dbEST Database Research Articles

IDENTIFICACIÓN DE cDNAs DE Agave tequilana Weber Var. azul SIMILARES A FUROSTANOL GLICÓSIDO 26-O-β-GLUCOSIDASAS

Furostanol glycoside 26-O-β-glucosidase (F26G) participates in the last step of steroidal saponin biosynthesis, cleaving the glucose bound to the C26 of the hydroxylin furostanol glucoside to allow the formation of spirostane. Despite the existence of numerous studies showing the structural diversity and biological activity of plant saponins, as well as their recognized biotechnological and pharmacological potential, few enzymes involved in the biosynthetic pathway of steroidal saponins have been studied and only one native F26G enzyme has been isolated and functionally characterized. In this study, by searching a database of Agave tequilana Weber var. azul (Agave DB), 46 ESTs (Expressed Sequence Tags) of complementary DNA (cDNAs) similar to the known F26G were identified through BLASTX, most come from cDNA from floral tissues (anthers and ovaries) or roots; both organs are saponins producers. Among them, six ESTs were identified whose alignment indicates that they represent three cDNAs different from each other. These ESTs were submitted to the dbEST database of NCBI (National Center for Biotechnology Information), being this the first record of native cDNA sequences putatively encoding F26G-like in Agave. The size of the cloned cDNAs and their alignment to the 5’ end of the known F26G suggest they are full-length cDNAs. Specific differential primers were designed for each type of cDNA and their expression in vegetative tissues of A. tequilana was analyzed by RT-PCR. Quantitative PCR protocols (qPCR) were established for future studies on gene expression regulation. The information reported here will serve as a basis for studying the function, regulation and enzymatic activity of F26G-like cloned from Agave.

Development of novel EST-SSR markers for Ephedra sinica (Ephedraceae) by transcriptome database mining.

Premise of the Study Ephedra sinica (Ephedraceae) is a gymnosperm shrub with a wide distribution across Central and Eastern Asia. It is widely cultivated as a medicinal plant, but its wild populations are monitored to determine whether protection is needed.Methods and ResultsThirty‐six microsatellite markers, including 11 polymorphic markers, were developed from E. distachya RNA‐Seq data deposited in the National Center for Biotechology Information dbEST database. Among 100 genotyped E. sinica individuals originating from five different population groups, the allele number ranged from three to 22 per locus. Levels of observed and expected heterozygosity ranged from 0 to 0.866 (average 0.176) and 0 to 0.876 (average 0.491), respectively. Allelic polymorphism information content ranged from 0.000 to 0.847 (average 0.333). Cross‐species amplifications were successfully conducted with two related Ephedra species for all 11 di‐ or trinucleotide simple sequence repeats.ConclusionsThis study provides the first set of microsatellite markers for genetic monitoring and surveying of this medicinal plant.

Computational study of putative functional variants in human kisspeptin.

Non-synonymous single nucleotide polymorphisms (nsSNPs) are a type of genetic mutations that result in amino acid substitution of the encoded proteins that may potentially affect its function and phenotype. An In Silico assay has been carried out by using bioinformatics prediction tools to identify nsSNPs which are responsible for important disorders in human kisspeptin (KISS1) gene. In this study, for the first time, KISS1 amino acid changes were discovered by tBlastn for EST database. A list of nsSNPs in human KISS1 gene from dbSNP, dbEST and UniProt databases were prepared. Computational analysis was performed using SIFT (Sorting Intolerant From Tolerant) and PolyPhen (Polymorphism Phenotyping) programs. Of the total 92 nsSNPs, 20 were found to be damaged by both servers. Six nsSNPs (P97L, G122R, W114C, R92C, R120H and N115K) are predicted with the highest damaging scores (SIFT = 0, PolyPhen = 1). These intolerant changes may suggest their functional significance in critical regions which may affect the function and stability of KISS1 protein. Identifying these nsSNPs among the thousands of them make an opportunity to screen only those predicted deleterious by programs.

Prognostic value of a newly identified MALAT1 alternatively spliced transcript in breast cancer.

