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PubMed1985

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

Amanitins↗
PubMed2002

Analysis of partial pol and env sequences indicates a high prevalence of HIV type 1 recombinant strains circulating in Gabon.

Forty-one HIV-1 strains from Gabonese patients were studied according to the following strategy: nested polymerase chain reaction were performed to obtain an approximately 1,100-bp fragment containing the protease gene and the 5' half of the reverse transcriptase gene. Additional amplifications were carried out to obtain an approximately 700-bp fragment encompassing the C2V3 env gene. Fragments of 600 to 1,200 bp in the gag gene overlapping the pol sequences were used for the study of recombination patterns. Phylogenetic analyses of the different fragments were used to investigate HIV-1 diversity in Gabon. Thirty-one strains were sequenced in the env and pol genes and phylogenetic analyses classified them as subtype A (n = 2), D (n = 4), G (n = 1), H (n = 1), CRF02 (n = 8), and CRF MAL-like (n = 6); in addition, there were 6 unique recombinant forms and 1 unclassified strain, and in 2 cases pol/env sequences classified strains as subtype D whereas gag phylogeny classified them as subtype A. In 10 cases only 1 fragment was available: 4 env (2 subtype D, 1 subtype H, and 1 subtype U) and 6 pol (1 subtype A, 1 subtype C, 2 subtype G, and 2 subtype U). Minor mutations associated with viral resistance to antiretroviral drugs were observed in more than 80% of analyzed strains. Our study confirms the extensive HIV-1 diversity found in Central Africa, with more than 70% of strains from Gabon exhibiting discordant clustering in pol and env genomic regions and less than 60% concordance between sequencing and heteroduplex mobility assay genotyping. These findings highlight the fact that Central Africa represents the epicenter for the origin of HIV-1. The strategy of sequencing pol in association with env has proved to be useful for analysis of the recombinant strains. The main advantage of this approach is that it also allows for evaluation of genotypic susceptibility to antiretroviral drugs without the need for supplementary analyses.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence and mutational analysis of an immunity repressor gene from Bacillus subtilis temperate phage phi 105.

We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host. Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment. We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map. Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39. The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3'. Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements. Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B. subtilis RNA polymerase, as would be expected for an early phage gene.

Amino Acid Sequence↗
PubMed2005

T-profiler: scoring the activity of predefined groups of genes using gene expression data.

One of the key challenges in the analysis of gene expression data is how to relate the expression level of individual genes to the underlying transcriptional programs and cellular state. Here we describe T-profiler, a tool that uses the t-test to score changes in the average activity of predefined groups of genes. The gene groups are defined based on Gene Ontology categorization, ChIP-chip experiments, upstream matches to a consensus transcription factor binding motif or location on the same chromosome. If desired, an iterative procedure can be used to select a single, optimal representative from sets of overlapping gene groups. T-profiler makes it possible to interpret microarray data in a way that is both intuitive and statistically rigorous, without the need to combine experiments or choose parameters. Currently, gene expression data from Saccharomyces cerevisiae and Candida albicans are supported. Users can upload their microarray data for analysis on the web at http://www.t-profiler.org.

Algorithms↗
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↗
PubMed2003

Evaluation of common gene expression patterns in the rat nervous system.

In the postgenomic era, integrating data obtained from array technologies (e.g., oligonucleotide microarrays) with published information on eukaryotic genomes is beginning to yield biomarkers and therapeutic targets that are key for the diagnosis and treatment of disease. Nevertheless, identifying and validating these drug targets has not been a trivial task. Although a plethora of bioinformatics tools and databases are available, major bottlenecks for this approach reside in the interpretation of vast amounts of data, its integration into biologically representative models, and ultimately the identification of pathophysiologically and therapeutically useful information. In the field of neuroscience, accomplishing these goals has been particularly challenging because of the complex nature of nerve tissue, the relatively small adaptive nature of induced-gene expression changes, as well as the polygenic etiology of most neuropsychiatric diseases. This report combines published data sets from multiple transcript profiling studies that used GeneChip microarrays to illustrate a postanalysis approach for the interpretation of data from neuroscience microarray studies. By defining common gene expression patterns triggered by diverse events (administration of psychoactive drugs and trauma) in different nerve tissues (telencephalic brain areas and spinal cord), we broaden the conclusions derived from each of the original studies. In addition, the evaluation of the identified overlapping gene lists provides a foundation for generating hypotheses relating alterations in specific sets of genes to common physiological processes. Our approach demonstrates the significance of interpreting transcript profiling data within the context of common pathways and mechanisms rather than specific to a given tissue or stimulus. We also highlight the use of gene expression patterns in predictive biology (e.g., in toxicogenomics) as well as the utility of combining data derived from multiple microarray studies that examine diverse biological events for a broader interpretation of data from a particular microarray study.

