• 대한수의학회지
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• 제33권1호
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• pp.43-51
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• 1993

Mitochondrial DNA(mt DNA) of Mammalian is the circular one which the 16.5K base pairs and show the maternal inheritance. Evolutional speed of nucleotide sequence is very fast. So that polymorphic analysis of mt DNA provide the useful informations to investigate the genetic relations of interspecies. Authors trials were focussed to compare with the polymorphic differences of mitochondrial DNA between Jindo and Japanese mongrel dogs. DNA was extracted from bloods of 21 head of Jindo dogs and 20 head of Japanese dogs and isolated using 10 kinds of restriction endonucleases(Apa I, BamH I, Bgl II, EcoR I, EcoR V, Hinc II. Hind III, Pst I, Sty I, Xba I) and then separated by the agarose gel electrophoresis. After sourthern blotting hybridization was completed using the mtDNA of Japanese mongrel dogs as a probe. Autoradiography was used to compare the polymorphism of mtDNA both dogs. The results obtained were as follows; 1. mt DNA of Jindo dog showed polymorphism resulting cleavage with four kinds of restriction endonuclease, Apa I, EcoR V, Hinc II, Sty I. While in the Japanese mongrel dogs observed the polymorphism in the five kinds of restriction endonuclease supplemented with EcoR I. 2. Compared with both dogs the frequency differences of DNA polymorphism were recognized in the specific restriction endonuclease Apa I. Consequently in the restriction endonuclease Apa I both dogs classified with three types as A, B, C however in the Jindo dogs frequency of C type was 71.5 percent but in Japanese mongrel dogs observed 45 percent in the A type. 3. DNA polymorphism obtained from the use of five kinds of restriction endonuclease were classified with seven types. In Jindo dogs frequency was highest in the type 6 as 71.4 percent but in the Japanese mongrel dogs showed 35 percent in the type 5. 4. Genetic distances calculated by NEI method showed 0.0089 in Jindo dogs and was 0.0094 in the Japanese mongrel dogs.

• 미생물학회지
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• 제30권1호
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• pp.30-36
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• 1992

물오리나무의 뿌리혹에서 분기한 Frankia sp. SNU 014201 공생균주의 게놈내에 13.5 kb의 EcoRI, 18.0 kb 의 BamHI, 10.5 kb의 Bg/II, 4.5 kb의 KpnI 절편에 nif-H, D 유전자가 존재함을 확인하였다. 람다 파아지 EMBI3-BamHI arm을 사용하여 제조한 genomic library 에서 nif유전자를 포함하고 있는 14개의 재조합 파아지 클론을 선별하였다. 이들 중 Ahnif-12번 클론은 nif 유전자를 포함하고 있는 18kb 의 삽입 DNA 를 가지고 있었으며, 이중 7.9 kb 의 BamHI 절편내에 nif-H. D. K가 3.6kb 의 HindIII/KpnI 절편내에 nif D 의 일부와 H 가 위치하고 있었다. 따라서 이등 절편을 각각 subcloning 하고 제한효소 지도를 작성한 결과, Frankia sp. SNU 01420의 nif-H. D. K 유전자는 6.5 kb 의 Hind III/Bam HI 절편과 5.2 kb Sal/IBamHI 절편내에 연속 배열하고 있다.

• KSBB Journal
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• 제6권3호
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• pp.289-297
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• 1991

The insecticidal protein gene in the pKL-20-1 clone derived from Bacillus thuringiensis serovar. kurstaki plasmid was subcloned in the plant shuttle vector, pGA643. The 7.3 kb fragment was cloned in the BglII and Hpal sites of pGA643 vector and expressed in E. coli S17-1, which produced insecticidal proteins killing Bombyx mori larvae. The clone was named pHL-20. The protoplast formation, calli induction and plantlet regeneration of Nicotiana tabacum was carried out. A tremendous number of mesophyll protoplasts of N. tabacum were formed, up to 7$\times$105 protoplast per ml, for 20 hours in darkness in the enzyme solution of 0.5% cellulase and 0.1% macerosin, pH 5.8. The viabilities of the protoplasts were maintained above 80% for 6 days in the media containing 2mg/1 of NAA and 1mg/1 of kinetin. Calli were induced from the protoplasts and leaves of the N. tabacum on MS medium containing 0.5mg/1 BAP. Under the culture conditions the protoplasts underwent repeated cell division into calli. Plantlets were regenerated from callus cultures derived from protoplast and leaves. Shoots were induced in a medium containing 1mg/1 of BAP.

• 미생물학회지
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• 제34권3호
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• pp.115-119
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• 1998

Pseudomonas sp. DJ77로부터 전보에서 클로닝한 pHENX7의 하류방향으로 약 3kb 정도를 포함하는 pYCS500을 클로닝하였다. PYCS500의 제한효소지도를 작성하고 부분적으로 염기서열을 분석한 결과 465 bp의 HindIII-ClaI절편에서 282 bp로 이루어진 하나의 open reading frame(ORF)을 발견하였다. phnM으로 명명된 이 ORF는 93개의 아미노산으로 구성된 polypeptide를 암호화하고 있었으며 계산된 분자량은 10,008 Da이었다. PhnM은 NahT, XylT, DmpQ, AtdS, PhlG, PhhQ, TbuW 등 plant-type ferredoxin 형태의 단백질과 37.7%-53.9%의 상동성을 나타내었으며 이들이 공통적으로 가지고 있는 motif가 일치하였다.

