Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum

Abstract We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans . Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans , 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.     

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.     

ES cell neural differentiation reveals a substantial number of novel ESTs

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease. Electronic Publication     

Characterization of a Human Fovea cDNA Library and Regional Differential Gene Expression in the Human Retina

The primate fovea is the region of the retina responsible for acute vision. This region constitutes less than 5% of the total area of the retina and has not been intensely studied at the molecular level. As a first step in the molecular characterization of the fovea, we have constructed a primary human fovea cDNA library. Experiments confirm that our cDNA library reflects a nonbiased distribution of foveal expressed sequences. Single-pass sequencing was performed on 209 randomly isolated clones from this library. Analysis of the sequences generated reveals that the distributions of fovea clones with either human mitochondrial gene sequences or repetitive elements are different than those observed in cDNA libraries made from other tissues. A significant number of the fovea expressed sequence tags (ESTs) (88, 42.1%) represent novel human ESTs. This suggests that the library will be useful in identifying new human genes. Northern analysis of specific fovea ESTs defined in this study suggests that there are significant quantitative differences in gene expression that distinguish the fovea from the rest of the retina.     

小麦抗病基因表达谱中的文库构建与筛选方法研究

以抗白粉病品系“百农 32 17×Mardler”BC5F4为材料 ,构建了白粉病菌诱导的普通cDNA文库和抑制消减杂交(SSH)cDNA文库。分别对两文库进行了一定规模的测序 ,获得普通cDNA文库不重复ESTs 387条和SSHcDNA文库ESTs 76 0条。将获得的ESTs与GenBank序列进行了BLASTn、BLASTx同源性分析。结果表明 :在普通文库中 ,一些参与光合作用与核糖体构成等的基因出现频率较高 ,而获得的抗病相关基因则较少。消减文库在构建方法、抗病相关基因的富集等方面具有明显的优越性 ,是目前抗病基因表达谱研究中的较好方法。利用高密度点阵膜杂交技术对两文库的筛选结果表明 ,该方法具有相对简便易操作、杂交膜可反复使用等优点 ;但也存在mRNA及同位素用量大等问题。经筛选 ,消减文库中有 5 4 1%的功能已知ESTs为抗病相关基因 ,被证明参与了小麦抗白粉病反应     

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eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。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％;光合作用相关的占笠％;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。     

Expressed sequence tags of Trichinella spiralis muscle stage larvae

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.     

Identification of preferentially expressed mRNAs in retina and cochlea

To search for genes that could be involved in genetic disorders primarily involving the retina and the cochlea, we tried to identify mRNAs preferentially expressed in retina and cochlea and to establish their chromosomal localization. Two approaches were employed. First, a mouse subtracted library (retina + cochlea against liver + brain) was generated. Randomly selected cDNA clones were sequenced and compared to databases. Tissue expression of some of them was analyzed by RT-PCR. Using radiation hybrid cell lines, the mouse chromosomal localization was determined for those showing the highest level in the retina and the cochlea. Second, human Expressed Sequence Tags (ESTs) with preferential expression in the retina and the cochlea were searched for in databases, and chromosomal localization was also established. From 171 sequenced clones, 73 were classified as known genes (with 17 clones coding for 6 genes), 86 were homologous to ESTs, and 12 were unidentified. Of 108 selected clones, 22 (18.5%) had the highest level of expression in the retina and/or the cochlea, while expression was higher in another tissue or ubiquitous for 60 (55.5%) and 22 (20.4%) of them, respectively. By RT-PCR, one clone similar to the mouse Asic3 cDNA (proton-gated channel) was found mainly in the retina and cochlea, but its human ortholog was widely expressed. We selected 17 ESTs from the UniGene database with restricted expression including in the retina and cochlea. We mapped 10 of these ESTs as well as four mouse clones from the subtracted library. Some of them localized to morbid intervals. The combined information from expression analysis and chromosomal localization allowed for the identification of potential candidate genes for retinal diseases (CORD8, CORD9) and syndromic blindness/deafness/renal defects.     

