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At least 739 records · Page 41Linked to original sources
PubMed2004

Molecular phylogenies of Parabasalia inferred from four protein genes and comparison with rRNA trees.

The molecular phylogeny of parabasalids has mainly been inferred from small subunit (SSU) rRNA sequences and has conflicted substantially with systematics based on morphological and ultrastructural characters. This raises the important question, how congruent are protein and SSU rRNA trees? New sequences from seven diverse parabasalids (six trichomonads and one hypermastigid) were added to data sets of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, alpha-tubulin and beta-tubulin and used to construct phylogenetic trees. The GAPDH tree was well resolved and identical in topology to the SSU rRNA tree. This both validates the rRNA tree and suggests that GAPDH should be a valuable tool in further phylogenetic studies of parabasalids. In particular, the GAPDH tree confirmed the polyphyly of Monocercomonadidae and Trichomonadidae and the basal position of Trichonympha agilis among parabasalids. Moreover, GAPDH strengthened the hypothesis of secondary loss of cytoskeletal structures in Monocercomonadidae such as Monocercomonas and Hypotrichomonas. In contrast to GAPDH, the enolase and both tubulin trees are poorly resolved and rather uninformative about parabasalian phylogeny, although two of these trees also identify T. agilis as representing the basal-most lineage of parabasalids. Although all four protein genes show multiple gene duplications (for 3-6 of the seven taxa examined), most duplications appear to be relatively recent (i.e., species-specific) and not a problem for phylogeny reconstruction. Only for enolase are there more ancient duplications that may confound phylogenetic interpretation.

Animals↗
PubMed2002

Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs.

A human immunodeficiency virus type 1 (HIV-1) particle contains approximately 1200 molecules of gag proteins and two copies of a 9.2-kb genomic RNA which has been reported to be dimerized and rapidly cleaved and to form a complex with a nucleocapsid protein, p7 (NCp7), during viral budding. These suggest that the cleavage can be reconstituted with gag proteins in vitro. Here we show that the p15(gag) coding region of viral RNA is fragmented in viral particles and that in vitro-synthesized RNA transcripts of HIV-1 undergo cleavage which is activated by NCp7 and other factors. Single-stranded oligoribonucleotides were cleaved between C and A or U and A, leaving 2',3'-cyclic phosphate and 5'-hydroxyl termini. These findings might explain the rapid degradation of genomic RNAs in HIV-1 particles.

Base Sequence↗
PubMed2002

cDNA macroarray analysis of gene expression in synoviocytes stimulated with TNFalpha.

Gene expression of synoviocytes stimulated with tumor necrosis factor-alpha (TNFalpha) was studied by macroarray analysis to elucidate the cellular response and identify new biological functions of known and unknown genes. 10035 cDNA clones were used to make cDNA macroarrays of representative genes. Synoviocytes expressed large amounts of fibronectin and collagen mRNA. Statistical analysis of the macroarray data revealed 26 genes, including six new genes, which underwent significant alteration of gene expression in response to TNFalpha stimulation. These findings suggest that the synoviocyte response to TNFalpha stimulation forms the basis of development of various aspects of the pathophysiology of rheumatoid arthritis.

Amino Acid Sequence↗
PubMed2002

Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings.

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (<five minutes of computational time) and are in complete agreement with corresponding NOE-based assignments. Orientational constraints derived from RDCs are used simultaneously to assemble sub-structures into an RNA tertiary conformation. Through enhanced speeds of application and reduced reliance on chemical shift dispersion, this RDC-based approach lays the foundation for rapidly determining RNA conformations in a structural genomics context, and may increase the size limit of RNAs that can be examined by NMR.

Base Sequence↗
PubMed2003

The nucleotide sequence and genome organization of Sclerophthora macrospora virus A.