Animals↗
PubMed2005

Distinction in gene expression profiles demonstrated in parathyroid adenomas by high-density oligoarray technology.

OBJECTIVES: Somatic deletion of chromosome 11q13 is the most frequent genetic aberration in parathyroid adenoma. To gain further insight into the genetic etiology of parathyroid tumor development, we examined a comprehensive gene expression profile of parathyroid adenomas and normal parathyroid tissues. The results were then evaluated with respect to differences between adenomas and normal parathyroid tissue, and to the presence of loss of heterozygosity (LOH) in chromosomal region 11q13. DESIGN AND METHODS: Sporadic parathyroid adenomas and normal parathyroids were hybridized against HG-U95Av2 oligonucleotide arrays (Affymetrix) containing a total of 12,625 probe sets. Quantitative real-time PCR (QRT-PCR) was performed in a larger series of parathyroid adenomas, in order to con-firm the microarray results. RESULTS: Cyclin D1 and c-Jun showed increased expression in adenomas vs normal parathyroids by microarray analysis and QRT-PCR, suggesting an oncogenic role of these genes in parathyroid tumor development. At unsupervised hierarchical clustering, the adenomas fell into two groups: Group I adenomas were characterized by 11q13 LOH, while Group II adenomas lacked this abnormality. In addition, a t-test analysis identified largely non-overlapping genes with differential expression in the tumors subgroups; e.g. in Group I tumors the putative oncogene ENC 1 was found highly over-expressed vs Group II adenomas. CONCLUSIONS: The microarray analyses revealed partly distinctive and partly common expression profiles in parathyroid adenomas with and without 11q13 LOH. In addition, approximately half of the under-expressed genes were mapped to chromosome 11, in agreement with a dose effect following loss of this chromosome.

Adenoma↗
PubMed2000

Ectodermally derived FGF8 defines the maxillomandibular region in the early chick embryo: epithelial-mesenchymal interactions in the specification of the craniofacial ectomesenchyme.

The most rostral cephalic crest cells in the chick embryo first populate ubiquitously in the rostroventral head. Before the influx of crest cells, the ventral head ectoderm expresses Fgf8 in two domains that correspond to the future mandibular arch. Bmp4 is expressed rostral and caudal to these domains. The rostral part of the Bmp4 domain develops into the rostral end of the maxillary process that corresponds to the transition between the maxillomandibular and premandibular regions. Thus, the distribution patterns of FGF8 and BMP4 appear to foreshadow the maxillomandibular region in the head ectoderm. In the ectomesenchyme of the pharyngula embryo, expression patterns of some homeobox genes overlap the distribution of their upstream growth factors. Dlx1 and Barx1, the targets of FGF8, are expressed in the mandibular ectomesenchyme, and Msx1, the target of BMP4, in its distal regions. Ectopic applications of FGF8 lead to shifted expression of the target genes as well as repatterning of the craniofacial primordia and of the trigeminal nerve branches. Focal injection of a lipophilic dye, DiI, showed that this shift was at least in part due to the posterior transformation of the original premandibular ectomesenchyme into the mandible, caused by the changed distribution of FGF8 that defines the mandibular region. We conclude that FGF8 in the early ectoderm defines the maxillomandibular region of the prepharyngula embryo, through epithelial-mesenchymal interactions and subsequent upregulation of homeobox genes in the local mesenchyme. BMP4 in the ventral ectoderm appears to limit the anterior expression of Fgf8. Ectopic application of BMP4 consistently diminished part of the mandibular arch.

Animals↗
PubMed1994

Cloning, analysis and expression of the HindIII R-M-encoding genes.

The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34,950 Da); the MTase is predicted to be 309 aa (35,550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible lambda pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.

Amino Acid Sequence↗
PubMed2006

Dynamics of a hepatitis B virus e antigen minus population ascribed to genotype F during the course of a chronic infection despite the presence of anti-HBs antibodies.

The in vivo evolution of genotype F HBV variants was recorded in a chronically infected patient throughout a 3-year observation period. Fluctuating levels of HBs Ag and anti-HBs antibodies were recorded, both of them cocirculating in peripheral blood samples at given times. Fifty S gene derived clones were sequenced and phylogenetically analyzed. As expected, some amino acid replacements within the S ORF were also observed within the P ORF while others were silent for the former. Such change was statistically significant for both S and P overlapping genes, which clearly indicates the appearance of a positive selection pressure. Supporting this notion, amino acid replacements were documented at both B and T cell epitopes in samples from 1997 and 1998. Several mutations were documented within and outside the "a" determinant in the major hydrophilic region. Such substitutions might have resulted from the attempt of HBV to evade both humoral and/or cellular immune response. To the best of our knowledge this unusual profile of HBV variants in presence of usually "neutralizing" anti-HBs antibodies was examined in vivo for the first time.

