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PubMed2001

The nuclear receptor Ftz-F1 and homeodomain protein Ftz interact through evolutionarily conserved protein domains.

The Drosophila homeodomain protein Fushi Tarazu (Ftz) and its partner, the orphan receptor Ftz-F1, are members of two distinct families of DNA binding transcriptional regulators. Ftz and Ftz-F1 form a novel partnership in vivo as a Hox/orphan receptor heterodimer. Here we show that the murine Ftz-F1 ortholog SF-1 functionally substitutes for Ftz-F1 in vivo, rescuing the defects of ftz-f1 mutants. This finding identified evolutionarily conserved domains of Ftz-F1 as critical for activity of this receptor in vivo. These domains function, at least in part, by mediating direct protein interactions with Ftz. The Ftz-F1 DNA binding domain interacts strongly with Ftz and dramatically facilitates the binding of Ftz to target DNA. This interaction is augmented by a second interaction between the AF-2 domain of Ftz-F1 and the N-terminus of Ftz via an LRALL sequence in Ftz that is reminiscent of LXXLL motifs in nuclear receptor coactivators. We propose that Ftz-F1 serves as a cofactor for Ftz by facilitating the selection of target sites in the genome that contain Ftz/Ftz-F1 composite binding sites. Ftz, on the other hand, influences Ftz-F1 activity by interacting with its AF-2 domain in a manner that mimics a nuclear receptor coactivator.

Amino Acid Motifs↗
PubMed1988

High-resolution analysis of a histone H1 binding site in a rat albumin gene.

Interaction of rat liver histone H1 fraction with the 5'-end of the rat serum albumin gene was localized within a 346 base pair (bp) restriction fragment. Sequence analysis of the fragment showed the fragment was 72 mol % adenosine-thymidine, which is significantly greater than the mole percent adenosine-thymidine composition of the rat genome. Gel retardation assays of the histone H1-DNA interaction indicate the complex formed behaves as previously characterized H1-DNA and shows a high-affinity H1 binding site within the enriched albumin restriction site. Deoxyribonuclease I (DNase I) protection assays on the H1 binding site define three protected regions only on the template strand of the DNA fragment. The three sites lie 55 and 110 bp apart (165 bp between the first and third binding site) with a consensus binding sequence of 5'-GA-ATA-CTGGCTT-C-TT-CTA-G-3'. The sequences between the protected DNA regions are highly enriched in adenosine-thymidine bases (79.3 and 86 mol % adenosine-thymidine, respectively). The functional significance is not understood.

Albumins↗
PubMed1996

Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter.

Oxidative stress (tert-butylhydroquinone) rapidly induced metallothionein-I gene expression in mouse Hepa cells, and this effect was mediated predominantly through metal response promoter elements in transient transfection assays. In vivo genomic footprinting of the mouse metallothionein-I promoter after treatment of Hepa cells with hydrogen peroxide, tert-butylhydroquinone, or zinc suggested a rapid increase in occupancy of the metal response elements. More subtle changes also occurred in the constitutive genomic footprint at the composite major late transcription factor/antioxidant response element. This element may, in part, mediate induction by hydrogen peroxide. Electrophoretic mobility shift assays demonstrated a rapid (30 min) increase in the DNA binding activity of metal-responsive transcription factor-1 in Hepa cells treated with any of these inducers. In control cells, upstream stimulatory factor binding with the major late transcription factor site, and a nuclear protein complex distinct from AP-1, but specific for the antioxidant response element, were detected. The amounts of these complexes were not altered after these treatments. These studies indicate that metal-responsive transcription factor-1 plays a role in activating mouse metallothionein-I gene transcription in response to reactive oxygen species.

Animals↗
PubMed2006

Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences.

A phylogenetic analysis of more than 350 multicopper oxidases (MCOs) from fungi, insects, plants, and bacteria provided the basis for a refined classification of this enzyme family into laccases sensu stricto (basidiomycetous and ascomycetous), insect laccases, fungal pigment MCOs, fungal ferroxidases, ascorbate oxidases, plant laccase-like MCOs, and bilirubin oxidases. Within the largest group of enzymes, formed by the 125 basidiomycetous laccases, the gene phylogeny does not strictly follow the species phylogeny. The enzymes seem to group at least partially according to the lifestyle of the corresponding species. Analyses of the completely sequenced fungal genomes showed that the composition of MCOs in the different species can be very variable. Some species seem to encode only ferroxidases, whereas others have proteins which are distributed over up to four different functional clusters in the phylogenetic tree.

Basidiomycota↗
PubMed1999

Common and specific characteristics of the high-pathogenicity island of Yersinia enterocolitica.