Sclerophthora macrospora virus A (SmV A) found in S. macrospora, the pathogenic fungus responsible for downy mildew of gramineous plants, is a small icosahedral virus containing three segments (RNAs 1, 2, and 3) of the positive-strand ssRNA genome. In the present study we report the complete nucleotide sequence of the SmV A genome. The viral genome RNA 1 consists of 2928 nucleotides (nt) and has two open reading frames (ORFs 1a and 1b). ORF 1a contains the motifs of RNA-directed RNA polymerase (RdRp). The function of ORF 1b is unknown. RNA 2 consists of 1981 nt and single ORF (ORF 2). ORF 2 encodes a capsid protein. RNA 3 consists of 977 nt but not any ORFs, suggesting it as a satellite RNA. The deduced amino acid sequence of ORF 1a shows some similarity to those of RdRp of certain positive-strand RNA viruses, especially to the members of the family Nodaviridae, and that of ORF 2 to CP of the members in the family Tombusviridae. The nucleotide sequence of RNA 3 shows a 40-nucleotide length of partial similarity to S. macrospora virus B (SmV B) RNA. The capsid of SmV A is composed of two capsid proteins, CP 1 (p43) and CP 2 (p39), both encoded in ORF 2. CP 2 is apparently derived from CP 1 via proteolytic cleavage at the N-terminus. The genome organization of SmV A is characteristic and distinct from those of other known fungal RNA viruses, including SmV B. These results suggest that SmV A should be classified into a new group of mycoviruses.

Amino Acid Sequence↗
PubMed1998

Characterization of Coxsackie B virus RNA in myocardium from patients with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products.

This study was performed to detect and characterize the enterovirus present in myocardium of some patients with heart muscle disease by nucleotide sequencing of polymerase chain reaction (PCR) products after amplification with enterovirus group-specific primers. Enterovirus sequences have been detected previously in myocardium of patients with myocarditis or dilated cardiomyopathy and seem causal, although the particular virus serotypes involved have not been identified. In a prospective study of endomyocardial biopsy specimens from 35 consecutive patients with suspected heart muscle disease, enterovirus sequences from the 5' nontranslated region were amplified by reverse transcription-nested PCR using group-specific primers. This region contains both conserved and variable sequence motifs, characteristic of particular enterovirus serotypes. The nucleotide sequences of individual PCR products were determined by cycle sequencing and compared with all known sequences (GenBank/EMBOL), using the GCG software package. Endomyocardial biopsy specimens from 9 of 21 (42.9%) patients with a histologically confirmed diagnosis of dilated cardiomyopathy were positive for enterovirus by PCR, compared with only 1 of 14 (7.1%) patients with other myocardial pathological conditions (Fisher's exact probability=0.0275: odds ratio=9.75; 95% confidence interval=1.31-72.78). The nucleotide sequence of the PCR products differed, indicating no cross-contamination. However, computerized comparison showed that each had greatest homology with the 5' nontranslated region of Coxsackie B virus but contained up to 11% sequence variations compared with the prototype Coxsackie B3 strain Nancy. Parallel investigation of tissue from our mouse model of Coxsackievirus B3-induced myocarditis showed that nucleotide sequence changes are not introduced by reverse transcription or PCR. These data support the link between enteroviral infection and dilated heart muscle disease and suggest that Coxsackie B serotypes are the enteroviruses most frequently involved.

Adolescent↗
PubMed2001

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

Amino Acid Sequence↗
PubMed2001

Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains.

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 41 nucleotide changes, 12 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.

5' Untranslated Regions↗
PubMed2003

Genetic typing of recent classical swine fever virus isolates from Croatia.

During a period of 5 years (1997-2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5'-non-translated region (5'-NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.

Animals↗
PubMed2002

Corynebacterium macginleyi isolation from conjunctival swab in Italy.

Corynebacterium macginleyi was isolated from conjunctival swabs of a farmer suffering from purulent conjunctivitis. This species has only recently been reported in Switzerland and Germany to be exclusively isolated from ocular surfaces. This represents the first isolation of C. macginleyi in Italy indicating that its circulation is not geographically limited.

Aged↗
PubMed2002

X-chromosome inactivation and the search for chromosome-wide silencers.