Amino Acid Sequence↗
PubMed2005

Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes.

We have conducted a comprehensive search for conserved elements in vertebrate genomes, using genome-wide multiple alignments of five vertebrate species (human, mouse, rat, chicken, and Fugu rubripes). Parallel searches have been performed with multiple alignments of four insect species (three species of Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven species of Saccharomyces. Conserved elements were identified with a computer program called phastCons, which is based on a two-state phylogenetic hidden Markov model (phylo-HMM). PhastCons works by fitting a phylo-HMM to the data by maximum likelihood, subject to constraints designed to calibrate the model across species groups, and then predicting conserved elements based on this model. The predicted elements cover roughly 3%-8% of the human genome (depending on the details of the calibration procedure) and substantially higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes. From yeasts to vertebrates, in order of increasing genome size and general biological complexity, increasing fractions of conserved bases are found to lie outside of the exons of known protein-coding genes. In all groups, the most highly conserved elements (HCEs), by log-odds score, are hundreds or thousands of bases long. These elements share certain properties with ultraconserved elements, but they tend to be longer and less perfectly conserved, and they overlap genes of somewhat different functional categories. In vertebrates, HCEs are associated with the 3' UTRs of regulatory genes, stable gene deserts, and megabase-sized regions rich in moderately conserved noncoding sequences. Noncoding HCEs also show strong statistical evidence of an enrichment for RNA secondary structure.

3' Untranslated Regions↗
PubMed2006

Reconstructing the evolutionary history of paralogous APETALA1/FRUITFULL-like genes in grasses (Poaceae).

Gene duplication is an important mechanism for the generation of evolutionary novelty. Paralogous genes that are not silenced may evolve new functions (neofunctionalization) that will alter the developmental outcome of preexisting genetic pathways, partition ancestral functions (subfunctionalization) into divergent developmental modules, or function redundantly. Functional divergence can occur by changes in the spatio-temporal patterns of gene expression and/or by changes in the activities of their protein products. We reconstructed the evolutionary history of two paralogous monocot MADS-box transcription factors, FUL1 and FUL2, and determined the evolution of sequence and gene expression in grass AP1/FUL-like genes. Monocot AP1/FUL-like genes duplicated at the base of Poaceae and codon substitutions occurred under relaxed selection mostly along the branch leading to FUL2. Following the duplication, FUL1 was apparently lost from early diverging taxa, a pattern consistent with major changes in grass floral morphology. Overlapping gene expression patterns in leaves and spikelets indicate that FUL1 and FUL2 probably share some redundant functions, but that FUL2 may have become temporally restricted under partial subfunctionalization to particular stages of floret development. These data have allowed us to reconstruct the history of AP1/FUL-like genes in Poaceae and to hypothesize a role for this gene duplication in the evolution of the grass spikelet.

Amino Acid Sequence↗
PubMed2001

Expression of zebrafish btg-b, an anti-proliferative cofactor, during early embryogenesis.

BTG/tob family proteins are thought to be a potential tumor suppressor due to their anti-proliferative activity. We cloned zebrafish btg-b, a member of the BTG1/2 subfamily, using in situ hybridization screening. The tissue-specific expression of btg-b is first observed in the organizer region at the early gastrula stage. Later in development, the forebrain, the hindbrain, the polster and the paraxial mesoderm transiently express btg-b. Recently, mouse Btg1 and Btg2 have been shown to be a cofactor for Hoxb9. Double in situ hybridization with zebrafish btg-b and hoxb9a indicates that the expression domains of these two genes overlap in the posterior paraxial mesoderm.

Amino Acid Sequence↗
PubMed1998

Identification of proteins and developmental expression of RNAs encoded by the 65A cuticle protein gene cluster in Drosophila melanogaster.

Proteins of the third instar larval cuticle of Drosophila melanogaster, LCP5-LCP9, were purified and their N-terminal sequences determined. Three of these proteins (LCP5, 6, and 8) were found to be encoded by two multicopy genes previously mapped to the gene cluster at 65A 5-6 on the left arm of the third chromosome. The analysis of the patterns of developmental expression of the 8 distinct genes at this site showed that all but two were expressed during larval life. The patterns fell into three groups: one where expression was all through larval life, one where expression was primarily in the third instar, and one only during the production of the adult cuticle. One duplicated gene was not expressed in the Canton S strain at any time from the embryo to adult ecdysis. These findings indicate that there is not a unique set of cuticle proteins in the third versus the first and second instar larval cuticles and indicates that overlapping gene sets in several different gene clusters encode the proteins of the cuticle of different developmental stages.

