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At least 1,315 records · Page 73Linked to original sources
PubMed2005

[Variety of differential display on gene and corresponding gene's clone following corneal alkali burns in rats].

OBJECTIVE: To study the condition of differential gene caused after corneal alkali burns in rats and clarify the molecular biological foundation of corneal denatured protein. METHODS: The animals were sacrificed on the 3rd day and the 2nd week after alkali burns. Total RNA was isolated from the excised corneas and then reverse-transcribed into cDNA. The differential gene was detected by mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) with two kinds of anchoring primer and 12 kinds of random primers after corneal alkali burns in the rats. The differential gene fragments were cloned, and their homogeneity was compared with each other in the Gene Bank. RESULTS: Compared with the normal cornea, the cornea of alkali burns on the 2nd week produced one differential gene fragment of 630 bp in the same reaction condition, and this differential gene was homologous to the rattus norvegicus mitochondrial cytochrome oxidase subunits I, II, III gene. CONCLUSIONS: It is found that there is differential gene in the cornea of alkali burns on the 2nd week in rats, and this differential gene is homologous to the rattus norvegicus mitochondrial cytochrome oxidase subunits I, II, III gene. It can be concluded that the occurrence of this differential gene is possible to be related with the action of the superoxide free radicals caused by alkali burns.

Animals↗
PubMed1996

The 5' ends of Thogoto virus (Orthomyxoviridae) mRNAs are homogeneous in both length and sequence.

Thogoto (THO) virus is a tick-borne member of the Orthomyxoviridae whose genome consists of six segments of linear, negative sense, single-stranded RNA. To gain insight into the mechanism by which viral mRNA transcripts are initiated, poly(A)+ RNA isolated from THO virus-infected cells was characterized by (i) primer extension experiments, (ii) immunoprecipitation studies with an anticap monoclonal antibody, (iii) direct sequencing analysis of the isolated RNA, and (iv) cloning and sequencing of individual mRNA molecules. The results indicated that THO virus mRNAs are capped and homogeneous in both length and sequence at their 5' end. These findings contrast with the situation found in all other segmented, negative sense or ambisense, single-stranded RNA viruses so far analyzed in which the 5' ends of viral mRNAs are heterogeneous in length and sequence. These results are discussed in terms of the mechanism used by THO virus to initiate mRNA synthesis.

Poly A↗
PubMed2004

IRIS: intermolecular RNA interaction search.

Here we present IRIS, a method for prediction of RNA-RNA interactions that is based on dynamic programming and extends current RNA secondary structure prediction approaches. Using this method we have found a number of interesting refinements to the structures of RNA-RNA complexes that have been studied previously and predicted novel targets for several known regulatory RNAs in E. coli. The computational time and memory usage of IRIS are O(n(3)m(3)) and O(n(2)m(2)), respectively, where n and m are the lengths of the input sequences. IRIS can be used for analysis of antisense regulatory systems in sequenced organisms and for the design of artificial riboregulators such as antisense drugs.

Base Sequence↗
PubMed2006

Phenotypic consequences of branch point substitutions.

The branch point sequence (BPS) is a conserved splicing signal important for spliceosome assembly and lariat intron formation. BPS mutations may result in aberrant pre-mRNA splicing and genetic disorders, but their phenotypic consequences have been difficult to predict, largely due to a highly degenerate nature of the BPS consensus. Here, we have examined the splicing pattern of nine reporter pre-mRNAs that have previously been shown to give rise to human hereditary diseases as a result of single-nucleotide substitutions in the predicted BPS. Increased exon skipping and intron retention observed in vivo were recapitulated for each mutated pre-mRNA, but the reproducibility of cryptic splice site activation was lower. BP mutations in reporter pre-mRNAs frequently induced aberrant 3' splice sites and also activated a cryptic 5' splice site. Systematic mutagenesis of BP adenosines showed that in most pre-mRNAs, the expression of canonical transcripts was lower for BP transitions than BP transversions. Differential splicing outcome for transitions vs. transversions was abrogated or reduced if introns were truncated to 200 nt or less, suggesting that the nature of the BP residue is less critical for interactions across very short introns. Together, these results improve prediction of phenotypic consequences of point mutations upstream of splice acceptor sites and suggest that the overrepresentation of disease-causing adenosine-to-guanosine BP substitutions observed in Mendelian disorders is due to more profound defects of gene expression at the level of pre-mRNA splicing.

Cell Line↗
PubMed1995

In vivo evidence for non-universal usage of the codon CUG in Candida maltosa.

