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At least 811 records · Page 45Linked to original sources
PubMed1996

The complete nucleotide sequence and genome organization of the M RNA segment of peanut bud necrosis tospovirus and comparison with other tospoviruses.

The M RNA of peanut bud necrosis virus (PBNV; synonym groundnut bud necrosis virus) is 4801 nucleotides in length. It comprised two ORFs in an ambisense organization and terminal inverted repeats. The 3' large ORF (3363 nucleotides in the virus-complementary strand) encoded a protein with a predicted size of 127.2 kDa which was identified as the glycoprotein precursor (GP) of the G1 and G2 glycoproteins. A comparison of the deduced amino acid sequence of GP revealed 37% identity and 58-59% similarity with that of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), and 21-23% identity and 44-47% similarity with those of other members of the genus Bunyavirus. The 5' small ORF (924 nucleotides in the virussense strand) encoded a 34.2 kDa protein which was identified as the non-structural (NSm) protein based on 41-43% identity and 60-63% similarity with that of TSWV and INSV. Defective RNA molecules derived from the genomic M RNA were detected during continuous passage of the virus by sap inoculations.

Amino Acid Sequence↗
PubMed1997

Nucleotide sequence and taxonomy of maize chlorotic dwarf virus within the family Sequiviridae.

The complete sequence of a Tennessee isolate of maize chlorotic dwarf virus (MCDV-TN) was determined from cDNA clones and by direct sequencing of the viral RNA. The genome is 11813 nucleotides (nt) in length and contains one large open reading frame between nt 435 and 10763 that encodes a polyprotein of 3443 amino acids. The N-terminal amino acid sequences were determined for the three capsid proteins. All three were adjacent, starting at nt 2526 and putatively ending at nt 3761. Comparison of the sequence of MCDV-TN with other viral sequences revealed similarities to several plant picorna-like viruses including members of the Sequiviridae, Comoviridae and Potyviridae. This work also provides evidence based on genome organization that MCDV-TN is a member of the genus Waikavirus within the family Sequiviridae.

Amino Acid Sequence↗
PubMed1992

Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing.

We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.

Aspartylglucosaminuria↗
PubMed2003

Molecular interactions between the specialist herbivore Manduca sexta (lepidoptera, sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates.

Evidence is accumulating that insect-specific plant responses are mediated by constituents in the oral secretions and regurgitants (R) of herbivores, however the relative importance of the different potentially active constituents remains unclear. Fatty acid-amino acid conjugates (FACs) are found in the R of many insect herbivores and have been shown to be necessary and sufficient to elicit a set of herbivore-specific responses when the native tobacco plant Nicotiana attenuata is attacked by the tobacco hornworm, Manduca sexta. Attack by this specialist herbivore results in a large transcriptional reorganization in N. attenuata, and 161 genes have been cloned from previous cDNA differential display-polymerase chain reaction and subtractive hybridization with magnetic beads analysis. cDNAs of these genes, in addition to those of 73 new R-responsive genes identified by cDNA-amplified fragment-length polymorphism display of R-elicited plants, were spotted on polyepoxide coated glass slides to create microarrays highly enriched in Manduca spp.- and R-induced genes. With these microarrays, we compare transcriptional responses in N. attenuata treated with R from the two most damaging lepidopteran herbivores of this plant in nature, M. sexta and Manduca quinquemaculata, which have very similar FAC compositions in their R, and with the two most abundant FACs in Manduca spp. R. More than 68% of the genes up- and down-regulated by M. sexta R were similarly regulated by M. quinquemaculata R. A majority of genes up-regulated (64%) and down-regulated (49%) by M. sexta R were similarly regulated by treatment with the two FACs. In contrast, few genes showed similar transcriptional changes after H(2)O(2)- and R-treatment. These results demonstrate that the two most abundant FACs in Manduca spp. R can account for the majority of Manduca spp.-induced alterations of the wound response of N. attenuata.

Amino Acids↗
PubMed2004

Combined transcript and metabolite analysis reveals genes involved in spider mite induced volatile formation in cucumber plants.

Many plants have an indirect defense against herbivores by emitting volatiles that attract carnivorous enemies of the herbivores. In cucumber (Cucumis sativus) the production of carnivore attractants can be induced by herbivory or jasmonic acid spraying. From the leaves of cucumber plants with and without spider mite infestation, two subtractive cDNA libraries were made that were enriched in cDNA fragments up- or down-regulated by spider mite infestation. A total of 713 randomly selected clones from these libraries were used to make a cDNA microarray. Subsequently, cucumber plants were sprayed with jasmonic acid, mechanically damaged, infested with spider mites, or left untreated (control). Leaf samples were taken at a range of different time points, and induced volatile compounds and mRNA (from the same leaves) were collected. cDNAs prepared from the mRNA were hybridized to the clones on the microarray. The resulting gene expression profiles were analyzed in combination with volatile production data in order to gain insight in the possible involvement of the studied genes in the synthesis of those volatiles. The clones on the microarray and the induced cucumber volatiles could be grouped into a number of clusters in which specific biosynthetic genes clustered with the product of that pathway. For example, lipoxygenase cDNA clones clustered with the volatile (Z)-3-hexenyl acetate and the volatile sesquiterpene (E,E)- alpha-farnesene clustered with an up-regulated sesquiterpene synthase fragment. This fragment was used to screen a cDNA library which resulted in the cloning of the cucumber (E,E)-alpha-farnesene and (E)-beta-caryophyllene synthases. The use of combined global gene expression analysis and metabolite analysis for the discovery of genes involved in specific biosynthetic processes is discussed.

