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At least 721 records · Page 40Linked to original sources
PubMed2002

Temperature-regulated transcription in the pathogenic fungus Cryptococcus neoformans.

The basidiomycete fungus Cryptococcus neoformans is an opportunistic pathogen of worldwide importance that causes meningitis, leading to death in immunocompromised individuals. Unlike many basidiomycete fungi, C. neoformans is thermotolerant, and its ability to grow at 37 degrees C is considered to be a virulence factor. We used serial analysis of gene expression (SAGE) to characterize the transcriptomes of C. neoformans strains that represent two varieties with different polysaccharide capsule serotypes. These include a serotype D strain of the C. neoformans variety neoformans and a serotype A strain of variety grubii. In this report, we describe the construction and characterization of SAGE libraries from each strain grown at 25 degrees C and 37 degrees C. The SAGE data reveal transcriptome differences between the two strains, even at this early stage of analysis, and identify sets of genes with higher transcript levels at 25 degrees C or 37 degrees C. Notably, growth at the lower temperature increased transcript levels for histone genes, indicating a general influence of temperature on chromatin structure. At 37 degrees C, we noted elevated transcript levels for several genes encoding heat shock proteins and translation machinery. Some of these genes may play a role in temperature-regulated phenotypes in C. neoformans, such as the adaptation of the fungus to growth in the host and the dimorphic transition between budding and filamentous growth. Overall, this work provides the most comprehensive gene expression data available for C. neoformans; this information will be a critical resource both for gene discovery and genome annotation in this pathogen.

Blotting, Northern↗
PubMed2005

Molecular characterization of thyroid toxicity: anchoring gene expression profiles to biochemical and pathologic end points.

Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PB- and PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5 -deiodinase I (5 -DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein-coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress-related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression.

Animals↗
PubMed1999

Prostate cancer expression profiling by cDNA sequencing analysis.

Prostate cancer is a frequently diagnosed solid tumor that is originated mostly from prostate epithelium. One of the key issues in prostate cancer research is to develop molecular markers that can effectively detect and distinguish the progression and malignancy of prostate tumors. Automated, single-pass cDNA sequencing was utilized to rapidly identify expressed genes in a number of cDNA libraries constructed from various normal and tumor prostatic tissues. These included cell lines as well as short-term epithelial culture. A total of 6604 expressed sequence tags (ESTs) were generated and searched against on-line nucleotide and protein databases. A relational database centric software system was constructed to process, store, and analyze EST data rapidly. cDNA contigs were also obtained by assembly of multiple EST sequences. Protein structural signatures were annotated using motif analysis tools including BLOCKS and an in-house-designed neural network. Cross-library comparisons revealed their unique gene expression profiles. Several differentially expressed cDNA clones were identified, and their expression patterns were confirmed by RNA dot blot and RT-PCR analyses.

Cell Line↗
PubMed2001

Engineering chromosomal rearrangements in mice.

The combination of gene-targeting techniques in mouse embryonic stem cells and the Cre/loxP site-specific recombination system has resulted in the emergence of chromosomal-engineering technology in mice. This advance has opened up new opportunities for modelling human diseases that are associated with chromosomal rearrangements. It has also led to the generation of visibly marked deletions and balancer chromosomes in mice, which provide essential reagents for maximizing the efficiency of large-scale mutagenesis efforts and which will accelerate the functional annotation of mammalian genomes, including the human genome.

Animals↗
PubMed2003

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt, TGFbeta, Hedgehog and JAK/STAT signaling pathways.

Cell-cell interactions play important roles in a variety of developmental processes, and therefore molecules involved in the signaling pathways have been studied extensively. Recently, the draft genome sequence of the basal chordate, Ciona intestinalis, was determined. Here we annotated genes for the signaling pathways of Wnt, transforming growth factor beta (TGFbeta), Hedgehog, and JAK/STAT in the genome of Ciona intestinalis. The Ciona genome contains ten wnt genes, six frizzled genes, four sFRP genes, ten TGFbeta family member genes, five TGFbeta-receptor genes, and five Smad genes; most of the genes were found with less redundancy than in vertebrate genomes. The other genes in the signaling pathways are present as a single copy in the Ciona genome. In addition, all of the identified genes for the signaling pathway, except for a few genes, have EST evidence, and their cDNAs are available from the Ciona intestinalis gene collection. Therefore, Ciona intestinalis may provide an experimental system for exploring the basic genetic cascade associated with the signaling pathways in chordates.

