共查询到20条相似文献,搜索用时 15 毫秒
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Hiroyuki Jikuya Jun Takano Reiko Kikuno Makoto Hirosawa Takahiro Nagase Nobuo Nomura Osamu Ohara 《DNA research》2003,10(1):49-57
To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility. 相似文献
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Dinh Truong Nguyen Younshin Oh Vijaya R. Dirisala Hojun Choi Keun-Kyu Park Jin-Hoi Kim Chankyu Park 《Biotechnology and Bioprocess Engineering》2010,15(5):817-821
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. 相似文献
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The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries. 相似文献
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T Kuno Y Ono M Hirai S Hashimoto H Shuntoh C Tanaka 《Biochemical and biophysical research communications》1987,146(2):878-883
Complementary DNA (cDNA) clones encoding the regulatory subunit of the type I cAMP-dependent protein kinase (R-I) were isolated by screening of rat brain cDNA libraries. A 1.5-kilobase (kb) cDNA insert containing the entire coding region was sequenced and full amino acid sequence has been deduced from the nucleotide sequence. The clone encodes for a protein of 380 amino acids that shows 97% homology to the bovine R-I subunit. Northern blot analysis demonstrated two major mRNA species (2.8 and 4.4 kb in size) in rat brain and liver. 相似文献
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Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen. 总被引:2,自引:0,他引:2
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cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen. 相似文献
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T Nagase K Ishikawa M Suyama R Kikuno M Hirosawa N Miyajima A Tanaka H Kotani N Nomura O Ohara 《DNA research》1999,6(1):63-70
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We have isolated from a rat brain lambda gt11 expression library two overlapping cDNA clones of sizes 2.5 and 3.0 kb corresponding to the heavy neurofilament polypeptide (NF-H). The 2.5 kb insert apparently represents virtually the whole of the C-terminal tail, the 3.0 kb insert also encodes the conserved epitope for the monoclonal antibody, anti-IFA. The identity of the cDNAs was established by comparison of the predicted amino acid sequence with the known partial amino acid sequence of porcine NF-H. A repeat peptide sequence that may be a multiphosphorylation site was identified in the C-terminal non-helical tail. 相似文献