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TAR cloning 법에 의한 인간 및 마우스의 상동성 HPRT 유전자의 분리
도은주,김재우,정정남,박인호,임선희,Do, Eun-Ju,Kim, Jae-Woo,Chung, Chung-Nam,Park, In-Ho,Leem, Sun-Hee 한국생명과학회 2006 생명과학회지 Vol.16 No.6
TAR (Transformation-Associated Recombination) cloning법은 복잡한 고등생물의 게놈으로부터 유전자나 특정 염색체 부위를 선별적 분리를 가능하게 한다. 이 방법은 목적으로 하는 염색체 부위의 주변에 존재하는 비교적 짧은 게놈 염기서열에 대한 정보를 필요로 한다. 이 기술은 출아효모의 spheroplasts 형질전환 동안 목적 유전자를 포함한 게놈 DNA와 그 유전자의 5' 또는 3' 말단 서열 (hook)을 포함하고 있는 TAR vector 사이에 일어나는 상동성 재조합에 의해 이루어진다. 본 연구에서는 TAR cloning 법을 상동성 유전자의 분리에 사용할 수 있는가를 조사하기 위해, 연간과 마우스 게놈의 HPRT 유전자를 선택하였다. 그 결과, 인간과 마우스의 게놈으로부터의 HPRT 유전자의 분리 빈도는 TAR vector로서 hHPRT hook 혹은 mHPRT hook을 사용한 경우에 거의 동일하게 나타났다. 또한 mHPRT 유전자의 gap 부분의 염기서열을 결정하여, 이 부분에 염기서열의 불안정의 요인이 되는 비정상적 특성을 발견하였다. 결론적으로 TAR cloning법을 이용하여 다른 이종 간의 게놈으로부터 상동성 유전자 즉 orthologue의 분리가 가능하였다. 더욱이 TAR cloning 시스템을 이용하여 고등동물 게놈 상에 남아있는 gap 부분을 메움으로서 고등동물의 모든 유전자들의 확인이 가속화될 수 있을 것으로 사료된다. The transformation-associated recombination (TAR) cloning technique allows selective isolation of chromosome regions or genes from complex genome. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosome region of interest. This method involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). To examine whether TAR cloning can be applied to the isolation of gene homologues, we chose the HPRT genes from human and mouse genome. As results, the yield of positive clones for HPRT gene from human and mouse genome when using a TAR vector containing mHPRT hook or hHPRT hook was almost same level. Analysis of the gap regions in mHPRT revealed that they contain abnormalities that could result in instability of the sequences. In conclusion, we were able to use the TAR cloning technology to isolate gene homologue (orthologue) from nonidentical genome. Moreover, the use of the TAR cloning system may accelerate work on closing the remaining gaps in mammalian genome to achieve the goal of annotation of all mammalian genes.
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MLL Gene Expression through Alternative RNA Splicing in Hematopoietic Lineage Specific Cells
Nam, Dong K. 아주대학교 1996 아주의학 Vol.1 No.2
The human MLL gene belongs to the trithorax gene family of which the Drosophila trithorax (trx) gene is known to regulate homeodc genes through alternative RNA splicing. MLL gene rearrangement or tandem duplication is known to be associated with subsequent development of leukemia and solid tumor in recent year. MLL rearrangement or tandem duplication results in the alteration of gene dosage of each putative regulatory domain of MLL gene. As a posttranscriptional gene expression, an alternative RNA splicing is a mechanism lo produce a diverse number of protein isoforms from a single gene by splicing out of intron sequence as well as certain exons which may encode an important regulatory domain. To test if such splicing mechanism operates in MLL during hematopoietic process, mRNA transcripts from human hematopoietic lineage specific cells were evaluated, making use of the reverse transcriptase polymerase chain reaction technique (RT-PCR), PCR cloning and DNA sequencing . It was indicated that cells from different lineage transcribe MLL mRNA species with spliced exons that generally encode putative regulatory domains such as AT hooks (exon 3), repression domain (exon 6), zinc finger motifs (exon 8) and activation domain (exon 18). Such findings suggest that posttranscriptional regulation by alternative RNA splicing may play an important role in MLL gene expression and provides the rationale for a mechanism by which this gene, with multiple functional domains, could produce discrete protein products that may prove critical in the regulation of human homeobox genes which in turn may regulate complex process of normal hematopoiesis.