Animals↗
PubMed2003

From annotated genomes to metabolic flux models and kinetic parameter fitting.

Significant advances in system-level modeling of cellular behavior can be achieved based on constraints derived from genomic information and on optimality hypotheses. For steady-state models of metabolic networks, mass conservation and reaction stoichiometry impose linear constraints on metabolic fluxes. Different objectives, such as maximization of growth rate or minimization of flux distance from a reference state, can be tested in different organisms and conditions. In particular, we have suggested that the metabolic properties of mutant bacterial strains are best described by an algorithm that performs a minimization of metabolic adjustment (MOMA) upon gene deletion. The increasing availability of many annotated genomes paves the way for a systematic application of these flux balance methods to a large variety of organisms. However, such a high throughput goal crucially depends on our capacity to build metabolic flux models in a fully automated fashion. Here we describe a pipeline for generating models from annotated genomes and discuss the current obstacles to full automation. In addition, we propose a framework for the integration of flux modeling results and high throughput proteomic data, which can potentially help in the inference of whole-cell kinetic parameters.

Algorithms↗
PubMed2002

Genetic and molecular analysis of region 88E9;88F2 in Drosophila melanogaster, including the ear gene related to human factors involved in lineage-specific leukemias.

We identified and characterized the Drosophila gene ear (ENL/AF9-related), which is closely related to mammalian genes that have been implicated in the onset of acute lymphoblastic and myelogenous leukemias when their products are fused as chimeras with those of human HRX, a homolog of Drosophila trithorax. The ear gene product is present in all early embryonic cells, but becomes restricted to specific tissues in late embryogenesis. We mapped the ear gene to cytological region 88E11-13, near easter, and showed that it is deleted by Df(3R)ea(5022rx1), a small, cytologically invisible deletion. Annotation of the completed Drosophila genome sequence suggests that this region might contain as many as 26 genes, most of which, including ear, are not represented by mutant alleles. We carried out a large-scale noncomplementation screen using Df(3R)ea(5022rx1) and chemical (EMS) mutagenesis from which we identified seven novel multi-allele recessive lethal complementation groups in this region. An overlapping deficiency, Df(3R)Po(4), allowed us to map several of these groups to either the proximal or the distal regions of Df(3R)ea(5022rx1). One of these complementation groups likely corresponds to the ear gene as judged by map location, terminal phenotype, and reduction of EAR protein levels.

Animals↗
PubMed2006

The Plasmodium selenoproteome.

The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms, Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS) elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein genes. These selenoproteins were incorrectly annotated in PlasmoDB, were conserved in other Plasmodia and had no detectable homologs in other species. We provide evidence that two Plasmodium SECIS elements supported Sec insertion into parasite and endogenous selenoproteins when they were expressed in mammalian cells, demonstrating that the Plasmodium SECIS elements are functional and indicating conservation of Sec insertion between Apicomplexa and animals. Dependence of the plasmodial parasites on selenium suggests possible strategies for antimalarial drug development.

Amino Acid Sequence↗
PubMed2002

Cloning and characterization of multiple glycosyl hydrolase genes from Trichoderma virens.

Trichoderma virens is a widely distributed soil fungus that is parasitic on other soil fungi. The mycoparasitic activity of T. virens is correlated with the production of numerous antifungal activities, including the secretion of a considerable repertoire of fungal cell wall-degrading enzymes. Here, we report the characterization of a diverse set of chitinase and glucanase genes from T. virens. In each case, full-length genomic clones were isolated and characterized, while sequencing of the corresponding cDNA clones and manual annotation provided a basis for establishing gene structure. Based on homology of the deduced amino acid sequences, we have identified three members of the 42Kd endochitinase gene family, two 33Kd exochitinases, two exochitinases with homology to N-acetylglucosaminidases, and three glucanase genes predicted to encode beta-1,3- and beta-1,6-proteins. The majority of these genes appear to encode signal peptides, suggesting an extracellular location for the corresponding proteins. Despite their overall similarity, the 42Kd class of chitinases can be subdivided, based on the presence of distinct N-terminal domains, suggesting that the proteins may have distinct cellular roles, while Northern blot analysis confirms that these genes possess distinct patterns of gene regulation. Similarly, one of the 33Kd chitinase genes is unique, because it is predicted to encode a protein C-terminus with high homology to the conserved family I cellulose-binding domain. The expression patterns of the chitinase genes were analyzed in both a wild-type strain and a strain disrupted for the major 42Kd chitinase gene of T. virens. The results of these transcript analyses, together with enzymatic assay of the extracellular proteins, suggest interdependent regulation of this important gene family in T. virens.

