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为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物,利用一种新的cDNA末端快速扩增方法(SMARTRACE)扩增该EST的5′末端,并进行克隆测序,与mRNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA.结果表明,SMARTRACE是一种简便、有效的克隆cDNA5′末端未知序列的技术.
Abstract:To clone the full-length cDNAs of genes related to spermatogenesis,ESTs obtained by mRNA differential display were used to design gene-specific primer.Then SMART RACE was performed to obtain the 5′ region of these ESTs.After cloning,sequencing and splicing with ESTs obtained by mRNA differential display,three full-length cDNAs were obtained.The results indicate that SMART RACE is a simple and an effective technique for cloning 5′-end unknown sequence of gene. 相似文献
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Otsuki Tetsuji; Ota Toshio; Nishikawa Tetsuo; Hayashi Koji; Suzuki Yutaka; Yamamoto Jun-ichi; Wakamatsu Ai; Kimura Kouichi; Sakamoto Katsuhiko; Hatano Naoto; Kawai Yuri; Ishii Shizuko; Saito Kaoru; Kojima Shin-ichi; Sugiyama Tomoyasu; Ono Tetsuyoshi; Okano Kazunori; Yoshikawa Yoko; Aotsuka Satoshi; Sasaki Naokazu; Hattori Atsushi; Okumura Koji; Nagai Keiichi; Sugano Sumio; Isogai Takao 《DNA research》2005,12(2):117-126
We have developed an in silico method of selection of humanfull-length cDNAs encoding secretion or membrane proteins fromoligo-capped cDNA libraries. Fullness rates were increased toabout 80% by combination of the oligo-capping method and ATGpr,software for prediction of translation start point and the codingpotential. Then, using 5'-end single-pass sequences, cDNAs havingthe signal sequence were selected by PSORT (signal sequencetrap). We also applied secretion or membrane protein-relatedkeyword trap based on the result of BLAST search againstthe SWISS-PROT database for the cDNAs which could not be selectedby PSORT. Using the above procedures, 789 cDNAs were primarilyselected and subjected to full-length sequencing, and 334 ofthese cDNAs were finally selected as novel. Most of the cDNAs(295 cDNAs: 88.3%) were predicted to encode secretion or membraneproteins. In particular, 165(80.5%) of the 205 cDNAs selectedby PSORT were predicted to have signal sequences, while 70 (54.2%)of the 129 cDNAs selected by keyword trap preservedthe secretion or membrane protein-related keywords. Many importantcDNAs were obtained, including transporters, receptors, andligands, involved in significant cellular functions. Thus, anefficient method of selecting secretion or membrane protein-encodingcDNAs was developed by combining the above four procedures. 相似文献
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目的:构建莱芜猪肝脏组织全长cDNA文库,以便研究与莱芜猪优良性状相关的基因。方法:采用改良的异硫氰酸酸胍一步法制备总RNA;利用SMART技术,以PrimeScript反转录酶逆转录合成第一链cDNA,通过LD-PCR扩增获得cDNA双链;经蛋白酶K消化和CHROMA SPIN-400柱分级分离后,收集500 bp以上的cDNA片段,并与pMD18-T载体连接,转化大肠杆菌DH5α感受态细胞,建成原始文库;随机挑取单菌落,用HindⅢ和EcoRⅠ进行双酶切鉴定重组子插入片段大小。结果:经鉴定,原始文库的滴度为2.8×105 cfu/mL,重组率约为98%,插入片段大小为0.5~2 kb,平均插入片段长度大于1 kb。结论:建立的cDNA文库质量良好,可以用于目的基因的筛选。 相似文献
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Identification of a Full-Length cDNA for an Endogenous Retrovirus of Miniature Swine 总被引:28,自引:3,他引:28
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Donna E. Akiyoshi Maria Denaro Haihong Zhu Julia L. Greenstein Papia Banerjee Jay A. Fishman 《Journal of virology》1998,72(5):4503-4507
Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attach- ment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation. 相似文献
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Rexroad CE Coleman RL Gustafson AL Hershberger WK Killefer J 《Marine biotechnology (New York, N.Y.)》2002,4(1):12-16
The efficiency of developing polymorphic microsatellite markers from 2 repeat enriched libraries was evaluated. Thirty-six
polymorphic microsatellite markers were developed for rainbow trout, 27 of which were informative in a mapping family. The
ability of each marker to amplify genomic DNA from other salmonids was also observed.
Received March 1, 2001; accepted May 1, 2001. 相似文献
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Flinn B Rothwell C Griffiths R Lägue M DeKoeyer D Sardana R Audy P Goyer C Li XQ Wang-Pruski G Regan S 《Plant molecular biology》2005,59(3):407-433
To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome
Project (CPGP) carried out 5′ end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar,
generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged
tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date
have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library
has enhanced the 3′ ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold
storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged
tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST
sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique
sequences.
Electronic Supplementary Material Electronic Supplementary material is available for this article at
and accessible for authorised users. 相似文献
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von Brunn A Teepe C Simpson JC Pepperkok R Friedel CC Zimmer R Roberts R Baric R Haas J 《PloS one》2007,2(5):e459
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies. 相似文献
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Dongliang Zhang Chungen Hu Yidan Ouyang Jialing Yao 《Plant Molecular Biology Reporter》2012,30(1):46-54
Eulaliopsis binata, which is a close relative of cereal crops, was recognized as an important research material owing to its high frequency
of apospory and autonomous endosperm formation. However, little information is known about its genomics and regulatory pathway
participating in reproductive development. For the first step to understand molecular basis in sabaigrass (E. binata), a SMART complementary DNA library from the inflorescence tissue was constructed and characterized. The titers of original
and amplified libraries were 5.53 × 106 and 1.49 × 1010 pfu/ml, respectively. The percentage of recombinants was 96% in the original library. Analysis of sequencing results of 398
out of 437 randomly picked clones showed that 271 (68.1%) expressed sequence tags (ESTs) exhibited significant similarity
with known putative functional nucleotide sequences in the GenBank databases, 25 (6.3%) ESTs have significant matches with
hypothetical proteins, putative proteins, and unknown proteins, and the other 25.6% ESTs had no significant similarity to
sequences in the public databases. Based on molecular function of GO annotation, the four most abundant terms are nucleotide
binding, hydrolase activity, ion binding, and protein binding, and these genes were involved in 61 different pathways using
the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Besides, simple sequence repeats detection in 398 ESTs was carried out, and several genes were chosen to
perform expression analysis. This report represents a first step in expanding molecular-genetic analyses in E. binata and can be used to optimally mine useful information from a relatively small data set. 相似文献