Background:Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known.Methods:We used RT–PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome.Results:MALAT1 was overexpressed in 14% (63/446) of the breast tumours. MALAT1-overexpressed tumour epithelial cells showed marked diffuse nuclear signals and numerous huge nuclear speckles. Screening of the dbEST database led to the identification of Δsv-MALAT1, a major alternatively spliced MALAT1 transcript, with a very different expression pattern compared with FL-MALAT1. This alternative Δsv-MALAT1 transcript was mainly underexpressed (18.8%) in our breast tumour series. Multivariate analysis showed that alternative Δsv-MALAT1 transcript is an independent prognostic factor. Δsv-MALAT1 expression was associated with alterations of the pre-mRNAs alternative splicing machinery, and of the Drosha-DGCR8 complex required for non-coding RNA biogenesis. Alternative Δsv-MALAT1 transcript expression was associated to YAP protein status and with an activation of the PI3K-AKT pathway.Conclusions:Our results reveal a complex expression pattern of various MALAT1 transcript variants in breast tumours, and suggest that this pattern of expressions should be taken into account to evaluate MALAT1 as predictive biomarker and therapeutic target.

Mining microsatellite markers from public expressed sequence tags databases for the study of threatened plants.

BackgroundSimple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest.MethodsHere, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea.ResultsWe explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics.ConclusionsThe present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2031-1) contains supplementary material, which is available to authorized users.

Mining of single nucleotide polymorphism (SNP) and simple sequence repeats (SSRs) from EST tropical fruits

The advancement in genomics technology has produced vast amount of expressed sequencetags (ESTs) sequence from tropical fruits. These resources have increased the publicavailability of ESTs sequence from year after year. Therefore, this effort permits mining ofsingle nucleotide polymorphism (SNP) and simple sequence repeat (SSR) from EST tropicalfruits. SNP and SSR are types of molecular marker which commonly used in modern geneticanalysis for wide application such as diversity analysis, linkage analysis and association study.In this study, a small scale EST sequences from tropical fruits (pineapple, mango, coconut andbanana) were retrieved from dbEST database (www.ncbi.nlm.nih.gov/dbEST/ ) as of March2013. Various bioinformatics tools were applied for rapid discovery of SNP and SSR markerfrom EST sequences. We analyzed 31,920 unigenes (contigs and singletons) representing atotal of 77,418 ESTs from four tropical fruits for their potential use in developing SNP and SSRmarkers. A total of 13,709 EST-SNP were discovered while a total of 4853 EST-SSR werediscovered from these four tropical fruits. The most abundant EST-SSR repeat is fromtrinucleotide (15,957 repeats) followed by dinucleotide (13,797 repeats) and tetranucleotide(973 repeats). Here, 1738 primers from SNP while 2033 primers from SSR were passedthrough the setting criteria and were selected for validation using genotyping platform. Thisstudy not only serves as a resource for marker development in tropical fruits but can provide abetter insight into the selection of candidate genes of interest.

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Computational identification and characterization of putative miRNAs in Heliothis virescens

Heliothis virescens, a polyphagous pest, is one of the most destructive pests of many crops and vegetables. Various insecticides and pesticides are used by agriculturalists to stop the growth and development of this pest. RNA interference is a new area for the management of pests/insects by inhibiting the growth related RNAs. This involves the miRNAs identification and its characterization. In the present study, computational approach is applied to predict putative miRNA candidates along with their possible target(s) in the Heliothis virescens. A total of 63,662 ESTs were downloaded from dbEST database and processed, trimmed and masked through EGassembler. The H. virescens contigs database obtained after assembly was now used to find the putative miRNA candidates by performing a local BLAST with the miRNAs of insects retrieved from miRBase. We have predicted putative miRNA candidates by homology search against all the reported insect miRNAs. These putative miRNAs candidates were further validated and filtered by different features. In addition, we have also attempted to predict the putative targets of these filtered miRNAs, by making use of 3' untranslated regions of mRNAs from B. mori. These miRNAs and their targets in H. virescens will help in improved understanding of molecular mechanisms of miRNA and development of novel and more precise techniques for better understanding some post transcriptional gene silencing.

