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At least 307 records · Page 17Linked to original sources
PubMed1998

The four cdc25 genes from the nematode Caenorhabditis elegans.

During eukaryotic evolution, multicellular organisms have evolved multiple members of gene families that may display unique, partially overlapping, or redundant functions during development. More than 75% of the C. elegans genome has been sequenced, which represents approximately 95% of the coding sequences. This provides a unique opportunity to identify most, if not all, of the members of a given gene family. We have searched the C. elegans genome database for members of a key family of cell cycle regulators, the CDC25 phosphatases, and have identified four genes. The four C. elegans genes represent a larger family within a single organism than has been reported so far in Drosophila, mice and humans. An amino acid comparison revealed a high degree of similarity and identity within the phosphatase domain. This analysis also identified an expanded consensus sequence that can be used to discover new members of the CDC25 phosphatase family. However, the four C. elegans sequences display a few novel amino acid substitutions in the residues surrounding the invariant catalytic motif CX5R. These data demonstrate the value of genome database searching for identifying new members of known gene families, understanding genetic diversity, and for studying gene structure.

Amino Acid Sequence↗
PubMed2005

Effects on promoter activity of base substitutions in the cis-acting regulatory element of HrpXo regulons in Xanthomonas oryzae pv. oryzae.

In Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, HrpXo is known to be a transcriptional regulator for the hypersensitive response and pathogenicity (hrp) genes. Several HrpXo regulons are preceded by a consensus sequence (TTCGC-N(15)-TTCGC), called the plant-inducible promoter (PIP) box, which is required for expression of the gene that follows. Thus, the PIP box can be an effective marker for screening HrpXo regulons from the genome database. It is not known, however, whether mutations in the PIP box cause a complete loss of promoter activity. In this study, we introduced base substitutions at each of the consensus nucleotides in the PIP box of the hrpC operon in X. oryzae pv. oryzae, and the promoter activity was examined by using a beta-glucuronidase (GUS) reporter gene. Although the GUS activity was generally reduced by base substitutions, several mutated PIP boxes conferred considerable promoter activity. In several cases, even imperfect PIP boxes with two base substitutions retained 20% of the promoter activity found in the nonsubstituted PIP box. We screened HrpXo regulon candidates with an imperfect PIP box obtained from the genome database of X. oryzae pv. oryzae and found that at least two genes preceded by an imperfect PIP box with two base substitutions were actually expressed in an HrpXo-dependent manner. These results indicate that a base substitution in the PIP box is quite permissible for HrpXo-dependent expression and suggest that X. oryzae pv. oryzae may possess more HrpXo regulons than expected.

Bacterial Proteins↗
PubMed2003

Total RNA yield and microarray gene expression profiles from fine-needle aspiration biopsy and core-needle biopsy samples of breast carcinoma.

BACKGROUND: Gene expression profiling should be applicable to needle biopsy samples if microarray technology is to become practically useful for clinical research or management of breast carcinoma. This study compared gene expression profiles derived from fine-needle aspiration biopsy (FNAB) and from core needle biopsy (CBX). METHODS: Total RNA was extracted from single FNAB and CBX samples. Corresponding pairs of FNAB and CBX were analyzed for similarity of gene expression profiles using cDNA microarrays that contain 30721 human sequences. A subset of genes that distinguished CBX samples from FNAB samples was evaluated in a larger group of needle biopsy samples and in a published genomic database derived from 78 sporadic breast carcinomas with known clinical outcome. RESULTS: Sixty-eight patients with newly diagnosed breast carcinoma were included in the current study. Sixty-five patients underwent FNAB (17 had both FNAB and CBX) and 3 underwent CBX only. Extracted RNA was of suitable quality for hybridization in 46 (71%) FNABs and 15 (75%) CBXs. Total RNA yield in those samples was similar for single-pass FNAB (mean = 3.6 microg and median = 2.2 microg; n = 46) and CBX (mean = 2.8 microg and median = 2.0 microg; n = 15), with 1 microg or more of total RNA in all cases. Transcriptional profiling was performed successfully in all cases when it was attempted, in a total of 50 samples (38 FNABs and 12 CBXs), including matched FNAB and CBX samples from 10 patients. There were differences in gene expression profiles in 10 matched FNAB and CBX sample pairs. Genes that were expressed differently in CBX samples, compared with FNAB samples, were recognized as being predominantly from the endothelium, fibroblasts, myofibroblasts or smooth muscle, and histiocytes. Corresponding microscopic cell counts from FNABs demonstrated means of 80% tumor cells, 15% lymphocytes, and 5% stromal cells, whereas CBXs contained 50% tumor cells, 20% lymphocytes, and 30% stromal cells. Considering that CBXs are approximately six-fold richer in nonlymphoid stromal cells than FNABs and that CBXs differentially express a set of recognized stromal genes, the authors used these biopsies to define a transcriptional profile of breast carcinoma stroma. A set of 120 genes differentially expressed in CBXs was assessed independently in a published breast carcinoma genomic database to classify breast carcinomas based on stromal gene expression. Subgroups of tumors with low or high stromal signal were identified, but there was no correlation with the development of systemic metastases within 5 years. CONCLUSIONS: Both FNAB and CBX yield a similar quality and quantity of total RNA and are suitable for cDNA microarray analyses in approximately 70-75% of single-pass samples. Transcriptional profiles from FNAB and CBX of the same tumor generally are similar and are driven by the tumor cell population. The authors concluded that each technique has relative advantages. The FNABs provide transcriptional profiles that are a purer representation of the tumor cell population, whereas transcriptional profiles from CBXs include more representation from nonlymphoid stromal elements. Selection of the preferred needle biopsy sampling technique for genomic studies of breast carcinomas should depend on whether variable stromal gene expression is desirable in the samples.

