cDNA Library를 이용한 생쥐 두개골 유래 조골세포의 유전자 발현 분석

정연관,정재환,최제용 대한골대사학회 2008 대한골대사학회지 Vol.15 No.1

Background: Large-scale sequencing of cDNA clones from cDNA library is a powerful approach for the discovery of novel genes, identification of novel gene family members and definition of gene expression profiles. Listing up of bone related genes is necessary for better understanding of molecular bone physiology and homeostasis. Methods: Primary cultured mouse calvarial osteoblast (MCO) was isolated from mouse embryo at day 17.5. Total RNA was purified from MCO and constructed cDNA library using Stratagen cDNA library contstruction kit. Random selected clones of cDNA library were purified and analyzed the sequence by sequencing analysis. Sequences were identified using BlastN in NCBI mouse EST database. Results: The high quality of MCO cDNA library was constructed showing low background (7.2%) such as no insert clone, mitochondrial DNA, E. coli gene, etc. Total 2361 clones were identified by random sequencing. Except low sequencing quality (207), 2154 sequences were further analyzed. In analyzed 1999 useable clones, 1137 clones were known gene, 231 clones were similar to known genes, 69 clones were novel genes, and 562 clones were expression sequence tag (EST). Majority of identified 1078 clones were detected only once, and the others were over two times. The most abundant genes were type I collagen, α2(I) and α1(I) that were detected 36 and 32 times each. Also osteopontin, actin, Thbs1, Fth and Lgals1 were detected highly. Conclusion: In NCBI EST library database, there is only one library related to mouse osteoblasts. Our results will provide a framework data for understanding the gene expression in osteoblasts.

누에 유충의 cDNA 유전자 은행 제작 및 cDNA 클론의 부분염기서열 분석

김상현,윤은영,강석우,김근영,강석권 한국잠사학회 1996 한국잠사곤충학회지 Vol.38 No.1

To secure the genetic resources of silkworm, Bombyx mori, the cDNA library was constructed with mRNA isolated from fifth instar larvae. Titer of the cDNA library was about1.3 X 106 plaques in total. We presumed that the titer covered all transcripts existed in Bombyx mori. Meanwhile, it is known that partial cDNA sequences, Expressed Sequence Tags (ESTs), have a good value for the discovery of novel genes and the elucidation of their structures. For this purpose, partial cDNA sequencing was carried out from randomly selected cDNA clones in the library. Partial cDNA sequences of 37 clones were determined and an average of 212 nucleotides of sequence can be read from the clone. The ESTs were searched in GenBank database and fifteen ESTs showed significant similarities to enlisted sequences. They included the genes of storage protein, heat shock protein, actin, catalase and so forth. We presumed that the 22 unmatched ESTs were novel genes.

Molecular Characterization of an Anther-Preferential Gene from Rice

Choi, Young-Ju,Ayahiko Shomura,Takuji Sasaki,An, Gynheung,Chung, Yong-Yoon 한국식물학회 2000 Journal of Plant Biology Vol.43 No.4

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones, RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.

민자주방망이버섯(Lepista nuda)의 생채학적특성과 cDNA에 관한 연구

이지선 ( Ji Sun Lee ),김종봉 ( Jong Bong Kim ) 대구가톨릭대학교 자연과학연구소 2014 자연과학연구논문집 Vol.12 No.1

