Transcriptome characterization of Ephedra sinica with 454 ESTs / 中国中药杂志

Using the latest 454 GS FLX platform and Titanium regent, a substantial expressed sequence tag (ESTs) dataset of Ephedra sinica was produced, and the profile of gene expression and function gene of which were investigated. A total of 48 389 reads with an average length of 373 bp were generated. These 454 reads were assembled into 18 801 unigenes, which were all 454 sequencing identified. A total number of 10 531 unigenes(56.0%) were annotated using BLAST searches (E-value≤1×10⁻⁵) against the Nr, Nt, TAIR, SwissProt and KEGG databases. With respect to genes related to ephedrine biosynthesis, 19 unigenes(encoding 9 enzymes) were found. A total of 97 putative genes encoding cytochrome P450s were also discovered. Data presented in this study will provide an important resource for the scientific community that is interested in the functional genomics and secondary metabolism of E. sinica.

Development and characterization of a high temperature stress responsive subtractive cDNA library in Pearl Millet Pennisetum glaucum (L.) R.Br.

Pearl millet (Pennisetum glaucum L. R. Br.) is an important cereal crop grown mainly in the arid and semi-arid regions of India known to possess the natural ability to withstand thermal stress. To elucidate the molecular basis of high temperature response in pearl millet, 12 days old seedlings of P. glaucum cv. 841A were subjected to heat stress at 46°C for different time durations (30 min, 2, 4, 8, 12 and 24 h) and a forward subtractive cDNA library was constructed from pooled RNA of heat stressed seedlings. A total of 331 high quality Expressed Sequence Tags (ESTs) were obtained from randomly selected 1050 clones. Sequences were assembled into 103 unique sequences consisting of 37 contigs and 66 singletons. Of these, 92 unique sequences were submitted to NCBI dbEST database. Gene Ontology through RGAP data base and BLASTx analysis revealed that about 18% of the ESTs showed homology to genes for “response to abiotic and biotic stimulus”. About 2% of the ESTs showed no homology with genes in dbEST, indicating the presence of uncharacterized candidate genes involved in heat stress response in P. glaucum. Differential expression of selected genes (hsp101 and CRT) from the SSH library were validated by qRT-PCR analysis. The ESTs thus generated are a rich source of heat stress responsive genes, which can be utilized in improving thermotolerance of other food crops.

Establishment of cDNA Library and EST Analysis from Leaves of Phyllanthus amarus.

DNA sequencing of randomly chosen clones from a cDNA library allows thousands of different transcripts to be identified. However, since the likelihood of observing a given transcript is proportional to the expression level of that transcript in the tissue from which the library is derived, often transcripts are represented by several EST sequences. An expressed sequence tags (EST) analysis was undertaken to identify the genes present in the leaves of Phyllanthus amarus, which is a small tropical, glabrous herb with several health benefits. Phyllanthin and hypophyllanthin, major bioactive components, present in highest amounts in the leaves, are of significant therapeutic importance like hepatoprotective, antioxidant, antiviral, hypoglycemic, etc. Taken together, sequencing of cDNA clones generated high-quality ESTs (Accession number: JK492908 to JK492964) with high similarities with genes from Ricinus communis, Onchocerca volvulus, Eucalyptus globules, Gossypium hirsutum, Nicotiana tabacum, Solanum spp. and many more. A BLASTN analysis along with BLASTX analysis of all the unique sequences was performed and was grouped according to the reported activities. Results represented here is the first reference collection of ESTs from this commercially important medicinal herb. This study indicated that the leaf transcriptome contains series of interesting sequences like ALBINO3, ribulose-1, 5 bisphosphate carboxylase/ oxygenase (RUBISCO), chloroplast photosystem II chlorophyll A/B-binding protein, stress-responsive proteins like methionine sulfoxide reductase type, etc.

Construction of an SSH cDNA library from symbiotic germinated seeds of Dendrobium officinale and sequence analysis / 中国药学杂志

OBJECTIVE: To investigate differential gene expression profiling of symbiotic germinated seeds of Dendrobium officinale. METHODS: cDNAs from 5-week symbiotic germinated seeds and 5-week aseptic cultivated seeds, taken as the tester and driver respectively, were used to construct a suppressive subtractive hybridization (SSH) cDNA library. RESULTS: By sequencing positive clones and BLASTx analysis against GenBank database, 100 expressed sequence tags (EST) homologous to plant known genes were obtained. Functional annotation revealed that they were grouped into serials of cellular processes including cell and chromosome structure, RNA synthesis, signal transduction, energy metabolism, protein synthesis and degradation, and cell defense, etc. Real-time quantitative RT-PCR analyses showed that the five randomly selected genes were all up-regulated in symbiotic germinated seeds. CONCLUSION: The symbiotic seed germination of D. officinale is involved in multiple pathways, and the results of this study will lay a foundation for further molecular elucidation of seed germination in Orchidaceae.