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Geleta Dugassa Barka,이준대 한국육종학회 2020 Plant Breeding and Biotechnology Vol.8 No.2
The production of chili pepper (Capsicum annuum L.) is hindered by several biotic factors even though stridingprogresses were made in genetic improvement in the last two decades. Among the advancements were the fast-track geneticimprovement of disease-resistant varieties by the use of marker-assisted selection (MAS) and the conventional breeding-based introgressionof major resistance genes. Marker development, marker-based identification and fine mapping have revealed a large numberof resistance genes, from which cloning of some candidate genes demonstrated the applicability and versatility of map-based cloningfor disease resistance. In some of the recent fine mapping of disease resistance QTLs, closely linked DNA markers were identified,which in turn resulted in the rapid introgression of target gene(s) into breeding lines. Also, progresses were made on the characterizationand map-based cloning of resistance genes conferring broad-spectrum resistance. As the number of identified and characterizedresistance genes and the DNA markers linked to resistance genes are steadily generated, the development of multiple/durable resistanceto major chili pepper diseases is accelerated by MAS. In the present review, the development of molecular markers, marker-basedmapping of genes conferring resistance to ten major chili pepper diseases were discussed, focusing on the recent advancements in majorand QTL-spanning resistance gene mapping. The review provides up-to-date insights into the development of DNA markers linked todisease resistance genes and the cloning of resistance genes, which are all so crucial in pepper breeding for disease resistance.
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Decriminalization Path of Gene Editing (Cloning) Embryo Implantation Experiments Related to Human
장광군(张,光君)(Zhang Guang-jun) 원광대학교 법학연구소 2021 의생명과학과 법 Vol.25 No.-
중화인민공화국 「형법」 초안은 “인체 관련 유전자 편집(복제) 배아이식 행위”에 대하여 “국가의 관련 규정을 위반”한 경우가 아니면 범죄에 해당하지 않는다는 조문해석의 여지를 남겼다. 하지만, 「형법」 개정안(11) 제39조는 “국가 관련 규정 위반”의 초안 규정을 삭제함으로써 동 행위는 불법이며, 어떠한 경우에도 허용될 여지가 없음을 명확히 하였다. 이로 인해 인체와 관련한 유전자 편집(복제) 배아이식실험도 당연히 범죄로 되어 처벌대상이 된다. 즉, 이러한 행위는 국가가 별도로 규정한 사항인 ‘유전자 조작 복제배아 불법이식죄’로 사법해석을 할 수는 없지만, 범죄의 불법적 요소를 근거로 실험의 합법을 유도할수도 없다는 것이다. 인체 관련 유전자 편집(복제) 배아이식실험으로 발생하는 죄는 입법기술에 있어 ‘응수형 입법’이자 ‘예방형 입법’으로, 결국 법적 규제의 공백을 막기 위해 어쩔 수 없는 일종의 ‘응급입법’이라 볼 수 있다. 이러한 행위에 대한 법적 규제가 인간의 유전적 안전확보라든지 유전자 편집기술의 연구 등의 질서를 유지하는데 도움이 되겠지만, 헌법상 학술연구의 자유를 침해하고, 유전자 편집 등 첨단기술 발전을 저해하며, 유전적 질병치료와 우생학 발전 등의 기회를 상실할 수도 있다. 따라서 적법성과 합리성인 해석방법을 통해 형사사법실천에서 선별적으로 비범죄화 할지의 여부는 중대한 이론적 문제이자 시급한 현실적 문제이다. 한편, “인체 관련 유전자 편집(복제) 배아이식 행위” 중 그 행위가 심각한 경우에 범죄를 구성한다는 것은 범죄구성의 총체적 규범평가요소로서 이 같은 실험을 비범죄화 할 수 있는 길을 열어준다. 사법해석에는 주관기관의 승인을 받지 않은 경우를 ‘사안의 심각’으로 규정하고 있어 일부 승인된 실험은 범죄가 되지 않을 수도 있다. 그러므로 ‘행위의 심각성’에 대하여 유전자 편집기술과 생명윤리 등을 고려하여 명확한 입법화를 통해 비범죄화 해석이 가능하도록 하여야 할 것이다. Article 39 of the amendment to the criminal law of the people s Republic of China (11) finally deleted the “violation of the relevant provisions of the state” in the draft, which means that the act of gene editing (cloning) embryo implantation related to human body itself is illegal, and there is no premise to be allowed. Gene editing (cloning) embryo implantation experiments related to human also belongs to the punishment scope of this crime. There is no such expression as “violating the relevant provisions of the state” in the