본 연구는 L. nuda의 생태학적 특징과 cDNA를 분석하여 자실체형성 및 유용물질과 관련 있는 유용유전자를 밝히고자 하였다. 생태학적 특징을 밝히기 위하여 채집지의 특징과 토양의 수분함유량, pH, 유기물과 무기물함량 등을 조사하였다. 민자주방망이버섯은 대부분 참나무, 소나무가 우거진 고도가 비교적 낮은 지역에서 낙엽이 쌓인 음지의 습한 장소에 군생을 하였다 수분함유율은 평균 27.84% 이었으며, 유기물함유량은 평균 32.58%로 일반토양의 6.9% 에 비해 매우 높은 함유량을 나타내었다. 조사지역의 토양의 산성도는 평균 pH가 4.26 있었다 토양 미량원소를 분석한 결과는 Mg 은 평균 0.05ppm, Ca은 평균 1. 81ppm, K와 Fe 는 각각 평균 194.61ppm, 1.097.81ppm, N의 함량은 7.08 ppm 을 나타내었다. 이 결과 토양은 비교적 산성을 나타내고 있으며, 유기물의 함량은 보통의 경우보다 상당히 높았다. 토양의 비옥도와 관련이 있는 양이온치환율은 일반적인 삼림토양보다 높아 비옥한 경향을 나타내었다. 또 L.nuda 의 cDNA library 제작 및 cDNA 염기서열 분석을 하였으며 cDNA library 로부터 밝혀낸 DNA 염기서열을 NCBI에서 제공하는 Blastx프로그램을 사용하여 유사성을 비교하였다. 유사성을 비교한 결과는 같은 송이과 버섯으로 재배환경이 비슷한 양송이버섯 (Agaricus bisporus) 의 serine proteinase 에 관여 하는 유전자와 61%의 유사도를 보였고, Histone H2B 에 관여하는 유전자와 89% 유사도를 보였다. 이 결과로 볼 때 L.nuda 의 cDNA 염기서열을 분석한 결과 유사성이 높은 여러가지 유전자를 확인하였다. In this research, ecological characteristics of L. nuda were investigated and its cDNA was analysed to find useful genes related with fruiting body formation and useful substances. The moisture content, pH, kind and content of organic compound of the soil on which L. nuda were collected were analysed. Most of L. nuda grew in moisty and shady spots with leaves of oak and pine trees. Moisture content of the soil was 27.84% on the average, and organic compound of the soil was very high (32.58% on the average) compared sith that of common soil. (6.9%). The pH was 4.36, and Contents of Mg, Ca, K, Fe and N were 0.05ppm, 1018ppm, 182.41ppm, 1036.72pp, and 6.95ppm respectively. These results showed that the soil was acidic relatively, and its organic compound was high compared with that of common soil. Also, its cation exchange capacity related sith soil fertility was high and showed a tendency of fertile soil, compared with that of common forest soil. The cDNA library was constructed and sequenced on L. nuda. Those DNA sequenced were search for homology with those of GenBank using Blastx program. A homology between one sequenced of L. nuda and a serine proteinase gene was 61%. Also, a homology between another sequence of L.nuda and histone H2B of Agaricus bisporus was 89%. These results showed that various gene could be identified using cDNA analysis

Construction of Complementary DNA Library and cDNA Cloning for Cy Strain of Odontoglossum Ringspot Virus Genomic RNA

Ki Hyun Ryu(류기현),Won Mok Park(박원목) 한국식물병리학회 1994 Plant Pathology Journal Vol.10 No.3

참나물 현탁배양세포 유래 배발생캘러스에서 HD-Zip 유전자, Phc5의 클로닝과 특성

손수인,김준철 한국식물생명공학회 1999 식물생명공학회지 Vol.26 No.2

참나물 (Pimpinella brachycarpa)의 엽병 (petiole)절편체로부터 캘러스가 MS배지 (0.5mg/L 2,4-D와 0.1mg/L BAP)에서 유도되었으며 이들 캘러스로부터 치밀하게 배열된 세포집단(cell cluster)을 선발하여 현탁배양하였다. 이들 현탁배양세포들은 0.1 mg/L NAA가 포함된 MS고체배지에 배양되어 배발생 (embryogenic) 캘러스로 성장하였다. 배발생캘러스는 연한 노란색을 띠며 체세포배로 분화되었으며 이들 체세포배는 MS액체배지에서 발아되어 식물체로 성장하였다. 참나물 현탁배양세포 유래 배발생캘러스로부터 분리한 mRNA로부터 cDNA library를 합성하여 PCR을 수행한 결과 제조된 library의 삽입절편의 크기가 대부분 500bp이상임을 확인하였다. 이들 cDNA library로부터 전체 1.5 $\times$$10^{6}$개의 plaque를 혼성화하여 일차의 screening을 통해 19개의 cDNA clone을, 이차의 screening을 통해 5개의 cDNA clone을 얻었으며 이중 4개의 cDNA clone은 참나물 shoot의 HD-Zip 유전자인 Phz4 유전자와 동일한 약 1.4 kb 정도인 것으로 나타났으나, 1개의 cDNA clone, Phc5는 약 1.5kb정도의 크기를 나타내었다. 1.5kb인 Phc5는 Phz4유전자의 5'쪽으로 163bp의 염기가 추가로 발견되어 총 1,531 bp에 해당하였으며 18개의 polyA tail을 가지고 있었다. Phc5는 284번째에 ATG개시코돈이 있고 302개의 아미노산을 암호화하는 906개의 단백질 암호화 부위와 Homeodomain을 갖고 있었다. Phc5로부터 추정된 단백질은 기존 전사조절자에서 많이 보고된 HD의 구조적 특징을 갖고 있었다. Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