Adult↗
PubMed2002

Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper.

To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-alpha motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Prothymosin alpha is not found in yeast.

According to published accounts, prothymosin alpha exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. FEBS Lett. 257, 247-250, 1989). We report here our failure to find evidence for prothymosin alpha in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin alpha coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin alpha coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no prothymosin alpha-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a prothymosin alpha gene in animals other than mammals is highly unlikely.

Amino Acid Sequence↗
PubMed2003

Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells.

Human CNT3 encodes the concentrative nucleoside transport N3 system. Previous expression studies in oocytes showed that the Km values for nucleosides of the cloned hCNT3 were 7- to 25-fold lower than the endogenous N3 transporter in HL60 cells. Therefore, in the present study we re-examined the kinetic properties of the cloned hCNT3 using mammalian cell expression systems by transient expression in Cos7L cells and stably expression in nucleoside transporter deficient PK15NTD cells. We demonstrated that hCNT3 is a Na-dependent, broadly-selective nucleoside transporter with affinities (<11 microM) for nucleosides closely resembling the endogenous N3 transporter. Pharmacological studies showed that phloridzin is a mixed-type inhibitor of hCNT3 (Ki=15 microM), and the dideoxyuridine analogs are poor substrates. By epitope-tagging, we further demonstrated that hCNT3 is N-glycosylated as PNGase F and Endo H deglycosylated hCNT3 from 67 kDa to 58 kDa. Searching the human genome database, we identified the genomic organization of hCNT3. This gene contains 19 exons and its exon-intron boundaries within the coding sequence exactly match with those of hCNT1 and hCNT2 with one additional exon in the N-terminus. Our data suggest that hCNT3 gene is evolutionarily conserved with hCNT1 and hCNT2. Physiologically, hCNT3 is a glycoprotein, which transports purine and pyrimidine nucleosides in a Na-dependent manner with high affinities.

Animals↗
PubMed2004

Visualizing the laboratory mouse: capturing phenotype information.

A concerted effort to develop myriad new phenotypic alleles through mutagenesis programs presents new challenges for the biomedical community and for the informatics infrastructure needed to support this work. To handle and co-ordinate large programs of treatment, breeding, and sequential or longitudinal testing for a variety of obvious and subtle traits requires sophisticated data management software. Further, trait analyses, heritability testing, and animal availability and status must be captured and disseminated to the wider community. The Mouse Genome Database (MGD) will serve as the central integration point for the various mutagenesis programs, registering new alleles, providing accession identifiers, and capturing phenotypic descriptions. In addition, MGD will provide public access to unified searches over all alleles with links to the centres of origin for detailed testing data.

Animals↗
PubMed2003

Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2.

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have diverse myotropic actions and are also initiating sex pheromone biosynthesis and embryonic diapause. Gene silencing, using the RNA-mediated interference technique, showed that CG8784 gene silencing caused lethality in embryos, whereas CG8795 gene silencing resulted in strongly reduced viability for both embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors to be identified.

Amino Acid Sequence↗
PubMed2004

Prediction and functional analysis of native disorder in proteins from the three kingdoms of life.