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNASerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.

Amino Acid Sequence↗
PubMed1997

Phylogenetic relationships of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) inferred from partial 18S rRNA gene sequences.

Aligned 265-bp sequences of partial 18S rRNA gene were used to infer phylogenetic relationships among entomopathogenic nematodes by using maximum parsimony and likelihood methods. Phylogenetic analyses support Heterorhabditidae and Steinernematidae belonging to different monophylies. There was more sequence divergence in Steinernema species than in Heterorhabditis species. These results are congruent with the phylogenies based on morphological, life cycle, and distributional evidence. Examination of all trees within 1% of the length of the most parsimonious trees and bootstrap analyses support most relationships among Steinernema species but the relationships among Heterorhabditis species were not supported. We suggest that the partial 18S rRNA gene sequences may be too conserved for phylogenetic inference among Heterorhabditis species, but are well suited for phylogenetic inference within and among closely related families and genera of entomopathogenic nematodes and for inferring phylogenetic relationships among Steinernema species.

Animals↗
PubMed1993

Template-directed and template-free RNA synthesis by Q beta replicase.

In the absence of extraneously added templates, Q beta replicase produces different RNA species after long lag times spontaneously in vitro. The sequences of the spontaneous products are short (30 to 45 nucleotides) and bear little sequence relation to one another and no detectable sequence homology to Q beta virus RNA or to the host. Their replication rates are much lower than those of optimized products. Incubation without template in long closed capillaries produces after long lag times many separate RNA growth foci with heterogeneous kinetics. The template-free reaction is strongly dependent on the conditions: lowering the enzyme or the triphosphate concentrations abolishes the template-free RNA synthesis without affecting the template-dependent synthesis. An explanation of the emerging RNA species in template-free reactions by residual RNA contaminants in the incubation mixture is very unlikely in the light of the experimental evidence; however, the experimental evidence is fully compatible with a de novo mechanism (which may include instruction by non-replicatable oligonucleotides).

Artifacts↗
PubMed1993

Role of protein conformation in the processing of dengue virus type 2 nonstructural polyprotein precursor.

The dengue virus type-2 (DEN-2) genome is a positive-strand RNA encoding a single polyprotein precursor, C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B- NS5, consisting of 3391 amino acids (aa). The N-terminal region of the polyprotein precursor, C-prM(M)-E, encodes the structural proteins and is processed cotranslationally by the host signal peptidase. The nonstructural region NS1-->NS5 is processed by the viral protease(s), as well as by the signal peptidase. A two-component viral protease consisting of NS2B and the serine protease domain of NS3 has been shown to be required for cleavages having the consensus sequence of dibasic aa (K-R, R-R, R-K, or Q-R). In this study, the region encoding all the nonstructural proteins, NS1-->NS5, was expressed using a recombinant vaccinia virus system. Cleavages at the consensus viral protease recognition sites, 2B-3 at the N terminus and 3-4A at the C terminus, are prerequisites to the release of mature NS3 protease. Although the 2B-3 site was cleaved readily in a variety of polyprotein precursors containing the intact NS2B and the NS3 protease domain, the 3-4A site was most efficiently cleaved, similar to that found in DEN-2-infected cells, only in the polyprotein precursor encoding the entire nonstructural region. Removal of NS1 at the N terminus or of NS5 coding sequences at the C terminus affected the cleavage at the 3-4A site to produce the processing intermediate, NS3-NS4A. These results indicate that the conformation of the nonstructural polyprotein precursor, NS1-->NS5, plays a major role in the efficient cleavage at the 3-4A site.

Animals↗
PubMed2004

Transcriptional response to glucose starvation and functional analysis of a glucose transporter of Neurospora crassa.

The response to glucose availability in Neurospora crassa was characterized by measuring transcript levels of 1335 genes represented on a cDNA microarray. We found that 19% of the 1335 genes were regulated at least twofold in response to glucose deprivation. Several sugar transporter homologs were found to be regulated by glucose. Functional analysis of one of these, hgt-1, indicates that it encodes a high-affinity glucose transporter. Comparing N. crassa profiling data with the published diauxic shift data from Saccharomyces cerevisiae revealed that the two fungi share similar, but not identical, transcriptional response patterns. Regulation of transcript levels measured by microarray analysis is consistent with previous measurements of central metabolism enzyme activities for N. crassa. Transcripts of genes for ethanol fermentation and the tricarboxylic acid cycle are abundant in glucose-rich medium, consistent with N. crassa carrying out a high rate of both fermentation and respiration simultaneously.