Computational Prediction of miRNA in Gmelina Arborea and their Role in Human Metabolomics

Micro RNA are the noncoding class of RNA which plays major role post transcriptional regulation of genes and 19-22 nt in length and endogenous in nature .In this study miRNA Gmelina arborea are predicted along with possible target genes , A total of 44 ESTs are taken from dbEST database and proceed and trimmed through EGassembler. This contig sequence was now used to find out the putative miRNA by performing a local alignment with miRNA of Arabidopsis thaliana retrieved from miRBase. The targets were scanned by hybridizing screened ESTs with the UTRs of human using DIANA tar software. Finally 6 putative miRNAs were found to hybridize with the various targets of signal transduction and apoptosis that play significant role in preventing disease like cancer, blood borne disease, and other urinary infections and various study indicated that is possesses antioxidants, diuretic, anthelmintic activity, cardio protective, anti diabetic, immunomodulatory, antipyretic and analgesic, antimicrobial in the treatment of jaundice, piles bases on their targets

In silico identification and validation of microsatellite markers from onion EST sequences

SummaryMicrosatellites or simple sequence repeats (SSRs) play an important role in plant genetics and breeding. The development of microsatellites is a time-consuming and expensive process. In silico mining of microsatellites from expressed sequence tags (ESTs), which are available in electronic molecular databases, is a cheaper alternative. In the present study, we used a computational approach for mining SSRs from 20,159 ESTs in Allium cepa. These onion ESTs represented a total length of 13.2 Mb and were downloaded from the dbEST database of the National Center for Biotechnology Information (NCBI) and subjected to various pre-processing steps. The pre-processed ESTs were clustered, resulting in non-redundant unigenes. These unigenes were analysed for their SSR content and distribution. In all, 1,464 SSRs consisting of di-, tri-, tetra-, penta- and hexa-nucleotide repeats were mined from the non-redundant ESTs (contigs and singletons). Tri-nucleotide SSRs were the most abundant, followed by tetra-, di-, hexa- and penta-nucleotide SSRs. Among the tri-coding repeats, leucine and serine codons were more abundant. The SSR-containing sequences were annotated and grouped into their respective functional categories. The predominant functional group among the annotated unigenes was “metabolism”, followed by “transcription factors” and “transporter proteins”. Primer pairs could be designed for 1,092 SSR-containing sequences. Of these, 51 primer pairs were validated in the laboratory. A database has been developed to store the unigenes, primer pairs, putative annotations, and BLAST results. After validation, the EST-derived microsatellite (SSR) markers can be used in studies related to marker-assisted selection, detection of polymorphism, DNA fingerprinting, and diversity analysis in onion.

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica, with the titer of 1.2×105 pfu/ml. The average insert size of the cDNA library is about 1.6 kb. From the cDNA library, 591 cDNA clones were randomly selected and sequenced. As a result, 574 EST (expressed sequence tag) sequences were generated. All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198. The cDNA library was screened with a α-32p labeled 453 bp TPS gene probe, which is a partial sequence yielded from Porphyra yezoensis. Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L. japonica TPS cDNA sequence. After PCR amplification, sequencing and assembling, the entire ORF (open reading frame) sequence of the TPS gene was obtained, which was named LjTPS. LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons. The LjTPS gene was successfully expressed in E. coli and rice. The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the TPS gene function and mechanism to stress tolerance.

Computational analysis of transcriptome of Indian major carp, Labeo rohita (Hamilton-Buchanan, 1822) for functional annotation.

A total of 1671 ESTs of Labeo rohita were retrieved from dbEST database and analysed for functional annotation using various computational approaches. The result indicated 1387 non-redundant (184 contigs and 1203 singletons) putative transcripts with an average length of 542 bp. These 1387 transcript sequences were matched with Refseq_RNA, UniGene and Swiss-Prot on high threshold cut-off for functional annotation along with help of gene ontology and SSRs markers. We developed extensive Perl programming based modules for processing all alignment files, comparing and extracting common hits from all files on a threshold, evaluating statistics for alignment results and assigning gene ontology terms. In this study, 92 putative transcripts predicted as orthologous genes and among those, 44 putative transcripts were annotated with gene ontology terms. The annotated orthologous gene of our result associated with some very important proteins of L. rohita involved in biotic and abiotic stresses and glucose metabolism of spermatogenic cells etc. The unidentified transcripts, if found important in expression profiling can be vital resource after re-sequencing. The predicted genes can further be used for enhancing productivity and controlling disease of L. rohita.

Identification of miRNAs in C. roseus and their potential targets.