Acetylglucosaminidase↗
PubMed2006

Sequence analysis of the protein kinase gene family in human testicular germ-cell tumors of adolescents and adults.

The protein kinase gene family is the most frequently mutated in human cancer. Previous work has documented activating mutations in the KIT receptor tyrosine kinase in testicular germ-cell tumors (TGCT). To investigate further the potential role of mutated protein kinases in the development of TGCT and to characterize the prevalence and patterns of point mutations in these tumors, we have sequenced the coding exons and splice junctions of the annotated protein kinase family of 518 genes in a series of seven seminomas and six nonseminomas. Our results show a remarkably low mutation frequency, with only a single somatic point mutation, a K277E mutation in the STK10 gene, being identified in a total of more than 15 megabases of sequence analyzed. Sequencing of STK10 in an additional 40 TGCTs revealed no further mutations. Comparative genomic hybridization and LOH analysis using SNP arrays demonstrated that the 13 TGCTs mutationally screened through the 518 protein kinase genes were uniformly aneuploid with consistent chromosomal gains on 12p, 8q, 7, and X and losses on 13q, 18q, 11q, and 4q. Our results do not provide evidence for a mutated protein kinase implicated in the development of TGCT other than KIT. Moreover, they demonstrate that the general prevalence of point mutations in TGCT is low, in contrast to the high frequency of copy number changes.

Adolescent↗
PubMed2004

Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements.

The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to approximately 1% of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

Alu Elements↗
PubMed2003

Structure and expression of the human oocyte-specific histone H1 gene elucidated by direct RT-nested PCR of a single oocyte.

Oocyte-specific histone H1 is expressed during oogenesis and early embryogenesis. It has been described in mice and some nonmammalian species, but not in humans. Here, we identified the cDNA in unfertilized human oocytes using direct RT-nested PCR of a single cell. Sequencing of this cDNA indicated an open reading frame encoding a 347-amino acid protein. Expression was oocyte-specific. Homology was closest with the corresponding gene of mouse (H1oo; 42.3%), and, to lesser extent, with that of Xenopus laevis (B4; 25.0%). The gene, named osH1, included five exons as predicted by the NCBI annotation project of the human genome, although the actual splicing site at the 3(') end of exon 3 was different by 48 nucleotides from the prediction. The presence of polyadenylation signals and successful amplification of cDNA by RT-PCR using an oligo(dT) primer suggested that the osH1 mRNA is polyadenylated unlike somatic H1 mRNA. Our technique and findings should facilitate investigation of human fertilization and embryogenesis.

Amino Acid Sequence↗
PubMed1997

IMGT, the international ImMunoGeneTics database.

IMGT, the international ImMunoGeneTics database, is an integrated database specializing in immunoglobulins, T-cell receptors (TcR) and major histocompatibility complex (MHC) of all vertebrate species, initiated and co-ordinated by Marie-Paule Lefranc, CNRS, Montpellier II University, Montpellier, France (lefranc@ligm.crbm.cnrs-mop.fr). IMGT includes two databases: LIGM-DB (for immunoglobulins and TcR) and MHC/HLA-DB. IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 19 000 immunoglobulin and TcR sequences from 78 species. MHC/HLA-DB contains class I and class II human leukocyte antigen alignment tables. An IMGT tool, DNAPLOT, developed for immunoglobulins, TcR and MHC sequence alignments, is also available. IMGT works in close collaboration with the EMBL database. IMGT goals are to establish a common data access to all immunogenetics data, including sequences, oligonucleotide primers, gene maps and other genetic data of immunoglobulins, TcR and MHC molecules, and to provide a graphical user-friendly data access. IMGT will have important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT can be accessed at http://imgt.cnusc.fr:8104 and http://www.ebi.ac.uk/IMGT

Amino Acid Sequence↗
PubMed2001

Interspecies conservation of gene order and intron-exon structure in a genomic locus of high gene density and complexity in Plasmodium.

