Cycle Sequencing에서 $[{\alpha}-^{32}P]$dATP를 Taq DNA Polymerase로 Primer에 표지하는 간편한 방법

김한집,김병국,Kim, Han-Jip,Kim, Byung-Kook 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.6

최근에 개발된 cycle sequencing은 thermal cycler를 사용하여 적은 양의 template DNA만으로 sequencing을 가능하게 하는 새로운 방법이다. 그러나 지금까지 소개된 방법에서는 ${\gamma}-labelled$ NTP와 polynucleotide kinase를 이용하여 end-labelled된 sequencing primer를 만들어서 사용하여야한다. end-labelled primer를 사용하여 cycle sequencing을 하면 효과적인 결과를 얻을 수 있으나 polynucleotide kinase의 구입이 필수적이다. 뿐만 아니라 국내에서 여러 종류의 동위원소를 손쉽고 저렴하게 구입하고 또한 보관하기 어렵다는 문제점을 개선하기 위해 필자들은 Taq DNA polymerase를 이용하여 ${\gamma}-labelled$ dATP로 pnmer를 표지하는 간편한 방법을 강구하였다. 즉, thermal cycler에서 Taq DNA polymerase에 의해 pnmer의 3' 말단을 단지 $[{\alpha}-^{32}P]dATP$만으로 신장하여 표지시켰다. 이때 다른 종류의 dNTP를 공급하지 않아 $[{\alpha}-^{32}P]dATP$로 표지된 primer는 신장이 중단되며 이들 primer로 cycle sequencing을 수행하였다. 이 간편한 primer 표지방법은 기존의 end-labelled primer방법만큼 효과적이므로 본 실험실에서는 end-labelling을 위한 ${\gamma}-labelled$ nucleotide와 polynucleotide kinase의 구입이 필요없게 되었다. 또한 end-labelled primer 방법과 같은 양의 Taq DNA polymerase로 primer의 표지와 sequencing의 연속반응을 할 수 있었다. 나아가서 이 방법으로 박테리아 colony로부터 직접 sequencing이 효율적으로 수행되어 sequencing을 위한 시간과 시약의 절약이 더욱 가능하게 되었다. A simplified method for cycle sequencing is introduced. Previously, sequencing primers for cycle sequencing were end-labelled with ${\gamma}-labelled$ NTPs by a polynucleotide kinase. The end-labelled primer method is effective but costly because a researcher has to purchase both the polynycleotide kinase and the ${\gamma}-labelled$ nucleotide. In our method, we labelled the 3' end of the primer with the $[{\alpha}-^{32}P]dATP$ using a Taq DNA polymerase. During the labelling process, extension of the primer was stopped and arrested after labelling because no other dNTPs were provided except the $[{\alpha}-^{32}P]dATP$. There was no need to add more Taq DNA polymerase to complete both primer-labelling and sequencing processes. In our laboratory, the method enabled us to routinely label primers for sequencing without the use of the polynucleotide kinase, and has eliminated the need to purchase the ${\gamma}-labelled$ nucleotide for end-labelling. The method was also effective for sequencing directly from a bacterial colony. This would further save time and materials for sequencing.

사람 신경 간세포에서 도파민 신경세포 분화유도에 대한 Nurr 1 유전자의 역할 규명

김한집(Han Jip Kim),이학섭(Haksup Lee),김현창(Hyon Chang Kim),민철기(Churl Ki Min),이명애(Myung Ae Lee),김승업(Seung Up Kim),한진(Jin Han),염재범(Jae Boum Youm),김나리(Nari Kim),박원선(Won Sun Park),김태호(Taeho Kim),김의용(Euiyong Kim 한국생물공학회 2005 KSBB Journal Vol.20 No.5

Peptide Nucleic Acid(PNA)를 이용한 antisense 기법에 적용할 병렬 컴퓨팅용 Bioinformatics tool 개발

김성조(Seongjo Kim),전호상(Hosang Jeon),홍승표(Seungpyo Hong),김현창(Hyon Chang Kim),김한집(Han Jip Kim),민철기(Churl K. Min) 한국정보과학회 2006 한국정보과학회 학술발표논문집 Vol.33 No.1

Unlike RNA interference, whose usage is limited to eukaryotic cells, Peptide Nucleic Acid (PNA) technique is applicable to both eukaryotic and prokaryotic cells. PNA has been proven to be an effective agent for blocking gene expressions and has several advantages over other antisense techniques. Here we developed a parallel computing software that provides the ideal sequences to design PNA oligos to prevent any off-target effects. We applied a new approach in our location-finding algorithm that finds a target gene from the whole genome sequence. Message Passing Interface (MPI) was used to perform parallel computing in order to reduce the calculation time. The software will help biologists design more accurate and effective antisense PNA by minimizing the chance of off-target effects.

