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PubMed1992

The nucleotide sequence of RNA-1 of raspberry bushy dwarf virus.

Raspberry bushy dwarf virus (RBDV) has isometric, 33 nm diameter particles and a bipartite RNA genome. Sequencing of the larger component (RNA-1) showed that it consists of 5449 nucleotides and contains one large open reading frame encoding a putative translation product with a calculated M(r) of 190,000. Comparisons of this polypeptide with non-structural proteins of other plant viruses revealed significant homologies with those of alfalfa mosaic virus (AlMV), brome mosaic virus (BMV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Thus RBDV belongs to the supergroup of 'Sindbis-like' plant viruses. The translation product of RBDV RNA-1 contains motifs characteristic of proteins with polymerase, methyltransferase and helicase activities, suggesting that this protein is involved in the replication of the viral RNA. Thus in RBDV, as in TMV, all three functional domains are combined in the single protein, whereas in AlMV, BMV and CMV these domains are distributed over the proteins encoded by RNA-1 and RNA-2. These findings support the idea that RBDV should be placed in a distinct virus genus for which the name idaeovirus has been proposed.

Amino Acid Sequence↗
PubMed2025

Developing and Benchmarking One Health Genomic Surveillance Tools for Influenza A Virus in Wastewater.

Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool. However, IAV is at low abundance in wastewater, making it difficult to sequence. We benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods. Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes. However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes, but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches, potentially enabling broader surveillance and detection of emerging strains. However, probes were costly, labor-intensive, and less sensitive than tiled-amplicon. When testing compatibility of sequencing methods with upstream virus concentration and extraction methods, ultrafiltration-based virus concentration outperformed large-volume direct extraction with all four sequencing methods. This set of benchmarking comparisons and custom panels provides needed information for the translation of IAV genomic sequencing into a routine component of wastewater surveillance.

Journal Article↗
PubMed2006

Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis.

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Regulation of Plastid Gene Expression during Photooxidative Stress.

We have used the carotenoid biosynthesis inhibitor norflurazon to study the relationship between chloroplast and nuclear gene expression and the mechanisms by which plastid mRNA accumulation is regulated in response to photooxidative stress. By treating 4-week-old hydroponic spinach plants (Spinacea oleracea), we were able to determine the response at two distinct stages of chloroplast development. For all parameters studied, differences were found between the norflurazon-treated young and mature leaves. Young leaves lost essentially all pigment content in the presence of norflurazon, whereas mature leaves retained more than 60% of their chlorophyll and carotenoids. The accumulation of plastid mRNA was determined for several genes, and we found a decrease in mRNA levels for all genes except psbA in herbicide-treated young leaves. For genes such as atpB, psbB, and psaA, there was a corresponding change in the relative level of transcription, but for psbA and rbcL, transcription and mRNA accumulation were uncoupled. In norflurazon-treated mature leaves, all plastid mRNAs except psaA accumulated to normal levels, and transcription levels were either normal or higher than corresponding controls. This led to the conclusion that plastid mRNA accumulation is regulated both transcriptionally and posttranscriptionally in response to photooxidative stress. Although direct photooxidative damage is confined to the plastid and peroxisome, there is a feedback of information controlling the transcription of nuclear-encoded plastid proteins. Considerable evidence has accumulated implicating a "plastid factor" in this control. Therefore, the expression of several nuclear-encoded plastid proteins and the corresponding mRNAs were determined. Although the levels of both the small subunit of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b-binding protein and corresponding mRNAs were reduced, a 28-kilodalton chloroplast RNA-binding protein and corresponding mRNA were at normal levels in norflurazon-treated plants. Changes in mRNA and protein levels were not the result of a general loss due to photooxidation but rather the result of selective stabilization of certain components. The response of both genomes to photooxidative stress is discussed in terms of the postulated plastid factor.

Journal Article↗
PubMed2006

Novel pathways for glycoprotein import into chloroplasts.