A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.

Alternative Splicing↗
PubMed2026

Dual Aberrant Splicing Caused by an Apparently Missense CHD7 Variant, c.5273A>G (p.Asp1758Gly), in CHARGE Syndrome.

CHARGE syndrome is a rare congenital disorder primarily attributed to heterozygous pathogenic variants of the CHD7 gene. Most pathogenic CHD7 variants are loss-of-function (LoF) variants, whereas the interpretation of missense variants remains challenging in the absence of functional evidence for their pathogenicity. We report a female infant presenting with clinical features characteristic of CHARGE syndrome. Targeted sequencing identified a heterozygous CHD7 variant (NM_017780.4:c.5273A>G), initially annotated as a missense substitution p.Asp1758Gly. This variant has been previously reported and registered with conflicting pathogenicity classifications; however, its transcript-level consequences remain unclear. Long-PCR-based RNA sequencing of total RNA from peripheral blood mononuclear cells revealed two aberrant splicing patterns associated with the variant: a predominant transcript carrying a 28-bp deletion due to cryptic donor splice-site activation, and a minor transcript with partial intron 24 retention. Both transcripts were predicted to result in premature termination codons. These findings demonstrate that c.5273A>G functions as a LoF variant through dual aberrant splicing rather than a simple missense substitution. This case underscores the importance of RNA-level splicing analysis for the accurate interpretation and classification of CHD7 missense variants.

CHD7↗
PubMed2006

Persistent biases in the amino acid composition of prokaryotic proteins.

Correspondence analysis of 28 proteomes selected to span the entire realm of prokaryotes revealed universal biases in the proteins' amino acid distribution. Integral Inner Membrane Proteins always form an individual cluster, which can then be used to predict protein localisation in unknown proteomes, independently of the organism's biotope or kingdom. Orphan proteins are consistently rich in aromatic residues. Another bias is also ubiquitous: the amino acid composition is driven by the G + C content of the first codon position. An unexpected bias is driven, in many proteomes, by the AAN box of the genetic code, suggesting some functional biochemical relationship between asparagine and lysine. Less-significant biases are driven by the rare amino acids, cysteine and tryptophan. Some allow identification of species-specific functions or localisation such as surface or exported proteins. Errors in genome annotations are also revealed by correspondence analysis, making it useful for quality control and correction.

Amino Acids↗
PubMed1997

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

Animals↗
PubMed2006

Correlated mutations: advances and limitations. A study on fusion proteins and on the Cohesin-Dockerin families.

Correlated mutations have been repeatedly exploited for intramolecular contact map prediction. Over the last decade these efforts yielded several methods for measuring correlated mutations. Nevertheless, the application of correlated mutations for the prediction of intermolecular interactions has not yet been explored. This gap is due to several obstacles, such as 3D complexes availability, paralog discrimination, and the availability of sequence pairs that are required for inter- but not intramolecular analyses. Here we selected for analysis fusion protein families that bypass some of these obstacles. We find that several correlated mutation measurements yield reasonable accuracy for intramolecular contact map prediction on the fusion dataset. However, the accuracy level drops sharply in intermolecular contacts prediction. This drop in accuracy does not occur always. In the Cohesin-Dockerin family, reasonable accuracy is achieved in the prediction of both intra- and intermolecular contacts. The Cohesin-Dockerin family is well suited for correlated mutation analysis. Because, however, this family constitutes a special case (it has radical mutations, has domain repeats, within each species each Dockerin domain interacts with each Cohesin domain, see below), the successful prediction in this family does not point to a general potential in using correlated mutations for predicting intermolecular contacts. Overall, the results of our study indicate that current methodologies of correlated mutations analysis are not suitable for large-scale intermolecular contact prediction, and thus cannot assist in docking. With current measurements, sequence availability, sequence annotations, and underdeveloped sequence pairing methods, correlated mutations can yield reasonable accuracy only for a handful of families.

Amino Acids↗