Adaptation, Physiological↗
PubMed2006

Cytokine levels in Crimean-Congo hemorrhagic fever.

BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans with a mortality reaching 30%. A CCHF outbreak took place in Albania in 2003. As in other viral hemorrhagic fevers cytokines may be involved and play a role in the pathogenesis and outcome of the disease. OBJECTIVES: To investigate the levels of TNF-alpha, sTNF-R, IL-6 and IL-10 in serum samples obtained from laboratory confirmed CCHF cases and relate them to the severity of the disease. STUDY DESIGN: A study population of 51 was divided into three groups: group A, consisting of PCR-positive cases; group B, consisting of PCR-negative and serology-positive cases; group C, consisting of doubly negative cases. Concentrations of serum TNF-alpha, sTNF-R, IL-6 and IL-10 were measured during the illness. RESULTS: High levels of all cytokines tested were present in one fatal case. Statistically significant differences between the groups were obtained for TNF-alpha and IL-6: TNF-alpha was detected in 3 cases in group A, and in none of the other groups, while IL-6 was elevated in 10/16 patients in group A, 4/9 in group B, and 4/26 in group C. sTNF-R was not significantly different for the three groups. High concentration of IL-10 was detected only in the fatal case. CONCLUSIONS: TNF-alpha and IL-6 are the cytokines most often detected during a CCHF viral infection. TNF-alpha was associated with the severe form of CCHF, while IL-6 was elevated in both severe and mild cases.

Adolescent↗
PubMed2003

Identifying splicing sites in eukaryotic RNA: support vector machine approach.

We introduce a new method for splicing sites prediction based on the theory of support vector machines (SVM). The SVM represents a new approach to supervised pattern classification and has been successfully applied to a wide range of pattern recognition problems. In the process of splicing sites prediction, the statistical information of RNA secondary structure in the vicinity of splice sites, e.g. donor and acceptor sites, is introduced in order to compare recognition ratio of true positive and true negative. From the results of comparison, addition of structural information has brought no significant benefit for the recognition of splice sites and had even lowered the rate of recognition. Our results suggest that, through three cross validation, the SVM method can achieve a good performance for splice sites identification.

Algorithms↗
PubMed2001

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli.

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.

Base Sequence↗
PubMed2004

Role of transposable elements in heterochromatin and epigenetic control.

Heterochromatin has been defined as deeply staining chromosomal material that remains condensed in interphase, whereas euchromatin undergoes de-condensation. Heterochromatin is found near centromeres and telomeres, but interstitial sites of heterochromatin (knobs) are common in plant genomes and were first described in maize. These regions are repetitive and late-replicating. In Drosophila, heterochromatin influences gene expression, a heterochromatin phenomenon called position effect variegation. Similarities between position effect variegation in Drosophila and gene silencing in maize mediated by "controlling elements" (that is, transposable elements) led in part to the proposal that heterochromatin is composed of transposable elements, and that such elements scattered throughout the genome might regulate development. Using microarray analysis, we show that heterochromatin in Arabidopsis is determined by transposable elements and related tandem repeats, under the control of the chromatin remodelling ATPase DDM1 (Decrease in DNA Methylation 1). Small interfering RNAs (siRNAs) correspond to these sequences, suggesting a role in guiding DDM1. We also show that transposable elements can regulate genes epigenetically, but only when inserted within or very close to them. This probably accounts for the regulation by DDM1 and the DNA methyltransferase MET1 of the euchromatic, imprinted gene FWA, as its promoter is provided by transposable-element-derived tandem repeats that are associated with siRNAs.

Arabidopsis↗
PubMed2001

Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements.

In the last decade, DNA fingerprint techniques have become available to study the interperson transmission of tuberculosis and other mycobacterial infections. These methods have facilitated epidemiological studies at a population level. In addition, the species identification of rarely encountered mycobacteria has improved significantly. This article describes the state of the art of the main molecular typing methods for Mycobacterium tuberculosis complex and non-M. tuberculosis complex (atypical) mycobacteria. Important new insights that have been gained through molecular techniques into epidemiological aspects and diagnosis of mycobacterial diseases are highlighted.

Animals↗
PubMed2005

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo.

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.

DNA, Viral↗
PubMed2001

Virologic and immunologic determinants of heterosexual transmission of human immunodeficiency virus type 1 in Africa.