An automatic method for recognizing natively disordered regions from amino acid sequence is described and benchmarked against predictors that were assessed at the latest critical assessment of techniques for protein structure prediction (CASP) experiment. The method attains a Wilcoxon score of 90.0, which represents a statistically significant improvement on the methods evaluated on the same targets at CASP. The classifier, DISOPRED2, was used to estimate the frequency of native disorder in several representative genomes from the three kingdoms of life. Putative, long (>30 residue) disordered segments are found to occur in 2.0% of archaean, 4.2% of eubacterial and 33.0% of eukaryotic proteins. The function of proteins with long predicted regions of disorder was investigated using the gene ontology annotations supplied with the Saccharomyces genome database. The analysis of the yeast proteome suggests that proteins containing disorder are often located in the cell nucleus and are involved in the regulation of transcription and cell signalling. The results also indicate that native disorder is associated with the molecular functions of kinase activity and nucleic acid binding.

Databases, Genetic↗
PubMed2005

Prion protein from Xenopus laevis: overexpression in Escherichia coli of the His-tagged protein and production of polyclonal antibodies.

Prion protein (PrP) and PrP-related proteins have been identified in reptiles, amphibians, and fishes by means of cDNA cloning, genome database searching and comparative genomics. However, no studies have been reported so far on the expression of PrP at the protein level in those animals. This report presents a procedure to obtain and purify recombinant PrP from Xenopus laevis expressed in Escherichia coli as a fusion protein in which mature PrP (residues 21-194) is linked to a 35-amino acid N-terminal extension containing a hexahistidine stretch. The protein was used to raise and purify by affinity chromatography anti-Xenopus PrP polyclonal antibodies which were suitable to detect the presence of PrP in Xenopus brain by Western blot. This is the first report of a positive identification of PrP in amphibian at the protein level. Anti-Xenopus PrP antibodies do not cross react with PrP from different sources (human, bovine, sheep, and turtle). Similarly, Xenopus PrP do not react with anti-turtle PrP(143-248) antibodies.

Amphibian Proteins↗
PubMed2001

Cloning and sequence of the gene encoding the muscle fatty acid binding protein from the desert locust, Schistocerca gregaria.

Muscle fatty acid binding protein (FABP) is a major cytosolic protein in flight muscle of the desert locust, Schistocerca gregaria. FABP expression varies greatly during development and periods of increased fatty acid utilization, but the molecular mechanisms that regulate its expression are not known. In this study, the gene coding for locust muscle FABP was amplified by PCR and cloned, together with 1.2 kb of upstream sequence. The sequence coding for the 607 bp cDNA is interrupted by two introns of 12.7 and 2.9 kb, inserted in analogous positions as the first and third intron of the mammalian homologues. Both introns contain repetitive sequences also found in other locust genes, and the second intron contains a GT-microsatellite. The promoter sequence includes a canonical TATA box 24 bp upstream of the transcription start site. The upstream sequence contains various potential myocyte enhancer sequences and a 160 bp segment that is repeated three times. In database searches in the genome database of Drosophila melanogaster, a gene with the same gene organization and promoter structure was identified, likely the dipteran homologue of muscle FABP. Upstream of both insect genes, a conserved 19 bp inverted repeat sequence was detected. A similar but reverse palindrome is also present upstream of all mammalian heart FABP genes, possibly representing a novel element involved in muscle FABP expression.

Amino Acid Sequence↗
PubMed2003

Functional genomics of intracellular peptide recognition domains with combinatorial biology methods.

Phage-displayed peptide libraries have been used to identify specific ligands for peptide-binding domains that mediate intracellular protein-protein interactions. These studies have provided significant insights into the specificities of particular domains. For PDZ domains that recognize C-terminal sequences, the information has proven useful in identifying natural binding partners from genomic databases. For SH3 domains that recognize internal proline-rich motifs, the results of database searches with phage-derived ligands have been compared with the results of yeast-two-hybrid experiments to produce overlap networks that reliably predict natural protein-protein interactions. In addition, libraries of phage-displayed PDZ and SH3 domains have been used to identify the residues responsible for ligand recognition, and also to engineer domains with altered specificities.

Databases, Protein↗
PubMed2000

Reverse transcription of an RNA genome from databasing paper (FTA(R)).

