Single Expressed Sequence Tag Research Articles

Chromosome‐specific physical localisation of expressed sequence tag loci in Corchorus olitorius L.

Jute (Corchorus spp.), as a natural fibre-producing species, ranks next only to cotton. Inadequate understanding of its genetic architecture is a major lacuna for genetic improvement of this crop in terms of yield and quality. Establishment of a physical map provides a genomic tool that helps in positional cloning of valuable genes. In this report, an attempt was initiated to study association and localisation of single copy expressed sequence tag (EST) loci in the genome of Corchorus olitorius. The chromosome-specific association of EST was determined based on the appearance of an extra signal for a single copy cDNA probe in mitotic interphase nuclei of specific trisomic(s) for fluorescence in situ hybridisation, and validated using a cDNA fragment of the 26S rRNA gene (600bp) as molecular probe. The probe exhibited three signals in meiotic interphase nuclei of trisomic 5, instead of two as observed in diploids and other trisomics, indicating its association with chromosome 5. Subsequent hybridisation of the same probe on the pachytene chromosomes of diploids confirmed that 26S rRNA occupies the terminal end of the short arm of chromosome 5 in C.olitorius. Subsequently, chromosome-specific association of 63 single copy EST and their physical localisation were determined on chromosomes 2, 4, 5 and 7. The study describes chromosome-specific physical localisation of genes in jute. The approach used here could be a step towards construction of genome-wide physical maps for any recalcitrant plant species like jute.

A Bayesian Nonparametric Approach for Comparing Clustering Structures in EST Libraries

Inference for Expressed Sequence Tags (ESTs) data is considered. We focus on evaluating the redundancy of a cDNA library and, more importantly, on comparing different libraries on the basis of their clustering structure. The numerical results we achieve allow us to assess the effect of an error correction procedure for EST data and to study the compatibility of single EST libraries with respect to merged ones. The proposed method is based on a Bayesian nonparametric approach that allows to understand the clustering mechanism that generates the observed data. As specific nonparametric model we use the two parameter Poisson-Dirichlet (PD) process. The PD process represents a tractable nonparametric prior which is a natural candidate for modeling data arising from discrete distributions. It allows prediction and testing in order to analyze the clustering structure featured by the data. We show how a full Bayesian analysis can be performed and describe the corresponding computational algorithm.

Transcript expression profiles of Takifugu rubripes spermatozoa and eggs by expressed sequence tag analysis

Two cDNA libraries from Takifugu rubripes spermatozoa and eggs were constructed and a total of 620 expressed sequence tag (EST) clones were generated from the two libraries: 300 clones are from the spermatozoa library and 320 clones are from the eggs library. The most abundant cDNA clones in the two libraries were identified. A total of 207 'contigs' (or single) EST clones were found to share significant sequence identity with known sequences in the GenBank database, representing at least 51 different genes. In order to understand the two types of germ cells further, the expression profiles of the identified clones in these cDNA libraries were analyzed. Furthermore, the presence of specific messenger RNAs in the spermatozoa and eggs has been demonstrated with BLAST analysis; the spermatozoa and egg library can supply unique and novel cDNA sequences in the Takifugu rubripes EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the spermatogenesis and oogenesis in Takifugu rubripes. Six potential clones (S1-3 from spermatozoa and E1-3 from eggs) were selected to analyze their expression patterns by reverse transcription (RT)-PCR analyses. Half of these showed a specific expression in the expected tissue. Two of the clones were found by RT-PCR and in situ hybridization to be expressed specifically in the testis or ovary, and they maybe suitable molecular markers for the analysis of spermatogenesis and oogenesis.

Evaluation of EST-data using the genome assembly

Using expressed sequence tag (EST) data for genomewide studies requires thorough understanding of the nature of the problems that are related to handling these sequences. We investigated how EST clustering performs when the genome is used as guidance as compared to pairwise sequence alignment methods. We show that clustering with the genome as a template outperforms sequence similarity methods used to create other EST clusters, such as the UniGene set, in respect to the extent ESTs originating from the same transcriptional unit are separated into disjunct clusters. Using our approach, approximately 80% of the RefSeq genes were represented by a single EST cluster and 20% comprised of two or more EST clusters. In contrast, approximately 25% of all RefSeq genes were found to be represented by a single cluster for the UniGene clustering method. The approach minimizes the risk for overestimations due to the amount of disjunct clusters originating from the same transcript. We have also investigated the quality of EST-data by aligning ESTs to the genome. The results show how many ESTs are not adequately trimmed in respect of vector sequences and low quality regions. Moreover, we identified important problems related to ESTs aligned to the genome using BLAT, such as inferring splice junctions, and explained this aspect by simulations with synthetic data. EST-clusters created with the method are available upon request from the authors.

Characterization of the brassinosteroid insensitive 1 genes of cotton.

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.

Characterization of a familial RCC-associated t(2;3)(q33;q21) chromosome translocation.

