Expressed sequence tags (ESTs) from the marine red alga Gracilaria gracilis

Expressed sequence tags (ESTs) are partial sequences of cDNAs, and can be used to characterize gene expression in organisms or tissues. We have constructed a 200-sequence EST database from vegetative thalli of Gracilaria gracilis, the first ESTs reported from any alga. This database contains recognizable ESTs corresponding to genes of carbohydrate metabolism (seven), amino acid metabolism (three), photosynthesis (five), nucleic acid synthesis, repair and processing (three), protein synthesis (14), protein degradation (six), cellular maintenance and stress response (three), other identifiable protein-coding genes (13) and 146 sequences for which significant matches were not found in existing sequence databases. We have already used this EST database to recover genes of carbohydrate biosynthesis from G. gracilis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sequence-tagged sites (STSs) for a set of mapped markers on chromosome 21.

Sequence tagged sites (STSs) have been proposed as a "common language" for comparing physical and genetic maps of the human genome produced by a variety of techniques. We have produced 44 STSs from 38 mapped loci on human chromosome 21. The STSs represent most of the loci designated as genetic reference or ordered physical framework markers, along with a number of others chosen to span all regions of 21q. Of the STSs, 12 are from gene segments, including 4 from exons of the APP gene encoding the amyloid beta protein precursor, and 32 mark anonymous DNA loci. These STSs make each of the corresponding loci readily accessible to the research community without the need for exchange of clones. These sites also represent multiple start points for the isolation of YAC clones that should permit overlapping the entire chromosome 21 long arm as cloned DNA.     

Sequence-tagged sites (STSs) as standard landmarkers in the rice genome

Generating sequence-tagged sites (STSs) is a prerequisite to convert a genetic map to a physical map. With the help of sequence information from these STSs one can also isolate specific genes. For these purposes, we have designed PCR primer sets, of 20 bases each, by reference to sequences of restriction fragment length polymorphism (RFLP) landmarkers consisting of rice genomic clones. These markers were evenly distributed over the 12 chromosomes and were shown to be single copy by Southern-blot analysis. With improved PCR protocols, 63 standard STS landmarkers in the rice genome were generated. Similarity searches of all partial sequences of RFLP landmarkers by the FASTA algorithm showed that 2 of the 63 RFLP landmarkers, G357 and G385, contained part of the ORFs of aspartate aminotransferase and protein kinase, respectively.     

Analysis of expressed sequence tags (ESTs) in the ciliated protozoan Tetrahymena thermophila

To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5' or 3' ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures. Of 116 total non-redundant clones, 8.6% represented genes previously cloned in Tetrahymena. Fifty-two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins. Intriguing matches include an opioid-regulated protein, a glutamate-binding protein for an NMDA-receptor, and a stem-cell maintenance protein. Eleven-percent of the non-Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae. Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome-scale gene discovery and functional analysis.     

A linkage map of the pea (Pisum sativum L.) genome containing cloned sequences of known function and expressed sequence tags (ESTs)

 A linkage map of the pea (Pisum sativum L.) genome is presented which is based on F₂ plants produced by crossing the marrowfat cultivar ‘Primo’ and the blue-pea breeding line ‘OSU442-15’. This linkage map consists of 209 markers and covers 1330 cM (Kosambi units) and includes RFLP, RAPD and AFLP markers. By mapping a number of anchor loci, the ‘Primo’בOSU442-15’ map has been related to other pea linkage maps. A feature of the map is the incorporation of 29 loci representing genes of known function, obtained from other laboratories. The map also contains RFLP loci detected using sequence-characterized cDNA clones developed in our laboratory. The putative identities of 38 of these cDNA clones were assigned by examining public-sequence databases for protein or nucleotide-sequence similarities. The conversion of sequence-characterized pea cDNAs into PCR-amplifiable and polymorphic sequence-tagged sites (STSs) was investigated using 18 pairs of primers designed for single-copy sequences. Eleven polymorphic STSs were developed. Received: 18 June 1997 / Accepted: 11 August 1997     

Analysis of genes expressed in head kidney of common carp Cyprinus carpio L treated with cortisol

We analyzed genes expressed from head kidney of common carp Cyprinus carpio L. treated with cortisol. The results of single-pass sequencing of expressed sequence tags (ESTs) from 188 clones (AU240288–AU240367, AU301120–AU301227) from kidney cDNA are presented. One-hundred-twenty-seven clones (67.6%) were completely unknown and are likely to represent newly described genes, whereas 61 clones (32.4%) were identified based on matches to sequences in the database. The putative genes contain several ribosomal proteins, cytochrome oxidase subunits. Immune related cDNA clones identified from kidney were immunoglobulin light chain (n=4), FK506/rapamycin-binding protein (FKBP), CXC chemokine receptor type 4, complement factor B/C2-A3, peptidylprolyl isomerase (cyclophilin; Cyp)-like1, cyclophilin S1, heat shock-70 kDa protein-4, stress-activated protein kinase-3 (n=2). FKBP and cyclophilin genes expressed in normal tissues (head kidney, spleen, liver, brain and heart). Expression of FKBP and cyclophilin genes were not detected in liver, brain and heart when treated with cortisol for 16 h.     

