Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.     

日本通草蛉cDNA文库构建及部分ESTs分析

本研究以日本通草蛉Chrysoperla nipponensis (Okamoto)为材料,采用Oligo(dT)引物定向克隆构建cDNA文库并进行EST序列测定,旨在以基因库的形式进行种质资源的保存,为其遗传改良奠定基础,并为探讨其分类地位提供分子依据。对该文库质量分析表明：库容量为1.0×10⁶,重组率为80.0％,平均插入片段为512 bp。测序后最终成功得到323条表达序列标签（expressed sequence tags,ESTs）序列,经Phrap程序聚类拼接后得到236条单基因簇（unigene）,包括86个重叠群（congtigs）和150个单拷贝（singlets）。使用NCBI中的BlastN和BlastX程序对236条ESTs进行本地化搜索,BlastN的结果表明：180条ESTs（76.3%）没有注解,56条ESTs（23.7%）与GenBank上公布的序列有较高的同源性,其中一条序列被确定为该种的16S rRNA基因,利用MEGA软件构建了基于该16S rRNA序列草蛉科的系统发育树,结果显示通草蛉属Chrysoperla与叉草蛉属Dichochrysa、玛草蛉属Mallada、草蛉属Chrysopa的亲缘关系比较近,这与传统分类相吻合。BlastX的比对结果为197条ESTs（83.5%）有功能注解,39条ESTs（16.5%）无注解或score值小于100。使用GO(gene ontology)数据库对236条ESTs序列进行功能注释,结果表明：142条ESTs（59.7％）有注解,并表达出40多种基因产物。     

高山植物黄管秦艽cDNA 文库构建与表达序列标签(EST) 分析

黄管秦艽( Gentiana officinalis) 是一种重要的藏药高山植物, 本研究构建了该物种开花期的cDNA 文库。经检测达到中等cDNA 文库水平, 文库滴度为1 . 2×107 pfu􊄯ml , 重组率95.9% , 插入片段平均长度大于500 bp。对343 个随机挑选的重组克隆进行部分测序, 获得的ESTs 经编辑后共有181 条有效序列。经生物信息学方法分析181 条表达序列标签(EST) 代表144 个单克隆序列, 其中55 个与已鉴定的基因同源, 35 个序列与未鉴定的EST 匹配, 54 个未找到同源序列; 后两者共有89 个EST 序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现, 相关基因主要编码以下蛋白: 与蛋白表达相关的占35%; 光合作用相关的占22%; 新陈代谢相关的占18%; 抗性相关的占11%; 质膜运输和细胞分裂相关的分别占5% ; 染色体变化和细胞信号转导的分别占2%。根据有效EST 序列设计引物, 通过RT-PCR 进一验证了所得EST 的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

Analysis of the expressed genome of the lone star tick, Amblyomma americanum (Acari: Ixodidae) using an expressed sequence tag approach

An expressed sequence tag (EST) approach was used to study the genome of two developmental stages of the lone star tick, Amblyomma americanum. cDNA libraries were constructed from the larval and adult stages of A. americanum. In total, 1942 ESTs were sequenced (1462 adult ESTs and 480 larval ESTs) and analyzed using bioinformatic programs. Contig assembly using the CAPII program revealed 11% and 15% redundancy of sequences in the larval and adult ESTs, respectively. Of the 1942 ESTs, 1738 sequences were considered quality sequences and of these, 771 or approximately 44.4% of the sequences were putatively identified based on amino acid identity using the protein Basic Local Alignment Search Tool (BLAST) algorithm. Putatively identified sequences were classified according to their predicted gene function. In total, 967 sequences, or 55.6% of the quality sequences, had limited or no protein similarity to previously identified gene products. Sequences lacking protein homology were analyzed using an automated sequence annotation system for predicted protein characteristics such as open reading frames, signal peptides, protein motifs, and transmembrane regions. In this paper we describe the sequencing of the largest number of ESTs obtained from an arachnid species to date and the subsequent detailed analysis of these sequences.