신 바이오디젤 원료 작물인 Camelina의 cDNA library 제작 및 유전자 특성

박원,장영석,안성주 한국작물학회 2010 Korean journal of crop science Vol.55 No.2

지금까지 양구슬냉이의 유전정보는 거의 연구되지 않았으므로 우리는 양구슬냉이의 잎으로부터 cDNA library를 제작하고 발현유전자의 종류와 기능별 분류를 조사하였다. 그 결과를 요약하면 다음과 같다. 1. cDNA library에서 1334개 의 클론들을 얻었고 삽입된 단편들의 염기서열의 평균길이는 736bp였다. 우리는 1269개의 high-quality expressed sequence tags (ESTs) 서열을 얻었다. 이러한 EST의 클러스터 분석결과 고유 염기서열(unigene)을 가진 유전자의 수는 851개를 나타냈다. 2. Unigene 476개는 GeneBank에 기능이 알려진 유전자들과 고도의 상동성을 나타내었다. 다른 375개의 unigene들은 기능이 알려지지 않은 것들이었다. 나머지 63개는 NCBI데이터베이스에 어떤 유전자와도 상동성을 보이지 않았고 이러한 유전자들은 아마도 양구슬냉이의 잎에서 발현되는 새로운 유전자일 것으로 보인다. 3. 데이터베이스에서 상동성을 나타낸 EST들을 기능별 주석에 따라서 17개의 카테고리로 분류하였다. 대표적으로 가장 분포도가 높은 카테고리는 결합 기능 또는 보조인자 요구의 단백질(27%), 대사(11%), 세포 소기관 위치(11%), 세포수송과 수송기관 그리고 수송 경로(7%), 에너지(6%), 대사와 단백질 기능의 조절(6%) 등이 있다. 이러한 우리의 연구 결과는 양구슬냉이의 유용한 유전적 자원과 전반적인 mRNA 발현 정보를 제공해 줌으로써 대체 에너지 작물로 떠오르는 양구슬냉이의 다양한 분자적 연구에 기여할 것으로 사료된다. Camelina sativa L., known as popular names "gold-of-pleasure" or "false flax" is an alternative oilseed crop that can be grown under different climatic and soil conditions. Up to date, however, the genomic information of Camelina has not been studied in detail. Therefore, a cDNA library was constructed and characterized from young leaves. The constructed cDNA library incorporated of 1334 cDNA clones and the size of the insertion fragments average was 736 base pair. We generated a total of 1269 high-quality expressed sequence tags (ESTs) sequences. The result of cluster analysis of EST sequences showed that the number of unigene was 851. According to subsequent analysis, the 476 (55.9%) unigenes were highly homologous to known function genes and the other 375 (44.1%) unigenes were unknown. Remaining 63 (7.4%) unigenes had no homology with any other peptide in NCBI database, indicating that these seemed to be novel genes expressed in leaves of Camelina. The database-matched ESTs were further classified into 17 categories according to their functional annotation. The most abundant of categories were "protein with binding function or cofactor requirement (27%)", "metabolism (11%)", "subcellular localization (11%)", "cellular transport, transport facilities and transport routes (7%)", "energy (6%)", "regulation of metabolism and protein function (6%)". Our result in this study provides an overview of mRNA expression profile and a basal genetic information of Camelina as an oilseed crop.