장자못의 생태학적 연구 - 제2보 춘계 장자못의 기초생산

엄규백(Kyu Back Uhm),김한집(Han Jip Kim) 한국식물학회 1974 Journal of Plant Biology Vol.17 No.2

A study was made on the primary production of Lake Changjamot during the spring season of 1973 by means of the oxygen method. The stratification of temperature and dissolved oxygen were formed in May with the stratified structure of Phytoplankton. The range of Secchi disc transparency was from 0.8m to 2.3m during the nine months of this investigation, which was begun in January, 1973. The value was lowest in early June when the phytoplankton blooming reached the peak. The concentration of PO_4-P, NH_3-N, NO_3-N and NO_2-N was reduced at the beginning of the phyoplankton blooming and increased again after May except PO_4-P. It might have been caused by the inflow of the nitrogenous fertilizer from the surrounding agricultural area since May when farming was started. The total amount of chlorophyll-a in the entire water column varied from 25㎎/㎡ to 277㎎/㎡ from January till September with the maximum value occurring in early June. These values show a considerable eutrophication of the lake in comparison with the data obtained in 1969. The daily gross production in the lake varied from a low of 655㎎ C/㎡ to a high of 2,859㎎ C/㎡ during the spring season and this corresponds to the variation of the amount of chlorophyll. The total amount of daily respiration varies from 650㎎ C/㎡ in winter to 2,307㎎ C/㎡ in late spring and exceeds gross primary production especially in late May showing the negative balance of daily production and consumption of organic material at that time. In conclusion, Lake Changjamot is a fairly productive and a moderately autotrophic lake and has been eutrophicated much during the past four years.

리블로스 1,5 - 이인산 탄산화효소 유전자의 분리 및 특성 규명

박성순(Sung Soon Park),김희진(Hee Jin Kim),김정호(Chung Ho Kim),김한집(Han Jip Kim),이종섭(Jong Seob Lee),이광응(Kwang Woong Lee),최양도(Yang Do Choi) 한국응용생명화학회 1994 Applied Biological Chemistry (Appl Biol Chem) Vol.37 No.5

To study the light-induced expression mechanism and protein transport into the chloroplast, a rice genomic clone (GrbcS) for the small subunit of ribulose 1,5-bisphosphate carboxylase (rbcS) was isolated and its nucleotide sequence was determined. Nucleotide sequence analysis of GrbcS revealed that the gene consists of two exons interrupted by an intron, encoding a protein of 175 amino acids including a transit peptide of 47 amino acids. These structural features of GrbcS are consistent with those of other rbcS genes from monocot species. Genomic Southern blot analysis suggested that the rbcS genes are present as a relatively small multigene family in the rice genome. Comparison of the nucleotide and deduced amino acid sequences to other rice rbcSs shows close sequence similaritiy. Conserved DNA sequences present in other light-responsive genes are also found in the 5′ upstream region of GrbcS such as G-box, 3AF1-binding site and GATA site. The possible function of these putative regulatory elements are discussed.

완두콩 ( Pisum sativum ) 에서 Ribulose - 1 , 5 - Bisphosphate Carboxylase Small Subunit 유전자 cDNA 클로닝과 광유도성 발현에 관한 연구

장무웅(Moo Woong Chang),구용범(Yong Bum Koo),김한집(Han Jip Kim) 한국식물학회 1989 Journal of Plant Biology Vol.32 No.1

Polysomal polyadenylated mRNAs which were purified from pea leaves were fractionated by sucrose grandient sedimentation. Fractions corresponding to the peak at 11.5S were found to contain mostly mRNA encoding the precursor polypeptide to the small subunit of ribulose bisphosphate carboxylase (rbcS) by in vitro translation in wheat germ extract. Double-stranded cDNA which was synthesized from the 11.5S mRNA was cloned into Hind Ⅲ site of plasmid pBR 325. A cDNA clone, H24, was identified to code for rbcS. In vitro translation product of the hybridization-selected mRNA was molecular weight 20,000, presumably the precursor of rbcS. The nucleotide sequences of the H24 showed almost complete homology with the sequences encoding the transit peptide of the rbcS-3A gene which was reported by Fluht et al. (1986).

