPD(random amplified polymorphic DNAs) was analyzed by PCR(polymerase chain reaction). After genetic similarity value from different breeds are analyzed, genetic similarity was estimated by UPGMA(unweighted pair-group method using average). The results obtained from this study can be summarized as follows: 1. When genomic DNA which was extracted from different breeds was subjected to electrophoresis on 1.5% agarose gel, bigger than 12.2kb was appeared. Ratio by absorbance of $A_{260}/A_{280}$ was 1.75~2.10, indicating that genomic DNA was quite pure for RAPD analysis. 2. Different band patterns by RAPD were appeared according to the breeds in cattle. The best primer used to distinguish Holstein from other breeds was 5'-GAC CGC TTG T-3'. 3. A 340bp fragment was amplified in $33.0^{\circ}C$ of annealing temperature for the Holstein and Charolais breeds, but any amplification was not occurred in this annealing temperature for Korean cattle and hybrid. In addition, a 340bp fragment was amplified in $37.5^{\circ}C$ of annealing temperature for the Holstein and Korean cattle, but any amplification was not occurred in this annealing temperature for Charolais and hybrid. For the reaction of PCR. $37.5^{\circ}C$ and $33.0^{\circ}C$ of annealing temperature was shown to be best for genetic marker identification from Holstein, Charolais, and hybrid between Korean cattle and Charolais. 4. When genetic similarity from different breeds are analyzed at the both temperature of $33.0^{\circ}C$ and $37.5^{\circ}C$, the genetic similarity value of Holstein and Korean cattle, Holstein and Charolais, Korean cattle and Charolais, and Korean cattle and hybrid were 0.666~0.777, 0.615~0.666, 0.400~0.461 and 0.857~0.888, respectively. 5. It could be concluded that different breeds are capable of distinguishing by RAPD used random primer 5'-GAC CGC TTG T-3', genetic similarity from different breeds was appeared the higher genetic similarity value of Korean cattle and Charolais than that of Holstein between Korean cattle and Charolais by UPGMA.

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