Technique of silico cloning and its application in medicine / 国际生物医学工程杂志

The strategy of silico cloning based on the express sequence tags sequence (ESTs)and genome database is a new technique developed in recent years,and the core of the technique is to use bioinformatics technique to assemble and extend ESTs to get part of cDNA and even all sequence.It has the virtue of low investment,high speed,low and well-targeted technical requirements,etc.The relationship between human disease and genes has been widely confirmed,therefore,silico cloning technology will become the important means of disease research.This paper aims at elaborating the progress of silico cloning in medicine.

Potential molecular targeting of splice variants for cancer treatment.

Array of new targets for investigation as cancer therapeutics has great potential to grow as new splice-variants are identified and characterized in cancer cell-lines and tumor samples. Tumor-specific splice variants are being discovered at an increasing rate and their functions are also investigated in cancer progression. The tumor-specific splice variants whose expression patterns and activities are successfully characterized may become attractive targets for ablation or splicing modification. The extreme specificity of their expression suggests that a variant-specific treatment may allow for targeting of cancerous cells with minimal impact to healthy tissues. Clinical investigation of applying antisense oligonucleotides to down-regulate mRNAs that contribute to cancer cell survival and to modify splicing patterns in muscular dystrophy has shown promising results. These results show that antisense therapy may be applied effectively and safely in humans. As these treatment strategies continue to improve and novel tumor-specific splice-variants are identified, modification of splicing patterns will become an important field of investigation to develop more effective and safe cancer therapies.

Normalized embryoid cDNA library of oil palm (Elaeis guineensis) / Biblioteca ADNc normalizada de embrioides en aceite de palma (Elaeis guineensis)

A normalized embryoid cDNA library (EON) was constructed based on reassociation kinetics reaction. Results from dot blot hybridization and sequencing of EON cDNA clones clearly indicated that the normalization process reduced the frequency of high abundance transcripts and increased the frequency of low abundance gene transcripts. A total of 553 non-redundant expressed sequence tags (ESTs) were identified, 325 of these were not observed in the standard oil palm cDNA libraries sequenced previously. A total of 10 EON cDNA clones were chosen for expression profiling across samples from different stages of the tissue culture process. Two of the genes exhibited promising expression patterns for predicting the embryogenic potential in callus. Some of these genes were also differentially expressed in the various tissues of oil palm. This study showed that normalization of the existing embryoid library improved the chances of identifying transcripts not captured in the standard libraries, some of which could be associated with embryogenesis. This collection of ESTs is particularly well suited for use as candidate genes for development of an oil palm DNA chip, which can be used to obtain a more comprehensive view of the molecular mechanism associated with oil palm tissue culture.

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.

An in silico analysis of the key genes involved in flavonoid biosynthesis in Citrus sinensis

Citrus species are known by their high content of phenolic compounds, including a wide range of flavonoids. In plants, these compounds are involved in protection against biotic and abiotic stresses, cell structure, UV protection, attraction of pollinators and seed dispersal. In humans, flavonoid consumption has been related to increasing overall health and fighting some important diseases. The goals of this study were to identify expressed sequence tags (EST) in Citrus sinensis (L.) Osbeck corresponding to genes involved in general phenylpropanoid biosynthesis and the key genes involved in the main flavonoids pathways (flavanones, flavones, flavonols, leucoanthocyanidins, anthocyanins and isoflavonoids). A thorough analysis of all related putative genes from the Citrus EST (CitEST) database revealed several interesting aspects associated to these pathways and brought novel information with promising usefulness for both basic and biotechnological applications.

Identification and in silico analysis of the Citrus HSP70 molecular chaperone gene family

The completion of the genome sequencing of the Arabidopsis thaliana model system provided a powerful molecular tool for comparative analysis of gene families present in the genome of economically relevant plant species. In this investigation, we used the sequences of the Arabidopsis Hsp70 gene family to identify and annotate the Citrus Hsp70 genes represented in the CitEST database. Based on sequence comparison analysis, we identified 18 clusters that were further divided into 5 subgroups encoding four mitochondrial mtHsp70s, three plastid csHsp70s, one ER luminal Hsp70 BiP, two HSP110/SSE-related proteins and eight cytosolic Hsp/Hsc70s. We also analyzed the expression profile by digital Northern of each Hsp70 transcript in different organs and in response to stress conditions. The EST database revealed a distinct population distribution of Hsp70 ESTs among isoforms and across the organs surveyed. The Hsp70-5 isoform was highly expressed in seeds, whereas BiP, mitochondrial and plastid HSp70 mRNAs displayed a similar expression profile in the organs analyzed, and were predominantly represented in flowers. Distinct Hsp70 mRNAs were also differentially expressed during Xylella infection and Citrus tristeza viral infection as well as during water deficit. This in silico study sets the groundwork for future investigations to fully characterize functionally the Citrus Hsp70 family and underscores the relevance of Hsp70s in response to abiotic and biotic stresses in Citrus.