입자사출기법에 (粒子射出技法) 의한 벼 세포로의 외래 유전자 도입과 그 발현

전종성(Jong Seong Jeon),정호성(Hou Sung Jung),성순기(Soon Kee Sung),이종섭(Jong Seob Lee),최양도(Yang Do Choi),김한집(Han Jip Kim),이광웅(Kwang Woong Lee) 한국식물학회 1994 Journal of Plant Biology Vol.37 No.1

For establishing a transformation system of rice, an efficient introduction of foreign genes into embryogenic cell suspension by particle bombardment was conducted. The particle inflow gun based on the acceleration of DNA-coated tungsten particles using pressurized helium was constructed for delivery of DNA into rice cells. Several bombardment parameters were optimized using the transient expression of GUS gene. The conditions that gave the highests GUS gene expression of about 1000 blue spots per a g fresh weight of bombarded cells include treatment of the cells with 0.5 M osmotic pressure, and use of the 410 kPa helium, 100 mm target distance, 13 mm syringe filter holder and 5 μL DNA/tungsten mixtures. It was also confirmed that rice actin promoter-intron constract gave the highest expression of all promoter-sequences studied. Eight weeks after the bombardment, stably transformed calluses were obtained on the selection medium containing 100 mg/L G418 and showed the strong activity in in situ GUS assay.

벼 엽록체 DNA 내의 151 bp 반복염기서열에 의한 유전자 재배열

김한집,남백희 한국농화학회 1993 Applied Biological Chemistry (Appl Biol Chem) Vol.36 No.3

To investigate the gene rearrangement via short repeated sequences in chloroplast DNA, the pattern of heterologous gene clusters containing the 151 by repeated sequence with the development of plastid was compared in rice and the homologous gene clusters from various plant sources were searched for comparative analysis. Southern blot analysis of rice DNA using rp12 gene containing 151 by repeated sequence as a probe showed the presence of heterologous gene clusters. Such heterologous gene clusters varied with the development of plastid. Also it was observed that the heterologous gene clusters were observed in all of the rice cultivars used in this work. Finally the comparative analysis of DNA sequence of the homologous gene clusters from various plants showed the evolutionary gene rearragngement via short repeated sequence among plants. These results suggest the possible relationship between the plastid development and gene rearrangement through short repeated sequences.

Heterosis in Winter Wheat F₁Hybrids Produced by the Chemical Hybridizing Agent System