Yersinia pestis, Y. pseudotuberculosis O:1, and Y. enterocolitica biogroup 1B strains carry a high-pathogenicity island (HPI), which mediates biosynthesis and uptake of the siderophore yersiniabactin and a mouse-lethal phenotype. The HPI of Y. pestis and Y. pseudotuberculosis (Yps HPI) are highly conserved in sequence and organization, while the HPI of Y. enterocolitica (Yen HPI) differs significantly. The 43,393-bp Yen HPI sequence of Y. enterocolitica WA-C, serotype O:8, was completed and compared to that of the Yps HPI of Y. pseudotuberculosis PB1, serotype O:1A. A common GC-rich region (G+C content, 57.5 mol%) of 30.5 kb is conserved between yersinia strains. This region carries genes for yersiniabactin biosynthesis, regulation, and uptake and thus can be considered the functional "core" of the HPI. In contrast, the second part of the HPI is AT rich and completely different in two evolutionary lineages of the HPI, being 12.8 kb in the Yen HPI and 5.6 kb in the Yps HPI. The variable part acquired one IS100 element in the Yps HPI and accumulated four insertion elements, IS1328, IS1329, IS1400, and IS1222, in the Yen HPI. The insertion of a 125-bp ERIC sequence modifies the structure of the promoter of the ybtA yersiniabactin regulator in the Yen HPI. In contrast to the precise excision of the Yps HPI in Y. pseudotuberculosis, the Yen HPI suffers imprecise deletions. The Yen HPI is stably integrated in one of the three asn tRNA copies in Y. enterocolitica biogroup 1B (serotypes O:8, O:13, O:20, and O:21), probably due to inactivation of the putative integrase. The 17-bp duplications of the 3' end of the asnT RNA are present in both Yersinia spp. The HPI attachment site is unoccupied in nonpathogenic Y. enterocolitica NF-O, biogroup 1A, serotype O:5. The HPI of Yersinia is a composite and widely spread genomic element with a highly conserved yersiniabactin functional "core" and a divergently evolved variable part.

Base Sequence↗
PubMed2025

Diagnostic performance of the Sanity 2.0 assay to detect resistance to rifampicin, isoniazid, and fluoroquinolones in tuberculosis.

UNLABELLED: Effective tuberculosis (TB) management relies on prompt diagnosis of Mycobacterium tuberculosis complex (MTBC) and associated drug resistance. The Sanity 2.0 assay is a high-resolution melting assay designed for direct respiratory sample testing, enabling simultaneous detection of MTBC and resistance to rifampicin (RIF), isoniazid (INH), and fluoroquinolones (FQ) in a single step. This study evaluated its diagnostic performance in two registered multicenter trials among bacteriologically confirmed TB patients. Diagnostic performance was evaluated for MTBC detection, as well as for the identification of resistance to RIF, INH, and FQ, using phenotypic drug susceptibility testing, whole-genome sequencing, and a composite reference standard. Agreement analyses were conducted between the Sanity 2.0 assay and Xpert MTB/RIF and Xpert MTB/XDR. Among 611 patients, the Sanity 2.0 assay detected MTBC in 563 patients, exhibiting a sensitivity of 92.1% (95% CI: 89.7-94.0). For detecting resistance to RIF, INH, and FQ, sensitivities exceeded 90%, with specificities of 95.8% (95% CI: 88.5-98.6), 100.0% (95% CI: 96.4-100.0), and 97.8% (95% CI: 93.8-99.3) against the composite reference standard, respectively. The agreement with Xpert MTB/RIF for RIF detection was 98.6% (95% CI: 96.9-99.3). For INH and FQ resistance, the agreement with Xpert MTB/XDR was 92.0% (95% CI: 88.5-94.5) and 94.3% (95% CI: 91.2-96.3), respectively. The Sanity 2.0 assay is a rapid and user-friendly platform capable of detecting both MTBC and key drug resistance. It demonstrated good diagnostic performance and could potentially be an effective alternative to guide individualized anti-TB treatment, especially in resource-limited settings. IMPORTANCE: Rapid and accurate detection of both Mycobacterium tuberculosis complex (MTBC) and key drug resistance is critical to improving tuberculosis treatment outcomes and reducing transmission. However, current molecular diagnostic workflows often require sequential testing, which can delay the initiation of effective and individualized therapy. We evaluated the Sanity 2.0 assay, an integrated high-resolution melting test that simultaneously detects MTBC and resistance to rifampicin, isoniazid, and fluoroquinolone resistance directly from respiratory samples in about 2-3 hours. The assay demonstrated excellent performance, with MTBC detection sensitivity of 92.1% and drug resistance sensitivities exceeding 90% and specificities over 95% against a composite reference standard, as well as strong concordance with World Health Organization-endorsed molecular assays. Implementation of the Sanity 2.0 assay could streamline TB diagnostic workflows; enable rapid, single-step resistance profiling; and facilitate timely, individualized treatment-particularly in resource-limited settings where rapid and comprehensive resistance testing remains a critical unmet need.