• 미생물학회지
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• 제30권3호
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• pp.149-153
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• 1992

Cloning of the gene encoding extracellular .betha.-lactamase from Streptomyces sp. SMF13 in a plasmid pIJ702 and expression of the gene in Streptomyces invidans were carried out. Optimal conditions for the formation of protoplasts of S.lividans and the regeneration of the protoplasts were evaluated. Streptomyces sp. SMF-13 was selected as a donor strain of .betha.-lactamase gene and totla DNA of the strain was partially digested with Sau3A I. DNA fragments ranged from 4kb to 10 kb were ligated to pIJ702 AT Bgl II site and then the ligated DNAs were transformed to the protoplasts of S, livivans. The transformation efficiency was $2 *10^{3}$ .$\mu$g DNA for the ligated DNA mixture. One colony among a thousand colonies regenerated showed extracellular .betha.-lactamase and the size of the inserted DNA fragment was estimated to be 3.94 kb. The .betha.-lactamase activity in the culture broth of the recombinant strain was maximum at 3 days culture to be 1.0 unit/ml.

• 한국미생물·생명공학회지
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• 제23권5호
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• pp.536-543
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• 1995

Multicopy integration vector is a very useful vector system in that they can be integrated into chromosomal DNA in several copies and stably maintained under non-selective conditions. To develop a multicopy integration vector system in the yeast Yarrowia lipolytica, P-type ribosomal DNA was cloned from Y lipolytica. A HindIII-BglII fragment of the cloned rDNA and a promoterless URA3 gene were inserted into pGEM1, generating multicopy integration vectors, pMIYL-1 and pMIYL-2. The rDNA fragment is for targeted homologous recombination between the vector and the chromosomal DNA of Y. lipolytica, and the promoterless URA3 gene is a defective selection marker for inducing multicopy integration. pMIYL-1 and pMIYL-2 have an unique restriction enzyme site, KpnI, and two unique restriction enzyme sites, KpnI and EcoRI, repectively, which can be used for targeting of the vectors into the rDNA of Y. lipolytica chromosomal DNA. After transformation of the vectors into Y. lipolytica, copy number and stability were analyzed by Southern hybridization. The vectors were found to be present in less than 5 copies per cell and were stably maintained during growth in non-selective media.

• 한국미생물·생명공학회지
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• 제13권3호
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• pp.297-302
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• 1985

미생물중 urease생성능이 아주 강한 B. pasteurii의 Hind III partial digest 된 chromosomal DNA를 E. coli-B. subtilis bifunctional plasmid vector pGR 71으로 E. coli RR1 균주에 cloning 하므로써 그 urease gene을 expression시킬 수 있었다. 그러나 B. subtilis에서는 insertion DNA fragment의 deletion으로 expression되지 않았다. Cloning된 E.coli RR1 균주로부터 분리 정제한 urease gene함유 Plasmid(pGU66)의 restriction map을 작성하여 본 결과 7.1 Mdal의 insertion fragment가 삽입된 12.6Mdal의 plasmid에 Hind III, Bgl II, Xba I, Sal I등 몇 개의 cleavage site 위치를 찾을 수 있었다. Cloning된 E. coli의 urease는 periplasmic space에 많은 비율로 축적되며, 그 효소학적 성질은 donor인 B.pasteurii의 그것과 매우 유사하였다.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.305-308
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• 1990

Shuttle plasmid vector인 pHN114를 이용하여 Kluyveromyces fragilis의 3-isopropylmalate dehydrogenase 유전자를 cloning 하였다. 그 결과 Saccharomyces cerevisiae의 leu2변이와 E.coli의 leuB변이를 상보하는 두가지 clone 체 pJK104와 pJK106을 얻었다. Restriction mapping 결과 이들은 서로 반대방향으로 삽입되어 있었으며 expression activity는 pJK104가 높았다. pJK104에 삽입된 유전자를 BglII와 SalI으로 끊은 1.6kb fragment를 probe 로 하여 Southern Hybridization 한 결과 유전자의 유래가 Kluyveromyces fragilis 임을 확인하였다.

• 원예과학기술지
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• 제35권3호
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

• 대한수의학회지
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• 제32권4호
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• pp.597-603
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• 1992

To elucidate the molecular genetical properties of the canine parvoviruses isolated from the diseased puppies in the regions of Kyunggi and Chungnam provinces, the replicative form (RF) DNA of four field isolates were compared with those of two attenuated vaccine strains and a reference strains of CPV by restriction endonuclease analysis (REA). REA by Hinf I showed that three CPV isolates except CPV-V15 had an identical banding pattern with two vaccine strains, one standard strain and feline panleukopenia virus (FPLV). In CPV-V15 strain the fourth fragment of DNA with 800 bp was deleted. REA by Bgl II and Pst I indicated that CPV-V15 and FPLV had a bigger second fragment than those of the other strains of CPV. Meanwhile REA by Bam HI revealed that all the field isolates and vaccine strains used in this experiment showed similar banding patterns.