A Polish family was identified in which multifocal clear cell renal carcinoma segregated with a balanced constitutional chromosome translocation, t(2:3)(q33;q21), similar to the renal cell cancer-associated t(2;3)(q35;q21) reported in a Dutch family. Bacterial artificial chromosome (BAC) contigs encompassing the 2q and 3q breakpoints were constructed and BACs crossing the breakpoints were partially sequenced. All known regional markers, genes, and expressed sequence tags (ESTs) were mapped relative to the contigs, as well as to the breakpoint sequences. Two single ESTs mapped within the 2q breakpoint BAC, whereas the repeat-rich 3q breakpoint region was gene poor. Physical mapping suggested that the 3q break was in 3q13, possibly near the border with 3q21. Physical mapping illustrated that the 2q break was closely telomeric to the 2q31 FRA2G site, consistent with the G-band assignment. Characterization of full-length cDNAs for the ESTs near the 2q break will determine if a gene(s) is altered by this familial translocation.

Refinement of the Smallest Commonly Deleted Segment of Chromosome 20 in Malignant Myeloid Diseases and Development of a PAC-Based Physical and Transcription Map

A deletion of the long arm of chromosome 20, del(20q), is a recurring abnormality in malignant myeloid diseases. In previous studies, we delineated a commonly deleted segment (CDS) of 5 Mb within band 20q12 flanked by D20S206 (proximal) and D20S481 (distal). We have generated a detailed physical map of P1 artificial chromosome (PAC) clones of this interval as well as a transcriptional map. The contig consists of 81 clones to which 152 markers (27 genes, 45 unique expressed sequence tags (ESTs) or UniGenes, 24 polymorphisms, and 56 sequence-tagged sites) have been mapped. Using PAC clones for fluorescence in situ hybridization analysis of myeloid leukemia cells with reciprocal translocations of 20q, or unbalanced rearrangements leading to loss of 20q, we have narrowed the CDS to an ∼250-kb interval encompassing two overlapping PACs, P201E16 and P29M7 (between EST AA368224 and D20S481). This interval is gene-rich and contains 5 characterized genes, 4 UniGenes, and 9 single ESTs. The development of a transcriptional map and the identification of the smallest CDS will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 20q.

UniBLAST and the EST Extractor: new WWW resources for EST data mining

Expressed sequence tags (ESTs) are nucleotide sequences generated from the ends of randomly selected cDNA clones. More than 1 500 000 entries generated from different tissues and species are now stored in the public EST database (dbEST) maintained at the National Center for Biotechnology[1]. The NCBI periodically analyzes all human ESTs and sequences of known genes to identify and cluster the ones belonging to the same transcript. The result of this effort is stored in the UniGene database[2], which, as of March 1998, contained more than 39 000 clusters. Both dbESt and UniGene are available from the NCBI anonymous FTP server as downloadable flat data files. Exploiting these two databases, we developed two WWW applications, UniBLAST and the EST Extractor, which can be useful to researchers involved in positional cloning projects or interested in extending the sequence information associated with a single EST. UniBLast[3]is a non interactive BLAST search server that queries a formatted version of the UniGene database maintained at Tigem. Using UniBLast, a researcher can easily assess the number of UniGene clusters (and consequently the number of gene transcripts) similar to a query sequence in a single step. The UniGene database at Tigem is synchronized with the one provided by the NCBI. The EST Extractor[4]performs a remote interactive BLAST search (either a BLASTN or a TBLASTN analysis) versus dbEST at NCBI and reformats the search output, adding the following possibilities:•Manually selecting the significant EST hits from the search output.•Retrieving the selected EST sequences from dbEST at Tigem.•Displaying the links with UniGene entries, both from NCBI and Tigem.•Assembling or aligning the selected EST sequences, using the Cap2 assembler[5]or the ClustalW multiple alignment[6]programs.•Repeating either the whole EST Extractor procedure or performing a remote BLAST search at NCBI with a selected EST sequence contig generated by the assembly program.

How to get the best of dbEST

Expressed sequence tags (ESTs) are nucleotide sequences generated from the ends of randomly selected cDNA clones. The remarkable expansion of EST efforts in the past seven years [1–3]has undoubtedly led to a revolutionary change in the strategies used by molecular geneticists for identifying and cloning novel genes. More than 1 200 000 entries generated from different tissues and species are now stored in the public EST database (dbEST) [4, 5], maintained at the National Center for Biotechnology Information (NCBI). Human and mouse sequences form the majority of the data held in this collection, with 833 000 and 237 000 entries, respectively (as of 25 October, 1997) [6]. For most of the clones, ESTs have been generated at both ends and the corresponding sequence traces can be easily retrieved for quality checking [7]; furthermore, it is possible to obtain in a few days most of the EST cDNA clones, such as the ones generated by the IMAGE consortium and The Institute for Genomic Research (TIGR) [1, 8], from international distributors [9]. EST clones represent useful molecular tools for gene characterization experiments, expression studies, and expression of recombinant proteins. In most cases, a single EST clone (average insert size around 1.5 kb) does not span the entire coding region of a gene. However, before starting any experimental procedure aimed at the isolation of the remaining part of the transcript, it must be pointed out that several bioinformatic tools exist that allow one to obtain full-length transcript information and refined chromosomal mapping assignment.