Enhanced expression of beta-thymosin mRNA in the ovary of GnRH analog or estradiol-17beta-treated paradise fish,Macropodus opercularis

cDNA clones were isolated as expressed sequence tags (ESTs) from the ovarian cDNA library of Macropodus opercularis. The EST sequences showed similarity with many housekeeping genes and ribosomal proteins. One of the ESTs showed similarity to beta-thymosin, a 5-kDa polypeptide expressed under different physiological conditions. The cDNA corresponding to beta-thymosin of M. opercularis is 368 bp in length and codes for a putative polypeptide of 42 amino acids. Multiple alignment of the deduced amino acid sequence showed 61% similarity with piscine beta-thymosins and 56% similarity with mammalian beta-thymosins. Administration of a gonadotropin releasing hormone analog or estradiol-17beta induced an increase in the gonadosomatic index, oocyte diameter and also enhanced expression of beta-thymosin m-RNA in the recrudizing ovary. This report indicates that both GnRH analog and E(2) might induce similar pathways for the differentiation of ovarian cells for the maturation of oocytes.     

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.     

Initial assessment of gene diversity for the oomycete pathogen Phytophthora infestans based on expressed sequences

A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes.     

ANALYSIS OF EXPRESSED SEQUENCE TAGS (ESTS) FROM THE POLAR DIATOM FRAGILARIOPSIS CYLINDRUS1

Analysis of expressed sequence tags (ESTs) was performed to gain insights into cold adaptation in the polar diatom Fragilariopsis cylindrus Grunow. The EST library was generated from RNA isolated 5 days after F. cylindrus cells were shifted from approximately +5° C to ?1.8°C. A total of 1376 ESTs were sequenced from a non‐normalized cDNA library and assembled into 996 tentative unique sequences. About 27% of the ESTs displayed similarity (tBLASTX, e‐value of ≤10^?4) to predicted proteins in the centric diatom Thalassiosira pseudonana Hasle & Heindal. Eleven additional algae and plant data bases were used for annotation of sequences not covered by Thalassiosira sequences (7%). Most of the ESTs were similar to genes encoding proteins responsible for translation, ribosomal structure, and biogenesis (3%), followed by genes encoding proteins for amino acid transport and metabolism and post‐translational modifications. Interestingly, 66% of all the EST sequences from F. cylindrus displayed no similarity ( e ‐value ≤10^?4) to sequences from the 12 non‐redundant databases. Even 6 of the 10 strong to moderately expressed sequences in this EST library could not be identified. Adaptation of F. cylindrus to freezing temperatures of seawater may require a complex protein metabolism and possibly also genes, which were highly expressed but still unknown. However, it could also mean that due to low temperatures, there might have been a stronger pressure to adapt amino acid sequences, making it more difficult to identify these unknown sequences and/or that there are still few protist sequences available for comparison.     

Identification of genes encoding mouse oocyte secretory and transmembrane proteins by a signal sequence trap

The oocyte plays a key role in follicular development. At all stages of follicular development, oocytes interact with surrounding granulosa cells and promote their differentiation into the types of cells that support further oocyte growth and developmental competence. These interactions suggest the existence of an oocyte-granulosa cell regulatory loop that includes both secreted proteins and cell surface receptors on both cell types. Factors involved in the regulatory loop will therefore contain a signal sequence, which can be used to identify them through a signal sequence trap (SST). A screen of an oocyte SST library identified three classes of oocyte-expressed sequences: known mouse genes, sequences homologous to known mammalian genes, and novel sequences of unknown function. Many of the recovered genes may have roles in the oocyte-granulosa cell regulatory loop. For several of the known mouse genes, new roles in follicular development are implied by identification of their expression, for the first time, in the oocyte. The future characterization of novel sequences may lead to the identification of novel proteins participating in the regulatory loop.     

DNA sequence-based "bar codes" for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.     

Census of orthologous genes and self-organizing maps of biologically relevant transcriptional patterns in chickens (Gallus gallus)

The launch of large-scale chicken expressed sequence tags (EST) projects has placed the chicken in the lead for the number of EST sequences in agriculturally important animals. More than 451,000 chicken ESTs derived from over 158 libraries have been deposited in the NCBI dbEST database as of December 2003. But how many genes these ESTs represent and how they are expressed in different chicken tissues/organs remain undetermined. In the present research, we developed a human gene-based strategy for census of chicken orthologous genes and identification of their expression patterns. Among 34,157 human coding genes used in the study, BLAST analysis revealed that 11,066 genes provisionally matched 248,628 chicken ESTs. Based on the average EST abundance of the orthologous genes, the current public repository of chicken ESTs could represent 20,000 provisional genes. Analysis of gene expression in 14 single tissues/organs showed that approximately 15% of genes were expressed exclusively in single tissue/organ whereas the remaining 85% of genes were co-expressed in two or more tissues/organs. A majority (91.15%) of genes expressed in chicken embryos were also expressed at post-hatch stages, indicating that most genes activated in chicken embryos could serve housekeeping functions. Self-organizing maps (SOM) analysis organized 8807 provisional genes in selected chicken tissues into 98 clusters with each cluster being indicative of common regulatory factors and pathways. A total of 969 provisional orthologous genes were identified as preferentially expressed genes (PEGs) in various chicken tissues/organs (LOD>3.0). No doubt, the present study on gene expression patterns will provide insight into dynamics of metabolic pathways and tissue/organ programming and reprogramming in chickens.     