Identification and phylogenetic analysis of the human endogenous retrovirus HERV-W pol in cDNA library of human fetal brain

Kim, Heui-Soo,Jeon, Seung-Heui,Yi, Joo-Mi,Kim, Tae-Hyung,Lee, Won-Ho Korean Society of Life Science 2003 생명과학회지 Vol.13 No.3

인간 내생 레트로바이러스 HERV-W는 다발성 경화증 환자로부터 탐지된 MSRV와 연루되어 있다. 인간 태아의 뇌로부터 유래된 cDNA library를 이용하여 PCR법으로 2개의 HERV-W 패밀리(HWP-FB10과 HWP-FB12)를 동정하고 분석하였다. 그들은 HERV-W (accession no. AF009668)와 89%의 염기서열의 유사성을 보였다. Pol 유전자를 아미노산의 서열로 분석해 본 결과 점돌연변이 또는 삽입/결실로 말미암아 frameshift 및 종결코돈을 나타내었다. 유전자정보의 데이터베이스를 이용하여 HERV-W 패밀리간의 분자계통분류도를 작성해 본 결과 HWP-FB10은 인간의 염색체 7q21-22로부터 유래된 AC000064와 매우 가깝게 관련되어 있음을 시사하였다. 이들의 새로운 HERV-W pol 패밀리가 이웃하는 어떤 유전자와 상호 연결되어 있으며, 어떠한 기능을 수행하는지에 대한 전망에 대해 토의하였다. A human endogenous retroviral family (HERV-W) has recently been described that is related to multiple sclerosis-associated retrovirus (MSRV) sequences that have been identified in particles recovered from monocyte cultures from patients with multiple sclerosis. Two pol fragments (HWP-FB10 and HWP-FBl2) of HERV-W family were identified and analysed by the PCR approach with cDNA library of human fetal brain. They showed 89 percent nucleotide sequence similarity with that of the HERV-W (accession no. AF009668). Deletion/insertion or point mutation in the coding region of the pol fragments from human fetal brain resulted in amino acid frameshift that induced a mutated protein. Phylogenetic analysis of the HERV-W family from GenBank database indicates that the HWP-FB10 is very closely related to the AC000064 derived from human chromosome 7q21-q22. Further studies on the genetic relationship with neighbouring genes and functional role of these new HERV-W pol sequences are indicated.

A Simple, Rapid, Efficient and Inexpensive Strategy for Sequencing Clones from cDNA Libraries

Nguyen, Dinh Truong,Oh, Youn-Shin,Dirisala, Vijaya R.,Choi, Ho-Jun,Park, Keun-Kyu,Kim, Jin-Hoi,Park, Chan-Kyu 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.5

In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 ~ 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.

큰28점박이무당벌레 cDNA libarary 제작과 dsRNA 주입을 통한 RNAi의 기능 탐색

김정희,김정규,권혜리,민지현,박상은,윤헌,장현주,조민규,유용만,윤영남 한국응용곤충학회 2016 한국응용곤충학회 학술대회논문집 Vol.2016 No.10

큰28점박이무당벌레(Henosepilachna vigintioctomaculata)는 감자, 토마토, 구기자 등 가지과 식물을 가해하는 저작형 해충이다. 한편, 최근에 RNA interference(RNAi)를 이용해 해충을 방제하려는 연구들이 많이 진행되고 있다. 따라서 이 실험은 RNAi를 이용하여 큰28점박이무당벌레 방제 및 유전자의 기능을 알아보고자 수행되었다. 먼저, RNAi에 이용할 target 유전자를 선발하기 위해 cDNA library를 제작하였다. 그 결과, 최종 titer 3.15×105의 cDNA library를 완성하였다. 완성한 cDNA library는 제한효소를 이용해 LITMUS 28i transcription vector에 cloning하였고, 이 후 insert의 크기와 중복성을 확인하였다. 그 결과, RNAi에 적합한 약 100~500bp 크기의 insert가 확인되었고, NCBI Blast search 결과, insert 사이에 중복성 없이 다양한 유전자가 cloning되었고, 대부분이 곤충 유전자임을 확인하였다. 확인된 유전자들은 T7 promoter를 이용해 dsRNA를 합성한 후, 0~2일된 큰28점박이무당벌레 4령 유충에 주입하였다. 앞으로의 실험에서는 RNAi 효과를 나타낸 유전자에 대해 Real-time PCR을 통해 큰28점박이무당벌레 체내 유전자 발현량 변화를 확인할 예정이다.