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PubMedDate not supplied

RFLP mapping of expressed sequence tags in the chicken.

Chicken cDNA probes were used to map 10 loci by restriction fragment length polymorphism (RFLP) analysis in progeny of the Jungle Fowl x White Leghorn chicken backcross gene mapping reference population. The cDNA probes were from a subset of a T-cell library whose sequence tags showed significant homology to other avian or mammalian sequences in the EMBL database. Placement of loci generated by Compton cDNA clones on the East Lansing linkage map contributes to development of a consensus from the two linkage maps and will help provide a framework to evaluate the phenotypic effects of these genes.

Animals↗
PubMedDate not supplied

Update of the gene discovery program in Schistosoma mansoni with the expressed sequence tag approach.

Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81%) had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor.

Animals↗
PubMed2002

Expressed sequence tag-based gene expression analysis under aluminum stress in rye.

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.

Adaptation, Physiological↗
PubMed2002

The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes.

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.

Animals↗
PubMed2005

Analysis of testis-specific transcripts in the chicken.

Sequences of candidate chicken testis-specific genes were analysed in order to develop a resource for functional genomic studies of the testis and male germ cells. Tentative consensus sequences (TC) containing expressed sequence tags expressed in testis libraries were selected from the TIGR Gallus gallus Gene Index, resulting in 292 TC. The transcriptional expression of these genes were evaluated in several chicken tissues, including testis and ovary. One hundred ten of the 292 TC were expressed in a testis-specific manner. Annotation of the TC using the Gene Ontology database terms showed that the proportion of testis-specific TC that were classified as having catalytic activity (within the molecular function branch) was higher than the proportion of total chicken TC classified in the same way. Our results may facilitate the investigation of testis-specific genes and their functional analysis in the chicken, as well as in other avian species.

Animals↗
PubMed1999

A comprehensive approach to clustering of expressed human gene sequence: the sequence tag alignment and consensus knowledge base.

The expressed human genome is being sequenced and analyzed by disparate groups producing disparate data. The majority of the identified coding portion is in the form of expressed sequence tags (ESTs). The need to discover exonic representation and expression forms of full-length cDNAs for each human gene is frustrated by the partial and variable quality nature of this data delivery. A highly redundant human EST data set has been processed into integrated and unified expressed transcript indices that consist of hierarchically organized human transcript consensi reflecting gene expression forms and genetic polymorphism within an index class. The expression index and its intermediate outputs include cleaned transcript sequence, expression, and alignment information and a higher fidelity subset, SANIGENE. The STACK_PACK clustering system has been applied to dbEST release 121598 (GenBank version 110). Sixty-four percent of 1,313, 103 Homo sapiens ESTs are condensed into 143,885 tissue level multiple sequence clusters; linking through clone-ID annotations produces 68,701 total assemblies, such that 81% of the original input set is captured in a STACK multiple sequence or linked cluster. Indexing of alignments by substituent EST accession allows browsing of the data structure and its cross-links to UniGene. STACK metaclusters consolidate a greater number of ESTs by a factor of 1. 86 with respect to the corresponding UniGene build. Fidelity comparison with genome reference sequence AC004106 demonstrates consensus expression clusters that reflect significantly lower spurious repeat sequence content and capture alternate splicing within a whole body index cluster and three STACK v.2.3 tissue-level clusters. Statistics of a staggered release whole body index build of STACK v.2.0 are presented.

Algorithms↗
PubMed2000

Human BAC ends quality assessment and sequence analyses.

End sequences from bacterial artificial chromosomes (BACs) provide highly specific sequence markers in large-scale sequencing projects. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length of >460 bp for a total of 141 Mb covering approximately 4.7% of the genome. Over 60% of the clones have BAC end sequences (BESs) from both ends representing more than fivefold coverage of the human genome by the paired-end clones. Our quality assessments and sequence analyses indicate that BESs from human BAC libraries developed at The California Institute of Technology (CalTech) and Roswell Park Cancer Institute have similar properties. The analyses have highlighted differences in insert size for different segments of the CalTech library. Problems with the fidelity of tracking of sequence data back to physical clones have been observed in some subsets of the overall BES dataset. The annotation results of BESs for the contents of available genomic sequences, sequence tagged sites, expressed sequence tags, protein encoding regions, and repeats indicate that this resource will be valuable in many areas of genome research.

Chromosome Mapping↗
PubMed2001

Peaks of linkage are localized by a BAC/PAC contig of the 6p reading disability locus.