Kim,Han Jip 嶺南大學校附設 基礎科學硏究所 1987 基礎科學硏究 Vol.7 No.-

本 實驗에서는 Rhom and Haas회사의 chemical hybridizing agent(gametocide RH0007)를 이용한 小麥의 F₁ hybrid의 收量과 收量構成要素의 雜種强勢를 연구하고자 하였다. 종례의 細胞質的 雄性不稔을 이용한 F₁ hybrid의 생산은 복잡하고 장기간을 요하며 小麥에 사용하는 稔性回復 유전자는 回復能力이 불완전하다. 이에 비하여 chemical hybridizing agent(CHA)를 이용하면 수술의 去勢가 용이하고 종자생산이 간편하므로 chemical hybridizing agent에 의한 F₁ hybrid 종자생산의 실질적인 효용을 연구하였다. 13개의 F₁ hybrid를 兩親인 純系品種들과 같이 1981년 11월부터 1982년 6월까지 Texas A & M 대학의 2個所의 實驗圃場에 供試하였다. 7개의 HRWW F₁ hybrid는 우수親에 비해 4~30%의 增收(heterobeltiosis)를 보였으며 6개의 SRWW F₁ hybrid는 -23~+37%의 heterobeltiosis를 보였다. 이들 13개 F₁ hybrid 중에서 SRWW F₁ hybrid인 McNair 3271×Blueboy 만이 SRWW 純系 중의 最多收量品種보다 15%(414kg/ha)의 增收를 나타내어 唯一하게 收量增加의 有意性을 보였다. 또한 收量構成要素中에서는 千粒種과 穗當粒數가 雜種强勢효과가 현저하였고 단위면적당 穗數의 雜種强勢는 그 有意性이 인정되지 않았다. Hybrid wheats will be competitive only if their yields exceed those of the best pure-line cultivars available in the productin area. This yield difference must be adequate to allow the recovery of the higher production costs for hybrids and an additional increment of profit to insure long-term popularity of hybrid wheats. The best yielding SRWW F₁ hybrid, McNair 3271 ⅹ Blueboy, showed 15% higher yield (6 bu/ac) than that of the best pure-line parent cultivar. A net increased profit of nine dollars per acre is estimated after allosing the reovery of the higher cost(estimated to be $10/ac) of hybrid seed compared to that of certified seed of pure-line cultivars. This increment of profit probably is marginal to insure long-term popularity of the F₁ cultivars. Although the results of this study indicated appreciable highparent heterosis for several F₁ hybrids, it is difficult to determine whether hybrid wheat will become economically competitive to the highest yielding pure-line in commercial production in the near future. The potential of hybrids reaches beyond yield increases alone. Effective techniques for producing hybrids by both cytoplasmic male sterility and chemical hybridization systems allow an avenue for more efficient breeding for disease and insect resistance. Resistance could be incorporated in a single generation if it were conditioned by a dominant gene or genes. In this regard, chemical hybridizing agents offer a greater advantage over the cytoplasmic male sterility system in allowing the rapid and easy manipulation of parental genotypes. For example, genes for disease resistance and easy manipulation of parental genotypes. For example. genes for disease resistance can be rapidly incorporated into F₁ hybrids for the target area. Also, it is quite likely that certain hybrids will produce yields superior to those of the best pure-line varieties. The highest yielding varieties will be crossed to various other varieties and experimental pure-lines to develop superior F₁ hybrids which have additional yield potential. A great many experimental hybrids can be produced for testing by the chemical hybridizing agents also should be very useful in "population development" programs based on intermation many parents, applying severe selection pressure, and continuing to intermate selection from the bulk populatin to further "concentrate" genes conditioning the desired trait(s). Heterosis of F hybrids used in this study generally was moderated. The results of this study compare favorably with those of previous studies conducted in closelyplanted ?? . Kernel weight and seeds per spike showed statistically sinificant heterosis in some F₁ hybrids. Heterosis for number of tillers was not found, contrary to previous results reported from space-planted populations. This finding brings into question the general belief that hybrid seeding rates could be somewhat lower than those for adapted parental cultivars and therefore partially compensate for the high cost of hybrid seed. Since this study was conducted in fullstand populations and did afford realistic competition among plants, it reveals more accurately the actual heterosis of wheat hybris which is likely to be obtained in commercial procuction fields than many previous research attempts. The study showed that heterosis for yield components is quite different than that reported from space-planted experiments. The prospect of an extremely lare and continuing market for hybrid wheat seed prompted several commercial seed companies to initiate hybrid wheat development pro-grams. The value of hybrid wheat undoubtedly can be enhanced by further development of improved parental lines. The development of high-yielding, strong-strawed, and disease resistant parental lines with high combining ability and good milling and baking quality will help insure the success of hybrid wheat. Combining of complementary parents to secure the best balance in yield components, and the incorporate of other desirable agronomic characteristics should be emphsized in developing successful F₁ hybrids.

SNuPE Assay 에 의한 겸형적혈구빈혈증 유전자와 L - myc 유전자의 점돌연변이 검증

김병국,최영진,김한집,나도선 한국유전학회 1997 Genes & Genomics Vol.19 No.4

Mutations on the alleles of sickle-cell anemia and human L-myc oncogene were characterized. The sickle cell anemia results from homozygosity of the sickle-cell allele (β^s) at the β-globin gene locus. The β^s allele is caused by A to T mutation at the second position of the sixth codon of the β chain gene, resulting in the replacement of a glutamic acid by a valine in the expressed protein. To detect the β^s allele, we synthesized a 20-oligonucleotide primer. The primer was complementary to the region right next to the point mutation site on the β-globin gene. Therefore, the 3' end of the primer ended right before the point mutation site of the β globin gene. The primer would be annealed with the DNA template and its 3' end would be extended/labeled with radiolabeled nucleotide using Taq DNA polymerase. By adding only [α-^(32)P]dATP, but omitting all dNTPs, the primer was extended/labeled when (S allele was served as template. By adding [α-^(32)P]dTTP instead of [α-^(32)P]dATP, the primer was extended/labeled when wild type allele (β^A) was served as template.