Amino Acid Sequence↗
PubMed2002

Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects.

Recent evidence indicates that transcriptional abnormalities may play an important role in the pathophysiology of polyglutamine diseases. In the present study, we have explored the extent to which polyglutamine-related changes in gene expression may be independent of protein context by comparing mouse models of dentatorubral-pallidoluysian atrophy (DRPLA) and Huntington's disease (HD). Microarray gene expression profiling was conducted in mice of the same background strain in which the same promoter was employed to direct the expression of full-length atrophin-1 or partial huntingtin transproteins (At-65Q or N171-82Q mice). A large number of overlapping gene expression changes were observed in the cerebella of At-65Q and N171-82Q mice. Six of the gene expression changes common to both huntingtin and atrophin-1 transgenic mice were also observed in the cerebella of mouse models expressing full-length mutant ataxin-7 or the androgen receptor. These results demonstrate that some of the gene expression effects of expanded polyglutamine proteins occur independently of protein context.

Animals↗
PubMed2003

Competitive promoter occupancy by two yeast paralogous transcription factors controlling the multidrug resistance phenomenon.

Highly flexible gene expression programs are required to allow cell growth in the presence of a wide variety of chemicals. We used genome-wide expression analyses coupled with chromatin immunoprecipitation experiments to study the regulatory relationships between two very similar yeast transcription factors involved in the control of the multidrug resistance phenomenon. Yrm1 (Yor172w) is a new zinc finger transcription factor, the overproduction of which decreases the level of transcription of the target genes of Yrr1, a zinc finger transcription factor controlling the expression of several membrane transporter-encoding genes. Surprisingly, the absence of YRR1 releases the transcriptional activity of Yrm1, which then up-regulates 23 genes, 14 of which are also direct target genes of Yrr1. Chromatin immunoprecipitation experiments confirmed that Yrm1 binds to the promoters of the up-regulated genes only in yeast strains from which YRR1 has been deleted. This sophisticated regulatory program can be associated with drug resistance phenotypes of the cell. The program-specific distribution of paired transcription factors throughout the genome may be a general mechanism by which similar transcription factors regulate overlapping gene expression programs in response to chemical stress.

Alleles↗
PubMed2005

Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2.

The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD(2)C(2). To assess the extent to which FlhD(2)C(2) directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42-44 bp inverted repeat FlhD(2)C(2) binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD(2)C(2) binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD(2)C(2) over the K(D) range 38-356 nM, and of the nine corresponding chromosomal promoter-lacZ fusions, those of the four genes b1904, b2446, wzz(fepE) and gltI showed up to 50-fold dependence on FlhD(2)C(2) in vivo. In comparison, four representative flagella class II promoters bound FlhD(2)C(2) in the K(D) range 12-43 nM and were upregulated in vivo 30- to 990-fold. The FlhD(2)C(2)-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted -35 motif of the FlhD(2)C(2)-activated sigma(70) promoters, as is the case with FlhD(2)C(2)-dependent class II flagellar promoters. The data indicate a wider FlhD(2)C(2) regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.

Artificial Gene Fusion↗
PubMed2006

Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages.

Although successful attempts to inhibit HIV-1 replication in T cells using RNAi have been reported, the effect of HIV-specific RNAi on macrophages is not well known. Macrophages are key targets for anti-HIV-1 therapy because they are able to survive long after the initial infection with HIV and can spread the virus to T cells. In this study, we identified a putative RNAi target of HIV, consisting of the portion of the nef gene overlapping the U3 region (Nef366), and generated a lentivirus-based short hairpin RNA (shRNA) expression vector (Lenti shNef366). We show that Lenti shNef366 inhibits (1) HIV-1 replication in a monocytic cell line and in primary monocyte-derived macrophages (MDMs), (2) reactivation of latent HIV-1 infection, and (3) the production of secondary HIV-1 from MDMs harboring a genomic copy of Nef366. Moreover, we found that the up-regulated production of macrophage inflammatory protein 1beta (MIP-1beta), but not MIP-1alpha, in MDMs by Nef expression was considerably suppressed by Lenti shNef366, which suggests that HIV-1 dissemination to T cells through its interaction with HIV-1-infected MDMs can also be controlled by Lenti shNef366. Thus, lentivirus-mediated shRNA expression targeting the U3-overlapping region of HIV nef represents a feasible approach to genetic vaccine therapy for HIV-1.

Cell Line↗