X-chromosome inactivation leads to divergent fates for two homologous chromosomes. Whether one X remains active or becomes silenced depends on the activity of Xist, a gene expressed only from the inactive X and whose RNA product 'paints' the X in cis. Recent work argues that Xist RNA itself is the acting agent for initiating the silencing step. Xist RNA contains separable domains for RNA localization and chromosome silencing. While no Xist RNA-interacting factors have been identified, a growing collection of chromatin alterations have been identified on the inactive X, including variant histone H2A composition and histone H3 methylation. Some or all of these changes may be critical for chromosome-wide silencing. As none of the silencing proteins identified so far is unique to X chromosome inactivation, the specificity must partly reside in Xist RNA whose spread along the X orchestrates general silencing factors for this specific task.

Animals↗
PubMed1998

Expression of abnormal transcripts of the FHIT (fragile histidine triad) gene in ovarian carcinoma.

To elucidate the role of the FHIT (fragile histidine triad) gene in ovarian carcinogenesis, the expression of the gene was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in 51 cases of ovarian carcinoma, 6 cases of borderline tumour and 4 cases of benign ovarian tumour. The concomitant expressions of normal and abnormal FHIT transcripts were detected in 39% of carcinomas and in 83% of borderline tumours, while benign tumours and normal ovarian tissues expressed only normal transcript. In addition, there were 4 (8%) cases of carcinoma lacking expression of normal FHIT transcript, all of which were in advanced stages (stage III-IV) and poorly differentiated. These results suggest that the expression of abnormal transcripts of the FHIT gene is a feature of ovarian malignant/borderline tumours and that the complete loss of normal FHIT expression is related to the progression of ovarian carcinoma in a subset of the cases. However, abnormal FHIT transcripts themselves were not associated with any clinicopathological parameters, such as clinical stage, histological subtype of tumour, grade of differentiation or outcome of the patient. Additionally, abnormal FHIT expression was not associated with the presence of loss of heterozygosity (LOH) at this locus, suggesting that abnormal FHIT transcripts are not derived from genetic alteration or that genetic alteration at this locus is complicated.

Acid Anhydride Hydrolases↗
PubMed2001

A tetracycline-binding RNA aptamer.

Aptamers are perfect tools to study the interaction of small ligands with RNA. To study the mode of interaction of tetracycline with RNA, we isolated aptamers with high affinity to this antibiotic via in vitro selection. One of the selected aptamers, cb28, which has a comparable affinity to tetracycline as the small ribosomal subunit, was characterised in more detail. Cb28 binds only to typical tetracyclines, while atypical tetracyclines are not recognised. The hydroxyl group at position 6 is an essential determinant for recognition, while modifications at positions 4, 5 and 7 do not interfere with RNA binding. Binding of tetracycline to cb28 is magnesium dependent. The secondary structure of cb28 was determined by lead cleavage and DMS modification. Upon tetracycline binding, nucleotides in J2/3 and the P5 stem-loop are protected from cleavage by lead, indicating a conformational change in the RNA. This conformational change was confirmed by tetracycline dependent changes in the DMS modification pattern. Photo-induced affinity incorporation of tetracycline into cb28 resulted in a crosslink to position G76, a residue in L5. The mode of binding of tetracycline to the cb28 aptamer resembles its interaction with the primary binding site on the small ribosomal subunit.

Anti-Bacterial Agents↗
PubMed2002

RNA-catalyzed RNA ligation on an external RNA template.

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside 5'-triphosphates onto the 3' end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

Directed Molecular Evolution↗
PubMed2002

Continuous in vitro evolution of a ribozyme that catalyzes three successive nucleotidyl addition reactions.

Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5'-->3' or a 3'-->5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

Base Sequence↗
PubMed2000

Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco.

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3' two-thirds of the GUS coding region. The 5' coding and the 3' noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.

Fluorescent Dyes↗
PubMed2000

In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection.

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.

Antiviral Agents↗