Humans↗
PubMed1981

Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays.

Animals↗
PubMed2006

Both spike and background genes contribute to murine coronavirus neurovirulence.

Various strains of mouse hepatitis virus (MHV) exhibit different pathogenic phenotypes. Infection with the A59 strain of MHV induces both encephalitis and hepatitis, while the highly neurovirulent JHM strain induces a fatal encephalitis with little, if any, hepatitis. The pathogenic phenotype for each strain is determined by the genetic composition of the viral genome, as well as the host immune response. Using isogenic recombinant viruses with A59 background genes differing only in the spike gene, we have previously shown that high neurovirulence is associated with the JHM spike protein, the protein responsible for attachment to the host cell receptor (J. J. Phillips, M. M. Chua, G. F. Rall, and S. R. Weiss, Virology 301:109-120, 2002). Using another set of isogenic recombinant viruses with JHM background genes expressing either the JHM or A59 spike, we have further investigated the roles of viral genes in pathogenesis. Here, we demonstrate that the high neurovirulence of JHM is associated with accelerated spread through the brain and a heightened innate immune response that is characterized by high numbers of infiltrating neutrophils and macrophages, suggesting an immunopathogenic component to neurovirulence. While expression of the JHM spike is sufficient to confer a neurovirulent phenotype, as well as increased macrophage infiltration, background genes contribute to virulence as well, at least in part, by dictating the extent of the T-cell immune response.

Animals↗
PubMed2002

Variables affecting in vivo performance of high-capacity adenovirus vectors.

In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.

Adenoviruses, Human↗
PubMed2000

Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications.

Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.

Amino Acid Sequence↗
PubMed2004

Quantitative trait loci associated with adventitious shoot formation in tissue culture and the program of shoot development in Arabidopsis.

Arabidopsis ecotypes, Columbia (Col) and Landsberg erecta (Ler), differ in their capacity to regenerate shoots in culture, as do many other cultivars and varieties of the same plant species. Recombinant inbred (RI) lines derived from a cross of Col x Ler were scored for shoot regeneration, and the Arabidopsis genome was scanned using composite interval mapping for loci associated with shoot regeneration. Three QTL were identified--a major one on chromosome 5 in which the Col parent contributed the superior allele and two minor QTL on chromosomes 1 and 4 in which the Ler parent contributed the superior alleles. The RI lines were binned into genotypic pools to isolate the effects of the major QTL on chromosome 5 while holding the minor QTL constant. To identify genes with expression levels that are associated with the allelic state of the major QTL on chromosome 5, oligonucleotide array expression patterns for genes in the LLC pool (Ler alleles at the minor QTL and a Col allele at the major QTL) were compared to those in the LLL pool (Ler alleles at all QTL). The genes that were significantly differentially expressed between the two pools included several encoding transcription factors and signaling or transposon-related proteins.

Arabidopsis↗
PubMed1996

Associations of individual genomic heterozygosity, estimated by molecular fingerprinting, and of dam major histocompatibility complex with growth and egg production traits in layer chickens.

Growth and egg production data from F2 layer chickens produced from a mating of MHC-heterozygous B15B44 sires to B15B44 and B15B43 dams were analyzed for associations with the level of the individual's genomic heterozygosity and dam MHC. The F1 sires and dams were produced from mating of birds from two genetically distinct, highly inbred lines. Genomic heterozygosity (expressed as composite band frequency, CBF) was estimated based on the average proportion of shared bands of individual birds compared to a composite pool of Hinf I-digested DNA samples from the two inbred lines. Birds from dams of genotype B15B43 had significantly greater BW than progeny from B15B44 dams at 6 wk and thereafter. Dam MHC genotype differences were also significant for number of eggs laid from 20 to 28 wk of age, with B15B43 progeny being superior. Negative regression coefficients at hatch, 32 and 44 wk were observed for the effect of CBF on BW of progeny from B15B43 dams and at hatch and 32 wk from progeny of pooled dam genotypes. Similarly, negative regression coefficients were observed for the association of CBF with number of eggs within progeny from the B15B44 dam MHC genotypes at 20 to 28 wk and total period recorded, and at 28 to 36 wk for progeny from B15B43 dams. The CBF regression coefficients and the differences among progeny based on dam MHC genotypes suggest that individual heterozygosity and MHC genotype may be associated with BW and egg production in chickens.

Animals↗
PubMed1987

Kinetics of DNase I digestion of interphase chromatin in differentiated cell nuclei of the mouse: a flow cytometric study.