An expression cDNA library for suppression cloning in yeast mutants, complementation of a yeast his4 mutant, and EST analysis from the symbiotic basidiomycete Hebeloma cylindrosporum.

An oriented expression library was constructed from the mycelia of the symbiotic model fungus Hebeloma cylindrosporum in the high-level yeast expression vector pDR196. DNA sequencing of approximately 500 expressed sequence tags (ESTs) showed that 15% correspond to known genes, two thirds contain sequences with unknown function, andthe remaining 20% showed no significant similarity to any known genes. The ESTs had a GC content between 44 and 56%, with most of them having a GC content of 52-54%, which could be correlated with GC contents of fungal genes. The library was successfully used to identify the Hebeloma HIS4 gene by functional complementation of a yeast his4 mutant. Thus, the library may serve as a powerful tool for identification and characterization of mycorrhizal genes by EST analysis and for the identification of ectomycorrhizal genes by means of suppression cloning.     

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

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.     

Expressed sequence tags from eyestalk of kuruma prawn, Marsupenaeus japonicus

We analyzed the expressed sequence tags (ESTs) obtained from a cDNA library of the eyestalk of the kuruma prawn, Marsupenaeus japonicus, to examine gene expression profile with special focus on female reproduction. The assembly of 1988 ESTs created 136 contigs from 738 ESTs; however 1250 ESTs remained singletons. Significant similarities (blast score > or = 50 bits) to the DNA sequences in the databank were found for only 16.7% of the 1386 sequences (136 contigs plus 1250 singletons), suggesting that the eyestalk library contains many unknown genes. Ribosomal RNA and mitochondrial respiration enzymes with significant similarities were found abundantly in the ESTs, whereas genes related to maturation or endocrine systems were scarce. Three ESTs were assumed to encode novel eyestalk hormones with marked similarities to pigment-dispersing hormone, molt-inhibiting hormone and crustacean hyperglycemic hormone. Sequences encoding a product highly homologous to farnesoic acid O-methyltransferase, an enzyme that produces methyl farnesoate, were also found.     

Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body

Insect immune processes are mediated by programs of differential gene expression. To understand the molecular regulation of the immune response in the tobacco hornworm, Manduca sexta, the relevant subset of differentially expressed genes of interest must be identified, cloned and studied in detail. In this study, suppression subtractive hybridization, a PCR-based method for cDNA subtraction was performed to identify mRNAs from fat body of immunized larvae that are not present (or present at a low level) in control larvae. A subtracted cDNA library enriched in immune-inducible genes was constructed. Northern blot analysis of a sample of clones from our subtracted library indicated that >90% of the clones randomly selected from the subtracted library are immune inducible. Sequence analysis of 238 expressed sequence tags (ESTs) revealed that 120 ESTs, representing 54 distinct genes or gene families, had sequences identical or similar to previously characterized genes, some of which have been confirmed to be involved in innate immunity. These ESTs were categorized into seven groups, including pattern recognition proteins, serine proteinases and their inhibitors, and antimicrobial proteins. 112 ESTs, about 47.5% of the library, showed no significant similarity to any known genes. The sequences identified in this M. sexta library reflect our knowledge of insect immune strategies and may facilitate better understanding of insect immune responses.     

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。     

从烟草EST微卫星标记中筛选合子胚中优势表达的基因

为获得烟草合子胚中的优势表达基因,利用CAP3程序对来自烟草公共数据库的EST序列进行组装,利用MISA程序从组装后的EST中筛选SSR位点,将多态性的SSR位点在烟草合子文库中进行扩增并对等位基因进行分析。结果表明:具有多态性的16个SSR标记中,有9个基因能从烟草的合子库中成功扩增得到。该研究为筛选烟草合子胚中优势表达基因提供新的途径。