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.     

Construction of a full-length cDNA library from young spikelets of hexaploid wheat and its characterization by large-scale sequencing of expressed sequence tags

The polyploid nature of wheat is a key characteristic of the plant. Full-length complementary DNAs (cDNAs) provide essential information that can be used to annotate the genes and provide a functional analysis of these genes and their products. We constructed a full-length cDNA library derived from young spikelets of common wheat, and obtained 24056 expressed sequence tags (ESTs) from both ends of the cDNA clones. These ESTs were grouped into 3605 contigs using the phrap method, representing expressed loci from each of the three genomes. Using BLAST, 3605 contigs were grouped into 1902 gene clusters, showing that loci of the three genomes are not always expressed. A homology search of these gene clusters against a wheat EST database (15964 gene clusters) and a rice full-length cDNA database (21447 gene clusters) revealed that a quarter of the wheat full-length cDNAs were novel. A protein database of Arabidopsis was used to examine the functional classification of these gene clusters. The GC-content in the 5 -UTR region of wheat cDNAs was compared to that of rice. Forty-three genes (3.5% of wheat cDNAs homologous to those of rice) possessed distinct GC-content in the 5 -UTR region, suggesting different breeding behaviors of wheat and rice.     

Analysis of expressed sequence tags from the antarctic psychrophilic green algae, Pyramimonas gelidicola

Expressed sequence tags (ESTs) from the Antarctic green algae Pyramimonas gelidicola were analyzed to obtain molecular information on cold acclimation of psychrophilic microorganisms. A total of 2,112 EST clones were sequenced, generating 222 contigs and 219 singletons, and 200 contigs and 391 singletons from control (4 degrees C) and cold-shock conditions (-2 degrees C), respectively. The complete EST sequences were deposited to the DDBJ EST database (http:// www.ddbj.nig.ac.jp/index-e.html) and the nucleotide sequences reported in this study are available in the DDBJ/EMBL/ GenBank. These EST databases of Antarctic green algae can be used in a wide range of studies on psychrophilic genes expressed by polar microorganisms.     

Expressed sequence tags-based identification of genes in the biocontrol agent <Emphasis Type="Italic">Chaetomium cupreum</Emphasis>

Chaetomium cupreum has a potential as biocontrol agent against a range of plant pathogens on the basis of production of antifungal metabolites, mycoparasitism, competition for space and nutrients, or various combinations of these. To explore genes expressed in C. cupreum, a cDNA library was constructed from mycelium and 3,066 expressed sequence tags (ESTs) were generated. Clusters analysis enabled the identification of 1,471 unigenes with 392 contigs and 1,079 singleton sequences. Putative functions were assigned to 874 unigenes that exhibited strong similarity to genes/ESTs in public databases putatively containing genes involved in cellular component, molecular function, and biological process. Other 597 ESTs representing novel genes showed no significant similarity to public database resource of NCBI. A proportion of genes was identified related to degradation of pathogen cell wall, antifungal metabolite production, as was estimated in the biocontrol fungus. The paper described is a first step towards the knowledge of the C. cupreum genome. The results present the useful application of EST analysis on C. cupreum and provide a preliminary indication of gene expression putatively involved in biocontrol.     

Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae)

To determine differential gene expression profiles in the venom gland and sac (gland/sac) of a solitary hunting wasp species, Orancistrocerus drewseni Saussure (1857), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 498 expressed sequence tags (EST) were clustered and assembled into 205 contigs (94 multiple sequences and 111 singletons). About 65% (134) of the contigs had matched BLASTx hits (E≤10^?4). Among these, 115 contigs had similarity to proteins with assigned molecular function in the Gene Ontology database, and most of them (112 contigs, 83%) were homologous to genes from Hymenoptera, particularly to Apis mellifera (98 contigs). The contigs encoding hyaluronidase and phospholipase A2, known to be main components of wasp venoms, were found in high frequencies (27 and 4%, respectively, as judged by the number of ESTs) in the gene ontology category of catalytic activity. Full‐length open reading frames of hyaluronidase and phospholipase A2 were characterized and their abundance in the venom gland/sac was confirmed by quantitative real‐time PCR. Several contigs encoding enzymes, including zinc‐metallopeptidases that are likely involved in the processing and activation of venomous proteins or peptides, were also identified from the library. Discovery of venom gland/sac‐specific genes should promote further studies on biologically active components in the venom of O. drewseni. © 2009 Wiley Periodicals, Inc.     

Comparative analysis of expressed sequence tags (ESTs) from Triticum monococcum shoot apical meristem at vegetative and reproductive stages

Triticum monococcum has recently drawn the attention of biologists to discover and utilize novel genes and alleles. To explore the molecular features of the genetic network governing floral transition in shoot apical meristem (SAM) of spring growth habit T. monococcum, two expressed sequence tag (EST) libraries containing 3,031 ESTs from vegetative SAM (VS) and 2,647 ESTs from early reproductive SAM (RS) were analyzed. Assembly of ESTs resulted in 2,303 unigenes for VS library (368 contigs and 1,935 singletons) and 1,890 unigenes (337 contigs and 1,553 singletons) for RS library. The 67.05 % of VS unigenes and 66.30 % of RS unigenes showed significant similarity with genes of known, putative and or unknown function, whereas the remaining 32.95 % of the VS unigenes and 33.7 % of RS unigenes displayed no significant match with the public protein database. The 1,064 and 866 unigenes of VS and RS libraries were assigned to functional categories using Pageman ontology tool. Further analysis revealed that the switch from VS to RS caused significant changes in the abundance of unigenes assigned to some functional categories. A total of 37 genes were identified which were significantly differentially expressed between vegetative and reproductive stages of T. monococcum SAM. Investigation of the differentially expressed genes revealed the importance of the genes involved in energy metabolism, ubiquitin/26S proteasome system, polyamines biosynthesis and signaling of reactive oxygen species in SAM differentiation towards floral transition in T. monococcum.     

Use of a large-scale Triticeae expressed sequence tag resource to reveal gene expression profiles in hexaploid wheat (Triticum aestivum L.).

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116,272 ESTs were produced, comprising 100,674 5' ESTs and 15 598 3' ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18,876 contigs and 23,034 singletons, or 41,910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the 1536 contigs that contained at least 10 5' EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.     

Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.     

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.