A gene for reading disability has been localized by nonparametric linkage to 6p21.3-p22 in several published reports. However, the lack of an uninterrupted genomic clone contig has made it difficult to determine accurate intermarker distances, precise marker order, and genetic boundaries and hinders direct comparisons of linkage. The search and discovery of the hemochromatosis gene (HFE) led to the creation of a bacterial artificial chromosome (BAC) and P-1 derived artificial chromosome (PAC) contig that extended physical maps 4 Mb from the MHC toward pter and localized new markers in that region [10-12]. Using this contig, we localized 124 sequence tagged sites, expressed sequence tags, and short tandem repeats including most of the markers in linkage with reading disability phenotypes, succinic semialdehyde dehydrogenase, GPLD1, prolactin, and 18 uncharacterized genes. This new contig joins and extends previously published physical maps to span the entire chromosome 6 reading disability genetic locus. Physical mapping data from the complete contig show overlap of the published linkage peaks for reading disability, provide accurate intermarker distances and order, and offer resources for generating additional markers and candidate genes for high resolution genetic studies in this region.

Bacteriophage P1↗
PubMed1998

Identification of sequence-tagged transcripts differentially expressed within the human hematopoietic hierarchy.

Hematopoiesis is regulated by a complex gene expression program. To gain further insight into the molecular mechanisms underlying this process in humans, we sampled the transcriptional activity of the CD34+ hematopoietic progenitor line KG1a by single-pass sequencing the 5' ends of 1018 clones from a unidirectional cDNA library. Searches of public databases with the resulting expressed sequence tags (ESTs) identified 101 clones that showed no sequence similarity to any of the existing entries and that were therefore considered to derive from previously undescribed genes. Of the remaining 917 ESTs, 553 (a total of 485 distinct transcripts) corresponded to known genes. A further 279 KG1a ESTs matched or exhibited sequence similarity to ESTs or genomic sequences from humans and other species. Among the latter were putative human orthologs of developmental and cell cycle control genes from Caenorhabditis elegans, Drosophila, and yeast, as well as genes whose predicted amino acid sequences showed similarity to mammalian transcription factors. Hybridization of selected novel KG1a ESTs to globally amplified cDNAs prepared from single primary human hematopoietic precursors and homogeneous populations of terminally maturing hematopoietic cells revealed transcripts that are expressed preferentially at a specific stage or in a particular lineage within the hematopoietic hierarchy. Thus, included in the KG1a EST dataset are candidates for new human genes that may play roles in hematopoietic differentiative progression and lineage commitment.

Animals↗
PubMed1998

Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetic disorder which is clinically characterized by rhizomelic shortening of the upper extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Patients with RCDP can be subdivided into three subgroups based on biochemical analyses and complementation studies. The largest subgroup contains patients with mutations in the PEX7 gene encoding the PTS2 receptor. This results in multiple peroxisomal abnormalities which includes a deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), peroxisomal 3-ketoacyl-CoA thiolase and phytanoyl-CoA hydroxylase, although there are differences in the extent of the deficiencies observed. Patients in the two other subgroups have been reported to be either deficient in the activity of DHAPAT (RCDP type 2) or alkyl-DHAP synthase (RCDP type 3) while no other abnormalities could be observed. To examine whether the gene encoding DHAPAT is mutated in patients with RCDP type 2, we determined the N-terminal amino acid sequence of the enzyme isolated from human placenta. Using this sequence as a query, we identified a 2040 bp open reading frame (ORF) in the human database of expressed sequence tags. Expression of this ORF in the yeast Saccharomyces cerevisiae showed that we have identified the DHAPAT cDNA. The deduced amino acid sequence revealed no PTS2 consensus sequence. In contrast DHAPAT appears to contain a putative PTS1 at the extreme C-terminus. All RCDP type 2 patients analyzed were found to contain mutations in their DHAPAT cDNA. This demonstrates that RCDP type 2 is the result of mutations in DHAPAT.

Acyltransferases↗
PubMed1997

A high-resolution STS, EST, and gene-based physical map of the hereditary paraganglioma region on chromosome 11q23.