MicroRNAs are small (20-22 nucleotides) none coding, regulatory RNAs, whose pivotal role in gene expression has been associated in number of diseases, therefore prediction of miRNA is an essential yet challenging field. In this study miRNAs of C. roseus are predicted along with their possible target genes. A total of 19,899 ESTs were downloaded from dbEST database and processed and trimmed through SeqClean. Nine sequences were trashed and 31 sequences were trimmed by the program and the resulting sequences were submitted to Repeatmasker and TGICL for clustering and assembly. This contig database was now used to find the putative miRNAs by performing a local BLAST with the miRNAs of B. rapa retrieved from miRBase. The targets were scanned by hybridizing screened ESTs with the UTRs of human using miRanda software. Finally, 7 putative miRNAs were found to hybridize with the various targets of signal transduction and apoptosis that may play significant role in preventing diseases like Leukemia, Arthritis and Alzheimer.

Analysis of the Asian Seabass Transcriptome Based on Expressed Sequence Tags

Analysis of transcriptomes is of great importance in genomic studies. Asian seabass is an important fish species. A number of genomic tools in it were developed, while large expressed sequence tag (EST) data are lacking. We sequenced ESTs from nine normalized cDNA libraries and obtained 11 431 high-quality ESTs. We retrieved 8524 ESTs from dbEST database and analyzed all 19 975 ESTs using bioinformatics tools. After clustering, we obtained 8837 unique sequences (2838 contigs and 5999 singletons). The average contig length was 574 bp. Annotation of these unique sequences revealed that 48.9% of them showed significant homology to RNA sequences in GenBank. Functional classification of the unique ESTs identified a broad range of genes involved in different functions. We identified 6114 putative single-nucleotide polymorphisms and 634 microsatellites in ESTs. We discovered different temporal and spatial expression patterns of some immune-related genes in the Asian seabass after challenging with a pathogen Vibrio harveyi. The unique EST sequences are being used in developing a cDNA microarray to examine global gene expression and will also facilitate future whole-genome sequence assembly and annotation of Asian seabass and comparative genomics.

Genomic and EST microsatellites for Rhododendron aureum (Ericaceae) and cross‐amplification in other congeneric species

Our objective of this study was to develop genomic and expressed sequence tag (EST)-derived microsatellites for the endangered species Rhododendron aureum and access their transferability in R. dauricum and R. brachycarpum. • Twelve genomic microsatellites were isolated in R. aureum using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol and seven EST-derived microsatellites were characterized by screening the Rhododendron dbEST database of GenBank. The number of alleles per locus ranged from one to eight. The observed and expected heterozygosity varied from 0.00 to 1.00 and from 0.00 to 0.73, respectively. • A total of 19 microsatellite loci were developed for R. aureum. Sixteen and 10 of these loci were successfully amplified in R. brachycarpum and R. dauricum, respectively. These microsatellite markers will have potential applications in genetic diversity and conservation genetics studies.

Functional analysis of expressed sequence tags from the liver and brain of Korean Jindo dogs

We generated 16,993 expressed sequence tags (ESTs) from two libraries containing full-length cDNAs from the brain and liver of the Korean Jindo dog. An additional 365,909 ESTs from other dog breeds were identified from the NCBI dbEST database, and all ESTs were clustered into 28,514 consensus sequences using StackPack. We selected the 7,305 consensus sequences that could be assembled from at least five ESTs and estimated that 12,533 high-quality single nucleotide polymorphisms (SNPs) were present in 97,835 putative SNPs from the 7,305 consensus sequences. We identified 58 Jindo dog-specific SNPs in comparison to other breeds and predicted seven synonymous SNPs and ten non-synonymous SNPs. Using PolyPhen, a program that predicts changes in protein structure and potential effects on protein function caused by amino acid substitutions, three of the non-synonymous SNPs were predicted to result in changes in protein function for proteins expressed by three different genes (TUSC3, ITIH2, and NAT2).

Analysis of 5′Untranslated Region in Trans‐Sialidase Annoted Sequences in the T.cruzi Genomes