Amino Acid Sequence↗
PubMed2006

Characterization of 43 non-protein-coding mRNA genes in Arabidopsis, including the MIR162a-derived transcripts.

Messenger RNAs that do not contain a long open reading frame (ORF) or non-protein-coding RNAs (npcRNAs) are an emerging novel class of transcripts. Their functions may involve the RNA molecule itself and/or short ORF-encoded peptides. npcRNA genes are difficult to identify using standard gene prediction programs that rely on the presence of relatively long ORFs. Here, we used detailed bioinformatic analyses of expressed sequence tag/cDNA databases to detect a restricted set of npcRNAs in the Arabidopsis (Arabidopsis thaliana) genome and further characterized these transcripts using a combination of bioinformatic and molecular approaches. Compositional analyses revealed strong nucleotide strand asymmetries in the npcRNAs, as well as a biased GC content, suggesting the existence of functional constraints on these RNAs. Thirteen of these transcripts display tissue-specific expression patterns, and three are regulated in conditions affecting root architecture. The npcRNA 78 gene contains the miR162 sequence in an alternative intron and corresponds to the MIR162a locus. Although DICER-LIKE 1 (DCL1) mRNA is known to be regulated by miR162-guided cleavage, its level does not change in a mir162a mutant. Alternative splicing of npcRNA 78 leads to several transcript isoforms, which all accumulate in a dcl1 mutant. This suggests that npcRNA 78 is a genuine substrate of DCL1 and that splicing of this microRNA primary transcript and miR162 processing are competitive nuclear events. Our results provide new insights into Arabidopsis npcRNA biology and the potential roles of these genes.

Alternative Splicing↗
PubMed2006

Highly specific gene silencing by artificial microRNAs in Arabidopsis.

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

Arabidopsis↗
PubMedDate not supplied

Efficient parameterized algorithms for biopolymer structure-sequence alignment.

Computational alignment of a biopolymer sequence (e.g., an RNA or a protein) to a structure is an effective approach to predict and search for the structure of new sequences. To identify the structure of remote homologs, the structure-sequence alignment has to consider not only sequence similarity, but also spatially conserved conformations caused by residue interactions and, consequently, is computationally intractable. It is difficult to cope with the inefficiency without compromising alignment accuracy, especially for structure search in genomes or large databases. This paper introduces a novel method and a parameterized algorithm for structure-sequence alignment. Both the structure and the sequence are represented as graphs, where, in general, the graph for a biopolymer structure has a naturally small tree width. The algorithm constructs an optimal alignment by finding in the sequence graph the maximum valued subgraph isomorphic to the structure graph. It has the computational time complexity O[k(t)N(2)] for the structure of N residues and its tree decomposition of width t. Parameter k, small in nature, is determined by a statistical cutoff for the correspondence between the structure and the sequence. This paper demonstrates a successful application of the algorithm to RNA structure search used for noncoding RNA identification. An application to protein threading is also discussed.

Algorithms↗
PubMed1997

Molecular characterization of the hepatitis G virus.

A novel virus associated with acute and chronic hepatitis has been cloned and characterized. The single-stranded RNA genome is approximately 9000 nucleotides long and has the structure of a virus in the Flaviviridae family. The genes of the virus have been identified and characterized by a number of molecular techniques and a genetic map has been determined.

Flaviviridae↗
PubMed1994

The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli.

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III.

Bacterial Outer Membrane Proteins↗
PubMed2005

Clear cell type of renal cell carcinoma with numerous hyaline globules: a diagnostic pitfall.

A unique and never-before-published example of clear cell renal cell carcinoma (CRCC) found among 9000 primary renal cell tumors is presented. The tumor contained such a dense concentration of glassy hyaline globules (GHG) in the cytoplasm of the neoplastic cells that it overshadowed the morphology of the neoplasm. The resulting appearance of the tumor was quite misleading and different from the conventional CRCC. The GHG were 7-30 microm in diameter. They were glassy and pale to slightly eosinophilic in color in hematoxylin-eosin. The GHG stained positively with periodic acid-Schiff and negatively with silver and Hale's colloidal iron. Immunohistochemically, they were also negative for anti Mycobacterium bovis antigen. At the ultrastructural level, the GHG were formed by an amorphous mass of stellate shape. The GHG were localized within rough endoplasmic reticulum. Molecular genetically, mutation of the distal part of exon 3 of the VHL gene causing elongation of the VHL protein was found in the tumor, thus confirming the diagnosis of CRCC.

Amino Acid Sequence↗
PubMed1999

Prevalence of GBV-C/HGV-RNA, virus genotypes, and anti-E2 antibodies in autoimmune hepatitis.