The genes responsible for hereditary paragangliomas (glomus tumors, MIM No. 168000) have been mapped to two distinct loci on the long arm of chromosome 11. Most of the informative families appear to be linked to the distal locus on chromosome 11q23 (PGL1), which has been previously confined to a 2-cM interval by haplotype analysis in an extended Dutch pedigree. To facilitate the identification of the PGL1 disease gene, we constructed an approximately 4-Mb ordered clone contig map of Sequence tagged sites, expressed sequence tags (ESTs), and known genes that spans the PGL1 critical region on chromosome 11q23. Among 29 new positional candidate ESTs, only two (EST100999 and EST241777) mapped within the PGL1 critical region. We further characterized the genomic organization of the promyelocytic leukemia zinc finger (PLZF) gene that maps within the PGL1 critical region and physically excluded the serotonin receptor type 3 (5HT3R) gene. Finally, we identified a common, silent, single-base substitution polymorphism in the 5HT3R gene and characterized the allele sets of two new highly polymorphic microsatellite repeats within the PGL1 critical region.

Alleles↗
PubMed2005

High-resolution mapping, cloning and molecular characterization of the Pi-k ( h ) gene of rice, which confers resistance to Magnaporthe grisea.

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ( h )) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F(2) individuals, we mapped the Pi-k ( h ) gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ( h ) gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ( h ) gene revealed that its expression is pathogen inducible.

Amino Acid Sequence↗
PubMed2001

Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice.

Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43 degrees C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.

Animals↗
PubMed2002

cDNA microarray analysis of vestibular schwannomas.

BACKGROUND: Vestibular schwannomas are known to harbor mutations in the neurofibromatosis type 2 tumor suppressor gene, but the mechanism of the neurofibromatosis type 2 tumor suppressor gene action is not well understood. Identification of genes differentially expressed in normal and diseased tissues through the use of a large-scale, cDNA microarray approach may lead to increased understanding of pathways that lead to tumor formation. OBJECTIVE: The objectives of this study were to evaluate the gene expression profiles in vestibular schwannomas in comparison with normal vestibular nerve tissues and to identify pathways that may be altered in schwannomas. METHODS: Total RNA was extracted from one normal vestibular nerve and seven vestibular schwannomas. The normal vestibular nerve was from one of the seven patients with small vestibular schwannomas. Radiolabeled cDNA was synthesized and hybridized to cDNA microarray filters that contained 25,920 known genes or expressed sequence tags. Expression profiles were imaged and analyzed. Selected genes that showed three-fold or greater difference in the intensity between the normal nerve and the schwannomas were further examined by real-time polymerase chain reaction and by immunohistochemical staining. RESULTS: Forty-two genes (0.2%) were upregulated 3-fold or more in at least 5 of the 7 tumors when the filter images were compared with a normal adjacent vestibular nerve. Among them, osteonectin, an angiogenesis mediator, and RhoB GTPase, which is important in cell signaling, were significantly upregulated in 5 of 7 tumors. Among genes that were downregulated, an apoptosis-related LUCA-15 gene was highly underexpressed in 6 of 7 schwannomas when compared with the normal nerve. Also, ezrin, a relative of the NF2 protein, was significantly downregulated in 5 of 7 tumors. Real-time PCR and immunohistochemistry data support the cDNA microarray findings. CONCLUSION: Our cDNA microarray analysis of schwannomas suggested several interesting and potentially important tumorigenesis pathways associated with vestibular schwannoma formation. Further in vivo study is necessary to define the roles of these identified genes and their potential relationships with the neurofibromatosis type 2 tumor suppressor gene.

Adult↗
PubMed2002

DNA microarray analysis of the expression profiles of luteinized granulosa cells as a function of ovarian reserve.

OBJECTIVE: To examine the expression profiles of luteinized granulosa cells isolated from women with normal or diminished ovarian reserve undergoing in vitro fertilization. DESIGN: Expression profiling by complementary DNA microarray analysis. SETTING: Women undergoing in vitro fertilization-embryo transfer in a university-based fertility clinic. PATIENT(S): Eighteen women with normal or decreased ovarian reserve. INTERVENTION(S): All patients were given gonadotropin stimulation in preparation for IVF with granulosa cells isolated at the time of follicular aspiration. MAIN OUTCOME MEASURE(S): Expression profiles of luteinized granulosa cells isolated from each woman were determined by using DNA microarray analysis. RESULT(S): Changes in patterns of gene expression in granulosa cells were observed between women with normal and diminished ovarian reserve. These genes included several anonymous expressed sequence tags and also expressed sequence tags that correspond to known genes. CONCLUSION(S): Expression profiling of granulosa cells by DNA microarray may yield signature patterns that reflect the status of ovarian reserve and the competence of granulosa cells.

Adult↗