The process of DNA digestion with DNase I was monitored in interphase chromatin of differentiated cells by flow cytometry after DNA staining with either the intercalating dye propidium iodide (PI) or the AT specific dye Hoechst 33258 (HO). Nuclei from the liver, kidney and spleen of the mouse were studied after different digestion times (0 to 120 min). During the first 30 min of treatment, a tissue specific digestion pattern was found after PI staining; from 60 min onward, the digestion curves ran parallel, with minor quantitative differences among the cell types. After HO staining, the digestion kinetics appeared to be similar for all the cell types; this is likely due to the peculiar base composition of the mouse genome, where inactive c-heterochromatin is exceptionally AT-rich. No quantitative correlation was found between interphase "heterochromatin" and chromatin DNA which is resistant to DNase I cleavage, while the amount of DNase-I-sensitive DNA does not correspond to the interphase "euchromatic" component. It was confirmed that the flow cytometric approach is a tool for quantifying relative changes in the functional state of chromatin in differentiated cell systems.

Animals↗
PubMed1993

Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase.

In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated. The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R. prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E. coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids. Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation. Both the E. coli and the R. prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription. However, the rickettsial RNA polymerase, in contrast to the E. coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds. These data indicate that the rickettsial RNA polymerase is weak, at least relative to E. coli, in the function required for the opening of DNA duplex. It appears that this operation in R. prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome.

Bacterial Proteins↗
PubMed2006

Nutrigenomics: from molecular nutrition to prevention of disease.

Until recently, nutrition research concentrated on nutrient deficiencies and impairment of health. The advent of genomics-interpreted broadly as a suite of high throughput technologies for the generation, processing, and application of scientific information about the composition and functions of genomes-has created unprecedented opportunities for increasing our understanding of how nutrients modulate gene and protein expression and ultimately influence cellular and organismal metabolism. Nutritional genomics (nutrigenomics), the junction between health, diet, and genomics, can be seen as the combination of molecular nutrition and genomics. The diverse tissue and organ-specific effects of bioactive dietary components include gene-expression patterns (transcriptome); organization of the chromatin (epigenome); protein-expression patterns, including posttranslational modifications (proteome); as well as metabolite profiles (metabolome). Nutrigenomics will promote an increased understanding of how nutrition influences metabolic pathways and homeostatic control, how this regulation is disturbed in the early phases of diet-related disease, and the extent to which individual sensitizing genotypes contribute to such diseases. Eventually, nutrigenomics will lead to evidence-based dietary intervention strategies for restoring health and fitness and for preventing diet-related disease. In this review, we provide a brief overview of nutrigenomics from our point of view by describing current strategies, future opportunities, and challenges.

Chronic Disease↗
PubMed2006

Dispensable genes and foreign DNA in Streptococcus mutans.

A range of properties, including the ability to utilize various sugars, bind macromolecules and produce mutacins, are known to vary in their occurrence in different strains of Streptococcus mutans. In addition, insertion-sequence elements show a limited distribution and sequencing of the genome of S. mutans UA159 has revealed the presence of putative genomic islands of atypical base composition indicative of foreign DNA. PCR primers flanking regions suspected of having inserted DNA were designed on the basis of the genome sequence of S. mutans UA159 and used to explore variation in a collection of 39 strains isolated in various parts of the world over the last 40 years. Extensive differences between strains were detected, and similar insertion/deletion events appear to be present in the genomes of strains with very different origins. In two instances, insertion of foreign DNA appears to have displaced original S. mutans genes. Together with previous results on the occurrence of deletions in genes associated with sugar metabolism, the results indicate that S. mutans has a core genome and a dispensable genome, and that dispensable genes have become widely distributed through horizontal transfer.

Bacterial Proteins↗
PubMed1981

Compositional heterogeneity of the chloroplast DNAs from Euglena gracilis and Spinacia oleracea.

The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using different experimental approaches. Euglena chloroplast DNA has a dG + dC content of 28%. Preparations averaging 20 x 10(6) in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content. Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity. From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC. Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT-rich sequences must occur in short stretches interspersed with dG + dC-rich stretches. Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile. But the higher resolution methods of melting, bouyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition. About 30% of genome is as low as 22% in dG + dC, while 10% is higher than 60% in dG + dC.

Base Composition↗
PubMed1999

Asymmetric substitution patterns: a review of possible underlying mutational or selective mechanisms.

In the absence of bias between the two DNA strands for mutation and selection, the base composition within each strand should be such that A = T and C = G (this state is called Parity Rule type 2, PR2). At a genome scale, i.e. when considering the base composition of a whole genome, PR2 is a good approximation, but there are local and systematic deviations. The question is whether these deviations are a consequence of an underlying bias in mutation or selection. We have tried to review published hypotheses to classify them within the mutational or selective group. This dichotomy is, however, too crude because there is at least one hypothesis based simultaneously upon mutation and selection.

DNA↗