Although the chloroplast contains its own genome, majority of its protein components are encoded by nuclear genes and must be imported post-translationally. In general, proteins synthesized by cytosolic ribosomes are post-translationally targeted to the chloroplast through interactions between their N-terminal transit sequence and protein translocon Toc/Tic complexes in the chloroplast membranes. An alternative pathway that mediates post-translational delivery of proteins to the chloroplast via the secretory pathway was recently described. This pathway provides new opportunities for complementation of the chloroplast protein maturation machinery with chaperones needing endoplasmic reticulum and/or Golgi typical maturations such as N-glycosylation for their biological activity or using chloroplasts as a storage compartment for glycoproteins.

Arabidopsis↗
PubMed2004

Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation.

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr- and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.

Acetate Kinase↗
PubMed1973

Characterization of nucleic acid of pichinde virus.

The nucleic acid of Pichinde virus was found to be single-stranded ribonucleic acid (RNA) as determined by sensitivity to ribonuclease, by alkaline degradation, by buoyant density in cesium sulfate, and by analysis of the base composition. The RNA of the virion could be separated into five components which had sedimentation coefficients corresponding to 31S, 28S, 22S, 18S and 4 to 6S. The 28S, 18S, and possibly the 4 to 6S RNAs appear to be derived from host cell components incorporated into the virion, whereas the 31S and 22S components appear to represent the genome of the virus.

Animals↗
PubMed1988

A multigene family encodes the human cytomegalovirus glycoprotein complex gcII (gp47-52 complex).

The HXLF (HindIII-X left reading frame) gene family is a group of five genes that share one or two regions of homology and are arranged in tandem within the short unique component of the human cytomegalovirus genome (K. Weston and B.G. Barrell, J. Mol. Biol. 192:177-208, 1986). These genes were cloned into an SP6 expression vector in both the sense and antisense orientations. An abundant 1.62-kilobase (kb) bicistronic mRNA, predicted to originate from HXLF1 and HXLF2, was detected in the cytoplasm of infected human fibroblast cells by Northern (RNA) blot analysis. Less abundant RNAs of 1.0 and 0.8 kb, predicted to originate from the HXLF5 and HXLF2 genes, respectively, were also detected. Monocistronic, bicistronic, and polycistronic RNAs synthesized in vitro by using SP6 polymerase were translated in rabbit reticulocyte lysates with or without canine pancreatic microsomal membranes. The HXLF1 or the HXLF1 and HXLF2 translation products were detected when the above mRNAs were used. The HXLF3, HXLF4, and HXLF5 gene products were not detected by in vitro translation of the SP6-derived polycistronic mRNA. Nonglycosylated or glycosylated HXLF1 and HXLF2 gene products were immunoprecipitated by monoclonal antibody 9E10, which is specific for a virion envelope glycoprotein complex designated gcII (gp47-52 complex). In addition, the monoclonal antibody 9E10 immunoprecipitated a diffuse glycoprotein band, designated gp47-52, from HCMV-infected cell lysates. The amino acid composition of gp47-52 purified from viron envelopes has the highest similarity to the predicted amino acid composition of the HXLF1 plus HXLF2 open reading frames, but it is more similar to HXLF2 than to HXLF1. The Northern blot results imply that gp47-52 is synthesized predominantly from the abundant 1.62-kb bicistronic mRNA encoded by the HXLF1 and HXLF2 genes. However, the glycoprotein could also be synthesized by the monocistronic 0.8-kb mRNA encoded by the HXLF2 gene as well as by the mRNAs predicted from the other HXLF genes.

Antibodies, Monoclonal↗
PubMed1990

Recombinogenic properties of herpes simplex virus type 1 DNA sequences resident in simian virus 40 minichromosomes.

In a previous work, it was demonstrated that the bacterial transposon Tn5 is capable of undergoing sequence inversion via recombination between its duplicated IS50 elements when replicated by the herpes simplex virus type 1 (HSV-1) origin oris but not by the simian virus 40 (SV40) origin orisv. Further analysis of the latter phenomenon indicated that this lack of recombination was the result of topological constraints imposed by the SV40 minichromosome, such that recombination events could be readily detected in Tn5 derivatives in which the IS50 elements were arranged in a direct rather than inverted orientation. With this information, a second set of experiments were carried out to examine how the highly recombinogenic sequences which mediate the inversion of the long (L) and short (S) components of the HSV-1 genome behave in an SV40 minichromosome. Tandem copies of the L-S junction of the HSV-1 genome were observed to promote deletions in an SV40 shuttle plasmid at a frequency that was considerably greater than that of duplicated bacterial plasmid vector DNA. However, the presence of superinfecting HSV-1 did not enhance the frequency of these recombination events. These results support our previous findings that HSV-1 genome isomerization is mediated by a homologous recombination mechanism which is intimately associated with the act of viral DNA synthesis. Moreover, they demonstrate that the sequences which comprise the L-S junction appear to be inherently recombinogenic and, therefore, do not contain specific signals required for HSV-1 genome isomerization.

