(41/3795) A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript.
Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA. (+info)
(42/3795) New experimental and computational approaches to the analysis of gene expression.
Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds. (+info)
(43/3795) Reed-Sternberg cell genome expression supports a B-cell lineage.
The malignant Reed-Sternberg cell of Hodgkin's disease, first described a century ago, has resisted in-depth analysis due to its extreme rarity in lymphomatous tissue. To directly study its genome-wide gene expression, approximately 11,000,000 bases (27,518 cDNA sequences) of expressed gene sequence was determined from living single Reed-Sternberg cells, Hodgkin's tissue, and cell lines. This approach increased the number of genes known to be expressed in Hodgkin's disease by 20-fold to 2,666 named genes. The data here indicate that Reed-Sternberg cells from both nodular sclerosing and lymphocyte predominant Hodgkin's disease were derived from an unusual B-cell lineage based on a comparison of their gene expression to approximately 40,000,000 bases (10(5) sequences) of expressed gene sequence from germinal center B cells (GCB) and dendritic cells. The data set of expressed genes, reported here and on the World Wide Web, forms a basis to understand the genes responsible for Hodgkin's disease and develop novel diagnostic markers and therapies. This study of the rare Reed-Sternberg cell, concealed in its heterogenous cellular context, also provides a formidable test case to advance the limit of analysis of differential gene expression to the single disease cell. (+info)
(44/3795) The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.
Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes. (+info)
(45/3795) A 3-Mb high-resolution BAC/PAC contig of 12q22 encompassing the 830-kb consensus minimal deletion in male germ cell tumors.
Cytogenetic and molecular genetic analyses have shown that the 12q22 region is recurrently deleted in male germ cell tumors (GCTs), suggesting that this site may harbor a tumor suppressor gene (TSG). Previous loss of heterozygosity (LOH) analyses identified a consensus minimal deleted region between the markers D12S377 and D12S296, and a YAC clone contig covering the region was generated. Here, we describe a high-resolution sequence-ready physical map of this contig covering a 3-Mb region. The map comprised of 52 cosmids, 49 PACs, and 168 BACs that were anchored to the previous YAC contig; 99 polymorphic, nonpolymorphic, EST, and gene-based markers are now placed on this map in a unique order. Of these, 61 markers were isolated in the present study, including one that was polymorphic. In addition, we have narrowed the minimal deletion to approximately 830 kb between D12S1716 (proximal) and P382A8-AG (distal) by LOH analysis of 108 normal-tumor DNAs from GCT patients using 21 polymorphic STSs. These physical and deletion maps should prove useful for identification of the candidate TSG in GCTs, provide framework to generate complete DNA sequence, and ultimately generate a gene map of this segment of the chromosome 12. [The sequence data described in this paper have been submitted to the Genome Survey Sequence under accession nos. AQ254896-AQ254955 and AQ269251-AQ269266. Online supplementary material is available at http://www.genome.org] (+info)
(46/3795) The molecular basis of malonyl-CoA decarboxylase deficiency.
We characterized a 2.1-kb human cDNA with a 1362-bp (454-amino acid) open reading frame showing 70.3% amino acid identity to goose malonyl-CoA decarboxylase (MCD). We have identified two different homozygous mutations in human MCD (hMCD) by using RT-PCR analysis of fibroblast RNA from two previously reported consanguineous Scottish patients with MCD deficiency. The first mutation is a 442C-->G transversion resulting in a premature stop codon (S148X) in the N-terminal half of the protein. The second is a 13-bp insertion in the mature RNA, causing a frameshift with predicted protein truncation. This insertion is the result of an intronic mutation generating a novel splice acceptor sequence (IVS4-14A-->G). Both mutations were found to segregate appropriately within the families and were not found in 100 normal unrelated individuals. These mutations would be predicted to cause MCD deficiency, thus confirming this transcript as the hMCD ortholog. The peptide sequence of hMCD revealed a C-terminal peroxisomal targeting sequence (-SKL). This targeting signal appears to be functional in vivo, since the distribution of MCD enzymatic activity in rat liver homogenates-as measured by means of subcellular fractionation-strongly suggests that MCD is localized to peroxisomes in addition to the mitochondrial localization reported elsewhere. These data strongly support this cDNA as encoding human MCD, an important regulator of fatty acid metabolism. (+info)
(47/3795) The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.
The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC. (+info)
(48/3795) Analysis of cDNAs expressed during first cell division of petunia petal protoplast cultures using expressed sequence tags.
A large-scale sequence analysis of randomly selected cDNA clones was performed to isolate numerous genes in petunia petal protoplast cultures. We have partially sequenced 1158 randomly selected genes of the cDNA library constructed from 2-6 d cultured petal protoplasts. Three hundred and sixty-five different genes were identified, 25% of which showed significant similarity to existing sequences in the petunia, and an array of other organisms. In this report, 90 independent genes are analyzed in detail. A functional categorization of the database-matched expressed sequence tags (ESTs) showed that defense- or stress-related genes, as well as genes involved in the primary metabolic pathways and in the transcriptional or translational apparatus are abundantly represented. In particular, ESTs were identified with apparent homologies to the cyclin-dependent kinase, histone, actin-depolymerizing factor, proteasome, and ubiquitin which are expected to be related to cell division or to cell cycle control. (+info)