Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.     

Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5’ EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests.     

Analyses of an expressed sequence tag library from Taenia solium, Cysticerca

Background

Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST) library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology.

Methods and Findings

Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52%) were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55%) were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed.

Conclusions

An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e.g. immune modulation.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

Tissue-Specific Expressed Sequence Tags from the Black Tiger Shrimp Penaeus monodon

Expressed sequence tag data were generated from complementary DNA libraries created from cephalothorax, eyestalk, and pleopod tissue of the black tiger shrimp (Penaeus monodon). Significant database matches were found for 48 of 83 nuclear genes sequenced from the cephalothorax library, 22 of 55 nuclear genes from the eyestalk library, and 6 of 13 nuclear genes from the pleopod library. The putative identities of these genes reflected the expected tissue specificity. For example, genes for digestive enzymes were identified from the cephalothorax library and genes involved in the visual and neuroendocrine system from the eyestalk library. A few sequences matched anonymous EST or genomic sequences, and others contained mini-satellite or microsatellite repeat sequences. The remainder, 31 from the cephalothorax library, 25 from the eyestalk library, and 5 from the pleopod library, were sequences of high nucleotide complexity with no matches in any database searched and thus may represent novel genes. Received February 12, 1999; accepted April 13, 1999     

Eighteen Novel Human Genes Regionally Mapped on Chromosome 11

Expression sequence tags (EST) obtained by sequencing a randomlyprimed cDNA library and gene signatures (GS) obtained by sequencinga 3'-directed cDNA library can identify genes that are activein the source cells. Eight ESTs and ten GSs which representnovel human genes, except for one GS, and which have been assignedto human chromosome 11 were used to select cosmids from a chromosome11-specific cosmid library. These cosmids were regionally mappedusing the fluorescence in situ hybridization technique.     

Searching the human genome database for novel relaxin- and insulin-like peptides

Summary In recent times, new members of the insulin/relaxin peptide superfamily have been identified by both differential cloning strategies as well as bioinformatic searching of the EST databases. We have used the public and Celera Genomics databases to search for novel members of this peptide family. No new members of the insulin/relaxin family were identified although the human (H3) and mouse (M3) relaxin 3 genes that we recently discovered in the Celera Genomics database were identified in the public database. We were able to confirm that there are no mouse equivalents of human INSL-4 or human gene 1 relaxin. Hence, as the two human relaxin genes (H1 and H2) are localized together with INSL6 and INSL4 on chromosome 9 it is probable that INSL4 and H1 relaxin are the result of a gene duplication which did not occur in non-primates. The discovery of a full relaxin 3 sequences in a new Zebrafish brain EST library, which retains a high homology in both A and B chain peptide sequence with the H3 peptide, indicate that this novel peptide has important conserved functions.     

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 tag analysis of Eimeria-stimulated intestinal intraepithelial lymphocytes in chickens

Intraepithelial lymphocytes (IELs) play a critical role in protective immune response to intestinal pathogens such as Eimeria, the etiologic agent of avian coccidiosis. A list of genes expressed by intestinal IELs of Eimeria-infected chickens was compiled using the expressed sequence tag (EST) strategy. The 14,409 ESTs consisted of 1851 clusters and 7595 singletons, which revealed 9446 unique genes in the data set. Comparison of the sequence data with chicken DNA sequences in GenBank identified 125 novel clones. This EST library will provide a valuable resource for profiling global gene expression in normal and pathogen-infected chickens and identifying additional unique immune-related genes.     

Expressed sequence tag analysis of blood cells in the vanadium-rich ascidian,Ascidia sydneiensis samea--a survey of genes for metal accumulation

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10(7) times that found in seawater. The vanadium ions are stored in vacuoles located within vanadium-containing blood cells, vanadocytes. To investigate the phenomenon, an expressed sequence tag analysis (EST) of a cDNA library of Ascidia sydneiensis samea blood cells was carried out. Three hundred clones were obtained and sequenced by EST analysis. A similarity search revealed that 158 of the clones (52.7%) were known genes, and 142 of the clones (47.3%) did not have any similarity to genes registered in the SwissProt database. According to the functions of their genes the identified EST clones were categorized into eight types of clones; these consisted of genes; metal-related proteins (29 clones), signal transduction (22 clones), protein synthesis (17 clones), nuclear proteins (17 clones), cytoskeleton and motility (14 clones), energy conversion (3 clones), hypothetical proteins (11 clones), and others (45 clones). The ferritin homologue has a high degree of similarity to that of mammals; the iron-binding sites of ferritin are well conserved including His-118 which is important for capturing Fe(2+), also works as a ligand for VO(2+).     

A Bayesian nonparametric method for prediction in EST analysis

Background  

Expressed sequence tags (ESTs) analyses are a fundamental tool for gene identification in organisms. Given a preliminary EST sample from a certain library, several statistical prediction problems arise. In particular, it is of interest to estimate how many new genes can be detected in a future EST sample of given size and also to determine the gene discovery rate: these estimates represent the basis for deciding whether to proceed sequencing the library and, in case of a positive decision, a guideline for selecting the size of the new sample. Such information is also useful for establishing sequencing efficiency in experimental design and for measuring the degree of redundancy of an EST library.     

Gene capture prediction and overlap estimation in EST sequencing from one or multiple libraries

Background  

In expressed sequence tag (EST) sequencing, we are often interested in how many genes we can capture in an EST sample of a targeted size. This information provides insights to sequencing efficiency in experimental design, as well as clues to the diversity of expressed genes in the tissue from which the library was constructed.     

Expressed sequence tag analysis of Eimeria-stimulated intestinal intraepithelial lymphocytes in chickens

Intraepithelial lymphocytes (IELs) play a critical role in protective immune response to intestinal pathogens such as Eimeria, the etiologic agent of avian coccidiosis. A list of genes expressed by intestinal IELs of Eimeria-infected chickens was compiled using the expressed sequence tag (EST) strategy. The 14,409 ESTs consisted of 1851 clusters and 7595 singletons, which revealed 9446 unique genes in the data set. Comparison of the sequence data with chicken DNA sequences in GenBank identified 125 novel clones. This EST library will provide a valuable resource for profiling global gene expression in normal and pathogen-infected chickens and identifying additional unique immune-related genes.     

Isolation and expression analysis of LEA genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.