OBJECTIVE: We investigated the prevalence of hepatitis G-RNA (GBV-C/HGV-RNA), a recently cloned new flavivirus, and of antibodies to the envelope 2 antigen (anti-E2), a marker of past infection, in patients with autoimmune hepatitis, and compared it with the prevalence in patients with chronic viral hepatitis and healthy control individuals. METHODS: Sera of 63 patients with autoimmune hepatitis were studied for the presence of GBV-C/HGV-RNA by reverse-transcription polymerase chain reaction and for anti-E2 by enzyme-linked immunosorbent assay. GBV-C/HGV genotypes were determined by genome sequencing. RESULTS: Patients with autoimmune hepatitis had a similar high prevalence of GBV-C/HGV-RNA and anti-E2 antibodies as patients with chronic viral hepatitis B or C. GBV-C/HGV-RNA was found significantly more often in patients with autoimmune hepatitis (11%, p = 0.045), hepatitis B (16%, p = 0.004), or hepatitis C (21%, p = 0.001) than in healthy controls (2%). The prevalence of anti-E2 antibodies in patients with autoimmune hepatitis was not different from healthy controls (17% vs 13%, NS). The various subtypes of autoimmune hepatitis had similar prevalence rates of GBV-C/HGV-RNA as patients with liver-kidney microsomal antibody-positive hepatitis C. All of our anti-E2+ (GBV-C/HGV-RNA-) patients were positive for anti-smooth-muscle antibody, whereas only 29% of GBV-C/HGV-RNA+ (anti-E2-) patients were positive (p = 0.025). All seven of the GBV-C/HGV-RNA+ patients with autoimmune hepatitis had genotype 2a, which is also the most prevalent genotype in our region. CONCLUSION: The prevalence of GBV-C/HGV-RNA is significantly increased in patients with autoimmune hepatitis, compared with healthy controls, and is similar to the increased prevalence seen in chronic hepatitis B or C patients. Anti-E2 positivity was associated with antibodies against smooth-muscle antigen in all cases. All GBV-C/HGV+ autoimmune hepatitis patients were infected with genotype 2a.

Adult↗
PubMed1993

The operon for the Fe-hydrogenase in Desulfovibrio vulgaris (Hildenborough): mapping of the transcript and regulation of expression.

The genes for the subunits of the Fe-only hydrogenase from Desulfovibrio vulgaris are transcribed as a 1.9 kb mRNA; the operon contains no other genes besides those encoding the two subunits. The transcriptional start site of the operon was mapped. Determination of hydrogenase activity and hydrogenase mRNA levels indicates a growth-phase dependent regulation of hydrogenase expression at transcriptional level. However, it has not yet been possible to localize the sequences required for regulation and expression of the genes.

Amino Acid Sequence↗
PubMed2001

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Base Sequence↗
PubMed1995

Pioneering work.

Explore the source record for details and available documents.

Genome, Viral↗
PubMed1997

A molecular view of microbial diversity and the biosphere.

Over three decades of molecular-phylogenetic studies, researchers have compiled an increasingly robust map of evolutionary diversification showing that the main diversity of life is microbial, distributed among three primary relatedness groups or domains: Archaea, Bacteria, and Eucarya. The general properties of representatives of the three domains indicate that the earliest life was based on inorganic nutrition and that photosynthesis and use of organic compounds for carbon and energy metabolism came comparatively later. The application of molecular-phylogenetic methods to study natural microbial ecosystems without the traditional requirement for cultivation has resulted in the discovery of many unexpected evolutionary lineages; members of some of these lineages are only distantly related to known organisms but are sufficiently abundant that they are likely to have impact on the chemistry of the biosphere.

Archaea↗
PubMed2000

An in vitro assay to study regulated mRNA stability.

The examination of posttranscriptional regulation of mRNA in mammalian cells is critical to discovering the role that mRNA plays in the initiation and maintenance of cellular processes. The complexity of the system defies a holistic approach and, therefore, we have devised an in vitro mRNA turnover assay that enables us to elucidate the factors involved in mRNA deadenylation and degradation. Our system, using an S100 HeLa extract and in vitro transcribed RNAs, accurately mimics the end products of mRNA turnover, which have been previously described using in vivo studies and, in addition, allows for the detailed study of factors that may play a role in regulated deadenylation and degradation. Another important aspect of our system is the ease with which it can be manipulated. We can provide any synthetic RNA molecule to the assay to test for specific sequence activity. Furthermore, the results are clear and accurately interpretable. We have demonstrated that our in vitro system accurately deadenylates and decays a capped and polyadenylated RNA molecule in a processive manner without nonspecific nuclease activity. Finally, we have demonstrated regulated instability in vitro using the AU-rich elements (AREs) from tumor necrosis factor-alpha (TNF-alpha) and granulocyte macrophage colony stimulating factor (GM-CSF) embedded within the RNA molecule. The presence of the AREs increased the deadenylation and the decay rates seen in vivo. We feel that this system can be expanded and adapted to examine a variety of mRNA regulatory events in mammalian cells.

HeLa Cells↗