家蚕母性基因的表达序列标签分析

为了研究家蚕Bombyx mori卵中储存的母性遗传信息,以未受精的肾脏型和正常型蚕卵为材料构建了cDNA文库,并对其随机测序,共获得391条表达序列标签(EST)序列,经拼接后得到374条非重复序列。生物信息学分析发现,172个非重复序列与国际数据库GenBank和silkbase中的已知基因具有较高的相似性。除3个已知母性基因外,其余主要与DNA复制、能量代谢、信号转导、转录、蛋白质结构以及胚胎发育中个体形成等相关。该结果可为进一步研究家蚕胚胎早期发育的调控提供基础数据。     

An evolutionary expressed sequence tag analysis of Drosophila spermatheca genes

This study investigates genes enriched for expression in the spermatheca, the long-term sperm storage organ (SSO) of female Drosophila. SSO genes are likely to play an important role in processes of sexual selection such as sperm competition and cryptic female choice. Although there is keen interest in the mechanisms of sexual selection at the molecular level, very little is known about the female genes that are involved. In the present study, a high proportion of genes enriched for expression in the spermatheca are evolving rapidly. Most of the rapidly evolving genes are proteases and genes of unknown function that could play a specialized role in the spermatheca. A high percentage of the rapidly evolving genes have secretion signals and thus could encode proteins that directly interact with ejaculate proteins and coevolve with them. In addition to identifying rapidly evolving genes, the present study documents categories of genes that could play a role in spermatheca function such as storing, maintaining, and utilizing sperm. In general, candidate genes discovered in this study could play a key role in sperm competition, cryptic female choice of sperm, and sexually antagonistic coevolution, and ultimately speciation.     

Microarray analysis identifies genes preferentially expressed in the lung schistosomulum of Schistosoma mansoni

The lung schistosomulum of Schistosoma mansoni is a validated target of protective immunity elicited in vaccinated mice. To identify genes expressed at this stage we constructed a microarray, representing 3088 contigs and singlets, with cDNA derived from in vitro cultured larvae and used it to screen RNA from seven life-cycle stages. Clustering of genes by expression profile across the life cycle revealed a number of membrane, membrane-associated and secreted proteins up-regulated at the lung stage, that may represent potential immune targets. Two promising secreted molecules have homology to antigens with vaccine and/or immunomodulatory potential in other helminths.     

Identification of key genes involved in root development of tomato using expressed sequence tag analysis

Root system of plants are actually fascinating structures, not only critical for plant development, but also important for storage and conduction. Due to its agronomic importance, identification of genes involved in root development has been a subject of intense study. Tomato is the one of the most consumed vegetables in the world. Tomato has been used as model system for dicot plants because of its small genome, well-established transformation techniques and well-constructed physical map. The present study is targeted to identify of root specific genes expressed temporally and also gene(s) involved in lateral root and profuse root development. A total of 890 ESTs were identified from five EST libraries constructed using SSH approach which included temporal gene regulation (early and late) and genes involved in morphogenetic traits (lateral and profuse rooting). One hundred sixty-one unique ESTs identified from various libraries were categorized based on their putative functions and deposited in NCBI-dbEST database. In addition, 36 ESTs were selected for validation of their expression by RT-PCR. The present findings will help in shedding light to the unexplored developmental process of root growth in tomato and plant in general.     

Heart-specific genes revealed by expressed sequence tag (EST) sampling

Background  

Cardiovascular diseases are the primary cause of death worldwide; the identification of genes specifically expressed in the heart is thus of major biomedical interest. We carried out a comprehensive analysis of gene-expression profiles using expressed sequence tags (ESTs) to identify genes overexpressed in the human adult heart. The initial set of genes expressed in the heart was constructed by clustering and assembling ESTs from heart cDNA libraries. Expression profiles were then generated for each gene by counting their cognate ESTs in all libraries. Differential expression was assessed by applying a previously published statistical procedure to these profiles.     

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.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Redundancy based detection of sequence polymorphisms in expressed sequence tag data using autoSNP

AutoSNP is a program to detect single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (indels) in expressed sequence tag (EST) data. The program uses d2cluster and cap3 to cluster and align EST sequences, and uses redundancy to differentiate between candidate SNPs and sequence errors. Candidate polymorphisms are identified as occurring in multiple reads within an alignment. For each candidate SNP, two measures of confidence are calculated, the redundancy of the polymorphism at a SNP locus and the co segregation of the candidate SNP with other SNPs in the alignment. AVAILABILITY: The program was written in PERL and is freely available to non-commercial users by request from the authors.     