The instability of RNA in solutions during storage and travel is an impediment to its utilization in routine diagnostics. A robust and simple approach to the problem of RNA protection and processing is offered by storage of RNA desiccated with processing procedures that do not solublize the RNA until the beginning of reverse transcription. The feasibility of this general approach was tested with coxsackievirus B4 (CVB-4) from blood or culture fluid held on a storage and transport medium (FTA(R)) and analysed by reverse transcriptase PCR (RT-PCR) without removing the RNA from the FTA(R) until reverse transcription. Phase-trapping techniques based on water-miscible solvents such as ethanol or phenol were compared with simple buffers and concentrated lithium chloride solutions. RT-PCR detection of viral RNA reached a sensitivity of approximately 0.1 fg, which is comparable with other non-nested PCR techniques. Whole blood as a virus vehicle significantly interfered with CVB-4 detection, but to an acceptable degree. Desiccation-storage of the RNA of CVB-4 appears to be unaffected by weeks on the storage medium under ambient conditions. These characteristics indicate that this approach forms a credible developmental base for RNA-based pathogen diagnostics with particular application to the problem of transporting potentially infectious body fluids to a centralized laboratory for analysis.

Blood Specimen Collection↗
PubMed1994

The GDB Human Genome Data Base anno 1994.

In 1991 the Genome Data Base at Johns Hopkins University School of Medicine was selected as the central repository for mapping data from the Human Genome Project, and was funded by NIH and DOE under a three year award. GDB has now finished 28 months of Federally funded operation. During this period a great deal of progress and many internal changes have taken place. In addition, many changes have also occurred in the external environment, and GDB has adapted its strategies to play an appropriate role in those changes as well. Recognizing the central role of mapping information in the genome project, it is important that GDB respond aggressively to the increasing demands of genomic researchers, as well as formulate a program of response to a number of long standing, but still unmet, needs of that community. It is even more important that GDB provide leadership in the genome informatics enterprise. Three themes described here are dominant in our future plans and represent the essence of the major changes made in the past year. They include: enhanced data acquisition, better map representation, and full integration into the collection of genomic databases.

Computer Communication Networks↗
PubMed1998

The mitBASE human dataset structure.

MitBASE is a comprehensive and integrated mitochondrial genome database funded within the EU BIOTECH PROGRAM. It is a project for the development and implementation of an integrated and comprehensive database of mitochondrial data which will collect all available information from different organisms and from intraspecies variants and mutants. The present paper describes the structure of the Human dataset in mitBASE where human molecular data are distinguished from clinical and pathological data. MitBASE home page address is: http://www.ebi.ac.uk/htbin/Mitbase/mitb ase.pl

Computer Communication Networks↗
PubMed2007

MaizeGDB's new data types, resources and activities.

MaizeGDB is the Maize Genetics and Genomics Database. Available at MaizeGDB are diverse data that support maize research including maps, gene product information, loci and their various alleles, phenotypes (both naturally occurring and as a result of directed mutagenesis), stocks, sequences, molecular markers, references and contact information for maize researchers worldwide. Also available through MaizeGDB are various community support service bulletin boards including the Editorial Board's list of high-impact papers, information about the Annual Maize Genetics Conference and the Jobs board where employment opportunities are posted. Reported here are data updates, improvements to interfaces and changes to standard operating procedures that have been made during the past 2 years. MaizeGDB is freely available and can be accessed online at http://www.maizegdb.org.

Chromosome Mapping↗
PubMed2006

Expanded protein information at SGD: new pages and proteome browser.

The recent explosion in protein data generated from both directed small-scale studies and large-scale proteomics efforts has greatly expanded the quantity of available protein information and has prompted the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) to enhance the depth and accessibility of protein annotations. In particular, we have expanded ongoing efforts to improve the integration of experimental information and sequence-based predictions and have redesigned the protein information web pages. A key feature of this redesign is the development of a GBrowse-derived interactive Proteome Browser customized to improve the visualization of sequence-based protein information. This Proteome Browser has enabled SGD to unify the display of hidden Markov model (HMM) domains, protein family HMMs, motifs, transmembrane regions, signal peptides, hydropathy plots and profile hits using several popular prediction algorithms. In addition, a physico-chemical properties page has been introduced to provide easy access to basic protein information. Improvements to the layout of the Protein Information page and integration of the Proteome Browser will facilitate the ongoing expansion of sequence-specific experimental information captured in SGD, including post-translational modifications and other user-defined annotations. Finally, SGD continues to improve upon the availability of genetic and physical interaction data in an ongoing collaboration with BioGRID by providing direct access to more than 82,000 manually-curated interactions.

Computer Graphics↗