Analysis of expressed sequence tags in prothallia of Adiantum capillus-veneris

The analysis of expressed sequences from a diverse set of plant species has fueled the increase in understanding of the complex molecular mechanisms underlying plant growth regulation. While representative data sets can be found for the major branches of plant evolution, fern species data are lacking. To further the availability of genetic information in pteridophytes, a normalized cDNA library of Adiantum capillus-veneris was constructed from prothallia grown under white light. A total of 10,420 expressed sequence tags (ESTs) were obtained and clustering of these sequences resulted in 7,100 nonredundant clusters. Of these, 1,608 EST clusters were found to be similar to sequences of known function and 1,092 EST clusters showed similarity to sequences of unknown function. Given the usefulness of Adiantum for developmental studies, the sequence data represented in this report stand to make a significant contribution to the understanding of plant growth regulation, particularly for pteridophytes.     

Mapping of genes expressed in activated porcine Peyer's patch

To determine the chromosomal locations for genes expressed in porcine Peyer's patches, polymerase chain reaction-based mapping of expressed sequence tags (ESTs) isolated from a porcine Peyer's patch-specific cDNA library was performed across a 6500-rad swine radiation hybrid panel. A total of 116 ESTs were mapped with LOD scores >6.0, and another 11 ESTs had LOD scores between 5.0 and 6.0. Of these 127 ESTs, 63% matched known genes (相似文献   

Generation of ESTs for flowering gene discovery and SSR marker development in upland cotton

Background

Upland cotton, Gossypium hirsutum L., is one of the world''s most important economic crops. In the absence of the entire genomic sequence, a large number of expressed sequence tag (EST) resources of upland cotton have been generated and used in several studies. However, information about the flower development of this species is rare.

Methodology/Principal Findings

To clarify the molecular mechanism of flower development in upland cotton, 22,915 high-quality ESTs were generated and assembled into 14,373 unique sequences consisting of 4,563 contigs and 9,810 singletons from a normalized and full-length cDNA library constructed from pooled RNA isolated from shoot apexes, squares, and flowers. Comparative analysis indicated that 5,352 unique sequences had no high-degree matches to the cotton public database. Functional annotation showed that several upland cotton homologs with flowering-related genes were identified in our library. The majority of these genes were specifically expressed in flowering-related tissues. Three GhSEP (G. hirsutum L. SEPALLATA) genes determining floral organ development were cloned, and quantitative real-time PCR (qRT-PCR) revealed that these genes were expressed preferentially in squares or flowers. Furthermore, 670 new putative microsatellites with flanking sequences sufficient for primer design were identified from the 645 unigenes. Twenty-five EST–simple sequence repeats were randomly selected for validation and transferability testing in 17 Gossypium species. Of these, 23 were identified as true-to-type simple sequence repeat loci and were highly transferable among Gossypium species.

Conclusions/Significance

A high-quality, normalized, full-length cDNA library with a total of 14,373 unique ESTs was generated to provide sequence information for gene discovery and marker development related to upland cotton flower development. These EST resources form a valuable foundation for gene expression profiling analysis, functional analysis of newly discovered genes, genetic linkage, and quantitative trait loci analysis.     

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.     

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.     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.     

The construction of Arabidopsis expressed sequence tag assemblies. A new resource to facilitate gene identification.

The generation of large numbers of partial cDNA sequences, or expressed sequence tags (ESTs), has provided a method with which to sample a large number of genes from an organism. More than 25,000 Arabidopsis thaliana ESTs have been deposited in public databases, producing the largest collection of ESTs for any plant species. We describe here the application of a method of reducing redundancy and increasing information content in this collection by grouping overlapping ESTs representing the same gene into a "contig" or assembly. The increased information content of these assemblies allows more putative identifications to be assigned based on the results of similarity searches with nucleotide and protein databases. The results of this analysis indicate that sequence information is available for approximately 12,600 nonoverlapping ESTs from Arabidopsis. Comparison of the assemblies with 953 Arabidopsis coding sequences indicates that up to 57% of all Arabidopsis genes are represented by an EST. Clustering analysis of these sequences suggests that between 300 and 700 gene families are represented by between 700 and 2000 sequences in the EST database. A database of the assembled sequences, their putative identifications, and cellular roles is available through the World Wide Web.