Animals↗
PubMed2006

Essential domains for ribonucleoprotein complex formation required for retrotransposition of telomere-specific non-long terminal repeat retrotransposon SART1.

Non-long terminal repeat (LTR) retrotransposons are major components of the higher eukaryotic genome. Most of them have two open reading frames (ORFs): ORF2 encodes mainly the endonuclease and reverse transcriptase domains, but the functional features of ORF1 remain largely unknown. We used telomere-specific non-LTR retrotransposon SART1 in Bombyx mori and clarified essential roles of the ORF1 protein (ORF1p) in ribonucleoprotein (RNP) formation by novel approaches: in vitro reconstitution and in vivo/in vitro retrotransposition assays using the baculovirus expression system. Detailed mutation analyses showed that each of the three CCHC motifs at the ORF1 C terminus are essential for SART1 retrotransposition and are involved in packaging the SART1 mRNA specifically into RNP. We also demonstrated that amino acid residues 555 to 567 and 285 to 567 in the SART1 ORF1p are crucial for the ORF1p-ORF1p and ORF1p-ORF2p interactions, respectively. The loss of these domains abolishes protein-protein interaction, leading to SART1 retrotransposition deficiency. These data suggest that systematic formation of RNP composed of ORF1p, ORF2p, and mRNA is mainly mediated by ORF1p domains and is a common, essential step for many non-LTR retrotransposons encoding the two ORFs.

Amino Acid Motifs↗
PubMed1997

Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae.

The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.

Base Sequence↗
PubMed2002

Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation.

Hibernators in torpor dramatically reduce their metabolic, respiratory, and heart rates and core body temperature. These extreme physiological conditions are frequently and rapidly reversed during the winter hibernation season via endogenous mechanisms. This phenotype must derive from regulated expression of the hibernator's genome; to identify its molecular components, a cDNA subtraction was used to enrich for seasonally upregulated mRNAs in liver of golden-mantled ground squirrels. The relative steady-state levels for seven mRNAs identified by this screen, plus five others, were measured and analyzed for seasonal and stage-specific differences using kinetic RT-PCR. Four mRNAs show seasonal upregulation in which all five winter stages differ significantly from and are higher than summer (alpha2-macroglobulin, apolipoprotein A1, cathepsin H, and thyroxine-binding globulin). One of these mRNAs, alpha2-macroglobulin, varies during the winter stages with significantly lower levels at late torpor. None of the 12 mRNAs increased during torpor. The implications for these newly recognized upregulated mRNAs for hibernation as well as more global issues of maintaining steady-state levels of mRNA during torpor are discussed.

Animals↗
PubMed2005

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive".

Centromere↗
PubMedDate not supplied

Role of complex carbohydrates and neurulation.

Extracellular and cell-coat complex carbohydrates play a major role in critical cellular functions, e.g., cell recognition and cell-to-cell adherence, which are involved in neurulation. To date, available evidence suggests that neurulation is a combination of intercellular and extracellular biochemical and morphological events which are under the direction of the genome. To define the carbohydrate components that are essential to neurulation, normal C-57 and abnormal splotch (Sp/Sp) mouse embryos were studied using fluorescein-labeled lectins and computer-aided microspectrophotometric analysis of hyaluronate lyase and chondroitin ABC lyase-sensitive Alcian blue staining. Preliminary results of these studies indicate that the neuroepithelium of the splotch Sp/Sp mutant, a genetic model of a primary neural tube defect, is characterized by alterations in the type and amount of glycoconjugates and in the concentration of individual glycosaminoglycans. This review of mammalian neurulation discusses the importance of complex carbohydrates on the cell surface of the neuroepithelium of the closing neural fold.