More than 80% of the world's HIV-infected adults live in sub-Saharan Africa, where heterosexual transmission is the predominant mode of spread. The virologic and immunologic correlates of female-to-male (FTM) and male-to-female (MTF) transmission are not well understood. A total of 1022 heterosexual couples with discordant HIV-1 serology results (one partner HIV infected, the other HIV uninfected) were enrolled in a prospective study in Lusaka, Zambia and monitored at 3-month intervals. A nested case-control design was used to compare 109 transmitters and 208 nontransmitting controls with respect to plasma HIV-1 RNA (viral load, VL), virus isolation, and CD4(+) cell levels. Median plasma VL was significantly higher in transmitters than nontransmitters (123,507 vs. 51,310 copies/ml, p < 0.001). In stratified multivariate Cox regression analyses, the risk ratio (RR) for FTM transmission was 7.6 (95% CI: 2.3, 25.5) for VL > or = 100,000 copies/ml and 4.1 (95% CI: 1.2, 14.1) for VL between 10,000 and 100,000 copies/ml compared with the reference group of <10,000 copies/ml. Corresponding RRs for MTF transmission were 2.1 and 1.2, respectively, with 95% CI both bounding 1. Only 3 of 41 (7%) female transmitters had VL < 10,000 copies/ml compared with 32 of 93 (34%) of female nontransmitters (p < 0.001). The transmission rate within couples was 7.7/100 person-years and did not differ from FTM (61/862 person-years) and MTF (81/978 person-years) transmission. We conclude that the association between increasing plasma viral load was strong for female to male transmission, but was only weakly predictive of male to female transmission in Zambian heterosexual couples. FTM and MTF transmission rates were similar. These data suggest gender-specific differences in the biology of heterosexual transmission.

Adolescent↗
PubMed2003

Technical advances: genome-wide cDNA-AFLP analysis of the Arabidopsis transcriptome.

cDNA-AFLP, a technology historically used to identify small numbers of differentially expressed genes, was adapted as a genome-wide transcript profiling method. mRNA levels were assayed in a diverse range of tissues from Arabidopsis thaliana plants grown under a variety of environmental conditions. The resulting cDNA-AFLP fragments were sequenced. By linking cDNA-AFLP fragments to their corresponding mRNAs via these sequences, a database was generated that contained quantitative expression information for up to two-thirds of gene loci in A. thaliana, ecotype Ws. Using this resource, the expression levels of genes, including those with high nucleotide sequence similarity, could be determined in a high-throughput manner merely by comparing cDNA-AFLP profiles with the database. The lengths of cDNA-AFLP fragments inferred from their electrophoretic mobilities correlated well with actual fragment lengths determined by sequencing. In addition, the concentrations of AFLP fragments from single cDNAs were highly correlated, illustrating the validity of cDNA-AFLP as a quantitative, genome-wide, transcript profiling method. cDNA-AFLP profiles were also qualitatively consistent with mRNA profiles obtained from parallel microarray analysis, and with data from previous studies.

Arabidopsis↗
PubMed2025

De novo clustering of large long-read transcriptome datasets with isONclust3.

MOTIVATION: Long-read sequencing techniques can sequence transcripts from end to end, greatly improving our ability to study the transcription process. Although there are several well-established tools for long-read transcriptome analysis, most are reference-based. This limits the analysis of organisms without high-quality reference genomes and samples or genes with high variability (e.g. cancer samples or some gene families). In such settings, analysis using a reference-free method is favorable. The computational problem of clustering long reads by region of common origin is well-established for reference-free transcriptome analysis pipelines. Such clustering enables large datasets to be split roughly by gene family and, therefore, an independent analysis of each cluster. There exist tools for this. However, none of those tools can efficiently process the large amount of reads that are now generated by long-read sequencing technologies. RESULTS: We present isONclust3, an improved algorithm over isONclust and isONclust2, to cluster massive long-read transcriptome datasets into gene families. Like isONclust, isONclust3 represents each cluster with a set of minimizers. However, unlike other approaches, isONclust3 dynamically updates the cluster representation during clustering by adding high-confidence minimizers from new reads assigned to the cluster and employs an iterative cluster-merging step. We show that isONclust3 yields results with higher or comparable quality to state-of-the-art algorithms but is 10-100 times faster on large datasets. Also, using a 256&#x2009;Gb computing node, isONclust3 was the only tool that could cluster 37 million PacBio reads, which is a typical throughput of the recent PacBio Revio sequencing machine. AVAILABILITY AND IMPLEMENTATION: https://github.com/aljpetri/isONclust3.

Algorithms↗