Cloning and functional annotation of rare mRNA species from drought-stressed hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

To better understand gene expression at very low levels, we have designed a method to eliminate cDNA clones representing abundant mRNAs. A cDNA library for drought-stressed hot pepper (Capsicum annuum) (Choi et al., 2002) underwent double-negative screening, once with probes made from a drought-stressed plant, the second time, with probes from a non-stressed plant. The cDNA clones that showed very weak or negative signals were isolated for further analysis, which resulted in 1399 cDNA clones from about 20,000 screened clones. When nucleotide sequences were determined, we obtained 1142 tentative unique genes, with a redundancy rate of 20.41%. An homology database search for the deduced amino acid sequences revealed that about 79% of the cDNA clones could not be matched for functioning with previously characterized sequences. However, when these uncategorized clones were subjected to classification based on functional domains, most could be cited. Notably, clones with possible functions in RNA transport, protein synthesis, and regulation of protein activity showed a dramatic increase in appearance while those coding for transposable elements, viral proteins, and plasmid proteins occupied a much smaller portion compared with those in theArabidopsis thaliana genome. In addition, those coding for proteins targeted to the endoplasmic reticulum were dramatically more abundant in our clones compared with theArabidopsis database.     

Expressed Sequence Tag Analysis of Kidney and Gill Tissues from Rainbow Trout (Oncorhynchus mykiss) Infected with Infectious Hematopoietic Necrosis Virus

Expressed sequence tags (ESTs) were obtained from the kidney and gill tissues of rainbow trout, Oncorhynchus mykiss, infected with infectious hematopoietic necrosis virus (IHNV). The results of single-pass sequencing of ESTs from 198 clones (AU081027–AU081192) from kidney complementary DNA and 45 clones (AU081193–AU081236) from gill cDNA are reported herein. Sequences of the cDNA clones were compared with sequences in the GenBank database. Fourteen clones (20%) appeared to be completely unknown and may represent newly described genes, whereas 158 clones (80%) were identified on the basis of matches to sequences in the database. Three of the unidentified sequences were isolated from both the kidney and the gill cDNA libraries. However, no sequences were identical between kidney and gill clones. Received December 7, 1999; accepted April 28, 2000.     

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR.

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs. Of these, 29 clones contained multiple simple sequence repeat units. PCR analysis with primers derived from unique sequences flanking the simple sequence repeat units in seven clones showed all to be polymorphic between various mouse strains. This novel approach to SSLP identification represents an efficient method for saturating a genomic interval with polymorphic genetic markers that may expedite the positional cloning of genes for traits and diseases.     

Identification and Characterization of GATA Gene Family in Castor Bean (Ricinus communis)

GATA proteins are considered to be broadly involved in yield associated biological process, such as photoresponse, chlorophyll biosynthesis, and carbon and nitrogen metabolism. Based on castor bean genome database, a total of 19 GATA genes were identified and classified into 4 subfamilies according to gene structure, protein structure and their phylogenetic relationships. Results exhibited that GATA factors were hydrophilic proteins. Analysis of gene structure and protein structure revealed the conserved structural features of GATA factors between castor bean and Arabidopsis thaliana. The high throughput RNA seq data were used to define the expressional profiles of GATA genes among tissues. The results showed that most of the castor GATA genes preferentially expressed in leaf and root in contrast to their expression in developing seeds. In particular, the expression of GATA genes responding to darkness treatment in leaves was detected using semi quantitative RT PCR. It was shown that expression of three genes was down regulated under darkness treatment, which suggests a role for GATA genes of castor bean in light mediated regulation. These results provide important theoretical basis to the functions identification of castor GATA genes and increase castor yields.     

Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways.

Jasmonates mediate various physiological events in plant cells such as defense responses, flowering, and senescence through intracellular and intercellular signaling pathways, and the expression of a large number of genes appears to be regulated by jasmonates. In order to obtain information on the regulatory network of jasmonate-responsive genes (JRGs) in Arabidopsis thaliana (Arabidopsis), we screened 2880 cDNA clones for jasmonate responsiveness by a cDNA macroarray procedure. Since many of the JRGs reported so far have been identified in leaf tissues, the cDNA clones used were chosen from a non-redundant EST library that was prepared from above-ground organs. Hybridization to the filters was achieved using alpha-33P-labeled single-strand DNAs synthesized from mRNAs obtained from methyl jasmonate (MeJA)-treated and untreated Arabidopsis seedlings. Data analysis identified 41 JRGs whose mRNA levels were changed by more than three fold in response to MeJA. This was confirmed by Northern blot analysis by using eight representatives. Among the 41 JRGs identified, 5 genes were JA biosynthesis genes and 3 genes were involved in other signaling pathways (ethylene, auxin, and salicylic acid). These results suggest the existence of a positive feedback regulatory system for JA biosynthesis and the possibility of crosstalk between JA signaling and other signaling pathways.     

Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants

Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions. Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively. Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR. The cDNAs were able to complement their respective E. coli auxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy. The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol. This implies that there must be transporters for pathway intermediates. KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures. We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E. coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue.     

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.     

Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: application to the mapping of human chromosome 7 using yeast artificial chromosomes.

Basic to the development of long-range physical maps of DNA are the detection and localization of landmarks within recombinant clones. Sequence-tagged sites (STSs), which are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR), can be used as such landmarks. Our interest is to construct physical maps of whole human chromosomes by localizing STSs within yeast artificial chromosome (YAC) clones. Here we describe a generalized strategy for the systematic generation of large numbers of STSs specific for human chromosome 7. These STSs can be detected by PCR assays developed following the sequencing of anonymous pieces of chromosome 7 DNA, which was derived from flow-sorted chromosomes or from lambda clones made from DNA of a human-hamster hybrid cell line. Our approach for STS generation is tailored for the development of PCR assays capable of screening a large YAC library. In this study, we report the generation of 100 new STSs specific to human chromosome 7.