Animals↗
PubMed2005

Emerging concepts in nutrigenomics: a preview of what is to come.

This article provides an overview of the fundamental principles of genetics and emerging concepts related to the ways in which nutrients and bioactive food components may interact with the genome and subsequently affect human health. This exciting area of research is likely to have far-reaching implications for the assessment and treatment of critically and chronically ill individuals that will affect nutrition standards of care and practice. A brief overview of some of the ethical, legal, and social implications of genomic research and genome-based health care and a list of genetics resources also are provided.

Biotechnology↗
PubMed2005

A novel mass spectrometry-based assay for GSK-3beta activity.

BACKGROUND: As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of gamma-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3beta) activity. RESULTS: Synthetic peptide substrates designed with a GSK-3beta phosphorylation site were assayed with both recombinant enzyme and GSK-3beta immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da.) was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) in a GSK-3beta target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3beta used in this assay, this shift was also inhibited by lithium chloride (LiCl), in a dose-dependent manner. CONCLUSION: We present here a novel method to sensitively measure peptide phosphorylation by GSK-3beta that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3beta in vivo, and to screen enzyme activity in relation to a variety of GSK-3beta related disorders.

Glycogen Synthase Kinase 3↗
PubMed2006

Phylophenetic properties of metabolic pathway topologies as revealed by global analysis.

BACKGROUND: As phenotypic features derived from heritable characters, the topologies of metabolic pathways contain both phylogenetic and phenetic components. In the post-genomic era, it is possible to measure the "phylophenetic" contents of different pathways topologies from a global perspective. RESULTS: We reconstructed phylophenetic trees for all available metabolic pathways based on topological similarities, and compared them to the corresponding 16S rRNA-based trees. Similarity values for each pair of trees ranged from 0.044 to 0.297. Using the quartet method, single pathways trees were merged into a comprehensive tree containing information from a large part of the entire metabolic networks. This tree showed considerably higher similarity (0.386) to the corresponding 16S rRNA-based tree than any tree based on a single pathway, but was, on the other hand, sufficiently distinct to preserve unique phylogenetic information not reflected by the 16S rRNA tree. CONCLUSION: We observed that the topology of different metabolic pathways provided different phylogenetic and phenetic information, depicting the compromise between phylogenetic information and varying evolutionary pressures forming metabolic pathway topologies in different organisms. The phylogenetic information content of the comprehensive tree is substantially higher than that of any tree based on a single pathway, which also gave clues to constraints working on the topology of the global metabolic networks, information that is only partly reflected by the topologies of individual metabolic pathways.

Chromosome Mapping↗
PubMed2005

Genotype-by-sex interaction in the regulation of high-density lipoprotein: the Framingham Heart Study.

Low levels of high-density lipoprotein (HDL) are widely documented as a risk factor for cardiovascular disease (CVD). Furthermore, there is marked sexual dimorphism in both HDL levels and the prevalence of CVD. However, the extent to which genetic factors contribute to such dimorphism has been largely unexplored. We examined the evidence for genotype-by-sex effects on HDL in a longitudinal sample of 1562 participants from 330 families in the Framingham Heart Study at three times points corresponding approximately to 1971-1974, 1980-1983, and 1988-1991. Using a variance component method, we conducted a genome scan of HDL at each time point in males and females, separately and combined, and tested for genotype-by-sex interaction at a quantitative trait locus (QTL) at each time point. Consistent findings were noted only for females on chromosome 2 near marker D2S1328, with adjusted LOD scores of 2.6, 2.2, and 2.1 across the three time points, respectively. In males suggestive linkage was detected on chromosome 16 near marker D16S3396 at the second time point and on chromosome 18 near marker D18S851 at the third time point (adjusted LOD = 2.2 and 2.4, respectively). Although the heritability of HDL is similar in males and females, sex appears to exert a substantial effect on the QTL-specific variance of HDL. When genotype-by-sex interactions exist and are not modeled, the power to detect linkage is reduced; thus our results may explain in part the paucity of significant linkage findings for HDL.

Adult↗