Dinoflagellate expressed sequence tag data indicate massive transfer of chloroplast genes to the nuclear genome

The peridinin-pigmented plastids of dinoflagellates are very poorly understood, in part because of the paucity of molecular data available from these endosymbiotic organelles. To identify additional gene sequences that would carry information about the biology of the peridinin-type dinoflagellate plastid and its evolutionary history, an analysis was undertaken of arbitrarily selected sequences from cDNA libraries constructed from Lingulodinium polyedrum (1012 non-redundant sequences) and Amphidinium carterae (2143). Among the two libraries 118 unique plastid-associated sequences were identified, including 30 (most from A. carterae) that are encoded in the plastid genome of the red alga Porphyra. These sequences probably represent bona fide nuclear genes, and suggest that there has been massive transfer of genes from the plastid to the nuclear genome in dinoflagellates. These data support the hypothesis that the peridinin-type plastid has a minimal genome, and provide data that contradict the hypothesis that there is an unidentified canonical genome in the peridinin-type plastid. Sequences were also identified that were probably transferred directly from the nuclear genome of the red algal endosymbiont, as well as others that are distinctive to the Alveolata. A preliminary report of these data was presented at the Botany 2002 meeting in Madison, WI.     

Analysis of expressed sequence tag loci on wheat chromosome group 4

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoeologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.     

Identification of genes up-regulated during conidiation of Fusarium oxysporum through expressed sequence tag analysis

Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. F. oxysporum produces microconidia and macroconidia in carboxymethyl cellulose-added liquid medium (CMCLM) and exhibits vegetative growth without conidiation in complete liquid medium (CLM). The cDNA libraries were constructed using mRNAs from CLM and CMCLM cultures. A total of 1288 and 1353 clones from CLM (vegetative growth) and CMCLM (conidiation) libraries, respectively, were sequenced, and 641 and 626 unique genes were identified. Of these unique genes, only 130 ( approximately 20%) were common in the two libraries, indicating different patterns of gene expression during vegetative growth and conidiation. The expression levels of 496 CMCLM-specific genes were compared during vegetative growth and conidiation by cDNA dot-blot differential hybridization and real-time quantitative PCR analyses, and 42 genes were identified to display >5-fold increases in mRNA abundance during conidiation. These genes provide ideal candidates for further studies directed at understanding fungal conidiogenesis and its molecular regulation.     

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.     

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.     

Schistosoma mansoni: stage-specific expression of muscle-specific genes

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, 1985). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, 1989). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.     

Analysis of an expressed sequence tag library from Dicrocoelium dentriticum

Dicrocoeliosis caused by Dicrocoelium dendriticum is an important liver disease, which affects ruminants all around the world. Despite the significant economic losses caused by this trematode, molecular knowledge is very scarce. In fact, there is no information in the expressed sequence tag (EST) database about the parasite. Furthermore, the immunological diagnosis of dicrocoeliosis remains unsatisfactory, and there aren’t available recombinant proteins that could be tested in the diagnosis. For this reason a cDNA library was constructed with mRNA extracted from D. dendriticum adults for first time. A random preliminary screening of 230 phage plaques from the library resulted in the identification of 173 new EST. The deduced proteins expressed by these genes have been described as possible vaccine targets in other trematodes, and/or as relevant diagnosis antigens. Then, our goal was to identify D. dentriticum diagnosis genes to be used as recombinant antigens in the specific immunological diagnosis of the trematodoses. A D. dendriticum cDNA encoding an 8-kDa recombinant protein has been cloned, expressed in Escherichia coli and evaluated in dicrocoeliosis diagnosis using both Western Blot and enzyme-linked immunosorbent assay (ELISA). The recombinant expression molecule has demonstrated its value as a diagnosis antigen of dicrocoeliosis, able to discriminate between positive and controls on day 30 post infection. This is the first research conducted for identification and characterization of D. dendriticum ESTs, which can serve as a starting point for future research on immunodiagnosis and immunoprofilaxis of dicrocoeliosis.