In silico analysis of ESTs from roots of Rangpur lime (Citrus limonia Osbeck) under water stress

CitEST project resulted in the construction of cDNA libraries from different Citrus sp. tissues under various physiological conditions. Among them, plantlets of Rangpur lime were exposed to hydroponic conditions with and without water stress using PEG6000. RNA from roots was obtained and generated a total of 4,130 valid cDNA reads, with 2,020 from the non-stressed condition and 2,110 from the stressed set. Bioinformatic analyses measured the frequency of each read in the libraries and yielded an in silico transcriptional profile for each condition. A total of 40 contigs were differentially expressed and allowed to detect up-regulated homologue sequences to well known genes involved in stress response, such as aquaporins, dehydrin, sucrose synthase, and proline-related synthase. Some sequences could not be classified by using FunCat and remained with an unknown function. A large number of sequences presented high similarities to annotated genes involved with cell energy, protein synthesis and cellular transport, suggesting that Rangpur lime may sustain active cell growth under stressed condition. The presence of membrane transporters and cell signaling components could be an indication of a coordinated morphological adaptation and biochemical response during drought, helping to explain the higher tolerance of this rootstock to water stress.

Expressed sequenced tags from Lygus lineolaris (Hemiptera: Miridae), the tarnished plant bug

Expressed sequenced tags (ESTs) were prepared to establish a baseline for molecular genetic studies of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). The largest class of identifiable ESTs (15.2%) was from genes involved in cellular metabolic functions, including physiological processes. Twenty-seven ESTs (9.8%) were from genes associated with transcription and translation, including ribosomal genes. One hundred and forty-two of the 276 unique ESTs were from genes not previously identified from any organism. Twelve sequences appear to be associated with feeding and digestion and may be targets for pest control studies

Oxidative stress response in Paracoccidioides brasiliensis

Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.

Bioinformatics of the Paracoccidioides brasiliensis EST Project

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an endemic mycosis of Latin America. This fungus presents a dimorphic character; it grows as a mycelium at room temperature, but it is isolated as yeast from infected individuals. It is believed that the transition from mycelium to yeast is important for the infective process. The Functional and Differential Genome of Paracoccidioides brasiliensis Project--PbGenome Project was developed to study the infection process by analyzing expressed sequence tags--ESTs, isolated from both mycelial and yeast forms. The PbGenome Project was executed by a consortium that included 70 researchers (professors and students) from two sequencing laboratories of the midwest region of Brazil; this project produced 25,741 ESTs, 19,718 of which with sufficient quality to be analyzed. We describe the computational procedures used to receive process, analyze these ESTs, and help with their functional annotations; we also detail the services that were used for sequence data exploration. Various programs were compared for filtering and grouping the sequences, and they were adapted to a user-friendly interface. This system made the analysis of the differential transcriptome of P. brasiliensis possible.

Cloning,identification,and cellular localization of a down-regulated gene fragment related with Down Syndrome / 北京大学学报(医学版)

Objective: To clone a novel gene and explore its expression patterns in tissues and cells,so as to find its role in the process of encephalopathy in DS.Methods: On the base of our previous microarray's result together with the tissue type,we chose EST AI480014 to carry out RACE,then analyzed its expression profiles in liver,spleen,kidney,heart,brain by multi-tissues Northern blot,after that semi-quantitive RT-PCR was used to reexamine the expression profiles.Furthermore,we used ISH to find whether aim gene expressed in neuroglial cells cultured in vitro.Finally we performed semi-quantitive RT-PCR to explore whether it expressed differently between DS and normal.Results: We gained a 682 bp new cDNA fragment(DQ275636)which expressed in all the tissues examined and had no alternative splices in them.It expressed highly in brain especially in frontal lobe and hippocampus.According to the ISH result we convinced that it expressed in neuroglial cells.Using bioinformatics we mapped DQ275636 to chromosome 5q14.Conclusion: We have obtained a new gene fragment based on the(above) results.According to its expression character and tissue type,it can be suggested that this gene has a probable role in the process of encephalopathy in DS.