Trans‐sialidase (TS) is a membrane glycoprotein belonging to a family of genes from multiple copies involved in the process of cell invasion of the vertebrate host by the Trypanosoma cruzi (T. cruzi). Previous studies have shown that 5′ untranslated region is responsible for controlling gene regulation. In this work, we evaluated segments from TS cDNAs deposited in the Genbank‐dbEST such as the trans‐splicing additional or multiple sites, and their respective signals. We also investigated the compositional variation and the size of the sequences among T. cruzi epimastigotes strains: Dm28c (TcI), Y and CL‐Brener (TcII) and 4167 (Zymodeme 3). To corroborate these theoretical sequences, we acquired new test sequences based on RT‐PCR and qPCR. The fragments obtained from the different T. cruzi strains were cloned, sequenced and compositionally analyzed. CLUSTAL X was used to edit and align both theoretical and obtained sequences. All the TS theoretical sequences were searched against the dbEST database using BLAST program, which resulted in 735 cDNAs sequences. We also estimated the size of the mini‐exon‐containing fragments as 312 base pairs found in Z3, 209 in TcI and 218 in TcII. No mutations in the gene duplication presented in the trans‐splicing site were observed in the different strains. The qPCR analysis detected no differences in TS genes expression, although these genes are involved in host infectivity.

RPN1, a new reference gene for quantitative data normalization in lung and kidney cancer

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different specimens with unknown mRNA/cDNA concentrations. For this purpose, reference genes with stable expression are used (e.g., GAPDH, ACTB, HPRT1, or TBP). The problem of choosing proper reference genes is still a topical issue, because well-known reference genes can be unsuitable for certain cancer types and their inappropriate use without additional testing can lead to wrong conclusions. A recently developed bioinformatical approach was employed to identify a new potential reference gene for lung and kidney tumors, RPN1, located on the long arm of chromosome 3. The method employed the mining of the dbEST and Oncomine databases and functional analysis of genes. RPN1 was selected from approximately 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI microarrays, we found no methylation, deletions, and/or amplifications in the RPN1-containing locus in 56 nonsmall cell lung and 42 clear cell renal cell cancer specimens. Real-time PCR showed that variation of RPN1 mRNA levels in nonsmall cell lung cancer and clear-cell renal cancer was low and comparable to that of the known reference genes GAPDH and GUSB, respectively. Expression levels of two hyalouronidase genes, HYAL1 and HYAL2, were assessed using the suggested references gene pairs (RPN1-GAPDH for lung cancer and RPN1-GUSB for kidney cancer), and these combinations were shown to produce accurate and reproducible data. These results suggest that RPN1 is a new, promising reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.

Identification of true EST alignments for recognising transcribed regions

Transcribed regions can be determined by aligning Expressed Sequence Tags (ESTs) with genome sequences. The kernel of this strategy is to effectively distinguish true EST alignments from spurious ones. In this study, three measures including Direction Check, Identity Check and Terminal Check were introduced to more effectively eliminate spurious EST alignments. On the basis of these introduced measures and other widely used measures, a computational tool, named ESTCleanser, has been developed to identify true EST alignments for obtaining reliable transcribed regions. The performance of ESTCleanser has been evaluated on the well-annotated human ENCyclopedia of DNA Elements (ENCODE) regions using human ESTs in the dbEST database. The evaluation results show that the accuracy of ESTCleanser at exon and intron levels is more remarkably enhanced than that of UCSC-spliced EST alignments. This work would be helpful to EST-based researches on finding new genes, complementing genome annotation, recognising alternative splicing events and Single Nucleotide Polymorphisms (SNPs), etc.

Identification of rice purple acid phosphatases related to posphate starvation signalling

Purple acid phosphatases (PAPs) are a family of metallo-phosphoesterases involved in a variety of physiological functions, especially phosphate deficiency adaptations in plants. We identified 26 putative PAP genes by a genome-wide analysis of rice (Oryza sativa), 24 of which have isolated EST sequences in the dbEST database. Amino acid sequence analysis revealed that 25 of these genes possess sets of metal-ligating residues typical of known PAPs. Phylogenetic analysis classified the 26 rice and 29 Arabidopsis PAPs into three main groups and seven subgroups. We detected transcripts of 21 PAP genes in roots or leaves of rice seedlings. The expression levels of ten PAP genes were up-regulated by both phosphate deprivation and over-expression of the transcription factor OsPHR2. These PAP genes all contained one or two OsPHR2 binding elements in their promoter regions, implying that they are directly regulated by OsPHR2. Both acid phosphatase (AP) and surface secretory acid phosphatase (SAP) activity assays showed that the up-regulation of PAPs by Pi starvation, OsPHR2 over-expression, PHO2 knockout or OsSPX1 RNA interference led to an increase in AP and SAP activity in rice roots. This study reveals the potential for developing technologies for crop improvement in phosphorus use efficiency.