accusations of the crime, although it can not be inferred that it is illegal for the judicial interpretation to summarize the crime as “the crime of illegal gene editing and embryo cloning”; However, we can not deduce the legality of such experiments according to the “illegal” elements added in the charges. The legislation of this crime is not only a kind of responsive legislation, but also a kind of preventive legislation. In the final analysis, it is an emergency legislation that must be adopted to prevent the “vacuum period” of legal regulation. Although it is conducive to ensuring the safety of human genetics and maintaining the management order of the research and application of gene editing technology, it violates the freedom of academic research in the constitution, and may also lose the opportunity to develop cutting-edge technologies such as gene editing, treat genetic diseases, develop liberal eugenics and expand basic rights. Therefore, it is an important theoretical issue as well as an urgent practical issue whether we can find a way of interpretation with both legitimacy and rationality to decriminalize such experiments selectively in criminal justice practice. The “serious circumstances” in the description of the crime, as the “integral standard evaluation element” of the crime constitution, can open up a path of decriminalization for such experiments. On the one hand, the situation of “not approved by the competent authority” can be included in the scope of “serious circumstances” by means of timely judicial interpretation, so as to decriminalize some approved experiments. On the other hand, “serious circumstances” is actually forcing the administrative legislation of gene editing (cloning) technology to legislate as soon as possible. In the future, according to the development of gene editing technology and ethics, we can gradually expand the scope of administrative license for such experiments, and find the path of decriminalization from the perspective of the unity of legal order and the relativity of illegal judgment.《中华人民共和国刑法修正案(十一)》第39条最终删除了草案中的“违反国家有关规定”,意味着与人体有关的基因编辑(克隆)胚胎植入行为本身即是非法的,不存在被允许的前提。与人体有关的基因编辑(克隆)胚胎植入实验当然也属于该罪的处罚范围。该罪罪状之中没有“违反国家有关规定”等等类似表述,虽然不能由此推断司法解释将其罪名概括为“非法植入基因编辑、克隆胚胎罪”不合法;但是,也不能根据罪名之中增加的“非法”要素,就推导出此类实验合法。 该罪的立法,既是一种回应型立法,也是一种预防型立法,归根结底是为了防止出现法律规制的“真空期”,而不得已采取的一种应急性立法。它虽然有利于确保人类遗传安全、维护基因编辑技术研究和应用的管理秩序,但是侵犯了宪法上的学术研究自由,也可能丧失发展基因编辑等前沿技术、治疗遗传性疾病、发展自由主义优生学扩大基本权利的机遇。因此,能否寻找到一条兼具合法性ߎ
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Jung, Jin-Sup,Kim, Joo-In,Oh, Sae-Ok,Park, Mi-Young,Bae, Hae-Rhan,Lee, Sang-Ho The Korean Society of Pharmacology 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.2
Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanims. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5'-flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.
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Jin Sup Jung,Joo In Kim,Sae Ok Oh,Mi Young Park,Hae Rhan Bae,Sang Ho Lee 대한생리학회-대한약리학회 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.2
<P> Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanims. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5 -flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.
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Park, Yong-Chjun,Shin, Hee-Jung,Kim, Young-Chang The Microbiological Society of Korea 1999 The journal of microbiology Vol.37 No.3
Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.
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