The Pattern of Differentially Expressed Genes in Biliary Atresia

Biliary atresia is a progressive obliterative cholangiopathy, but the etiology of this disorder remains uncertain. Identifying genes specifically expressed in biliary atresia and analyzing the pattern of expression may lead to a better understanding of the pathogenesis. Liver tissues were taken from a recipient with biliary atresia and a normal donor during liver transplantation. Total RNA was extracted from each sample and reversely transcribed to cDNA. Then radiolabeled cDNA probe pools were made by random primed DNA labeling method and used for screening of differentially expressed genes by hybridizing with expressed sequence tags (EST) dot blot panel. Northern blot hybridization was done to confirm that these genes are also differentially expressed in other liver tissues. Among 1,730 EST clones, 26 cDNA clones were significantly overexpressed in biliary cirrhosis, while 2 clones were significantly decreased in biliary atresia. By Northern blot hybridization, the results of tissue inhibitor of metalloproteinase (TIMP)-1 and IGFBP-2 were well correlated with differential EST screening (DES). This study identified the pattern of differentially expressed genes in the biliary cirrhosis due to biliary atresia using DES technique.

Obtaining, analysis and cloning of a novel gene of Schistosoma japonicum / 中华传染病杂志

Objective To obtain and characterize a novel gene of Schistosoma japonicum. Methods Looking for a novel gene through expressed sequence tags(EST),then predicting its function with the help of software on line. Finally the novel gene was cloned into an eukaryotic plasmid pEGFP N3. Results A novel gene with complete ORF was obtained. It has homogeneity with Defender Against Apoptotic Death 1 of human and pig. Conclusions EST is a good method to find new genes and analyse them with the help of software on line and characterize their function.

Identification of some expressed sequence tags in cerebellum of rat / 中国病理生理杂志

AIM: To obtain differentially expressed cDNA fragments in the cerebellum of rats and screen unknown expressed sequence tag (EST). METHODS: Suppression subtractive hybridization (SSH) was carried out, in which the cDNA fragments of cerebellum were taken as "tester" and correspondingly that of cerebrum and brain stem as "driver". The homogeneous sequences between the tester and driver were excluded and the rare sequences in cerebellum were enriched by SSH. The differentially expressed cDNA fragments were further cloned for the construction of subtracted cDNA libraries and sequencing. RESULTS: 32 clones were selected and 34 cDNA fragments were sequenced. 8 of 32 were proved to be true positive with reverse Northern assay, 13 of 34 fragments were identified to be new cDNA fragment and given the gene sequence numbers by GenBank (AW288461-AW288474). CONCLUSION: SSH is very useful method for screening differentially expressed genes. Our data may be helpful to understand the molecular mechanism of brain function.

Acquiring and analyzing of ornithine aminotransferase gene of Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To screen and identify the novel genes from the adult worm cDNA library of Schistosoma japonicum by the expressed sequence tags (EST) strategy.Methods The cDNA clones were selected randomly and the ESTs were obtained.The novel gene was searched for homologue identity with NCBI blast programmer.The homologue of the two sequences with a high identity was compared at amino acid and nucleotide level with the BLAST programmer,and the analysis of protein was carried out with the pcgene software. Results There was a novel gene of Schistosoma japonicum which included 1 677 bp coding for 424 amino acid residues, and it was given the accession number of sequence in Genbank(AY336497).The cDNA sequence was homologous to the known ornithine aminotransferase gene of Xenopus laevis. The identity of amino acid sequence was 66%. The academic pI was 8.52, and the antigen determinant was probably from 795 to 846 on the cDNA sequence. Conclusion EST strategy is an effective measure to discover new genes of Schistosoma japonicum. The novel gene is homologous to the ornithine aminotransferase gene of Xenopus laevis.This study is helpful to further sequential and functional search of the gene.[

The in Silico Elongation and Analysis of the EST from Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To construct a platform for in silico elongation and batch analysis of Schistosoma japonicum(Sj) ESTs, acquire the potential novel genes and research the expression profile of the genes. \ Methods\ On the basis of Linux operating system and local ESTs database of Sj, the BLAST and PHRAP softwares were used to construct a program to achieve the elongation of ESTs. Stand\|alone BLAST search against the nr database helped analyze the elongated sequence. After finishing the batch analysis script, the platform was used to research the Sj gene expression profile and acquire the potential novel genes. \ Results \ The platform showed satisfactory efficiency and fidelity. 487 elongated sequences obtained from 552 and 307 elongated sequences showed high homology within the nr database downloaded from NCBI. Furthermore, 104 elongated sequences displayed significant homology but showed no homology before elongated. 27 potential novel genes were filtered out. \ Conclusion \ An effective platform for Sj ESTs data mining was accomplished and further information on the potential novel genes was acquired.