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At least 721 records · Page 40Linked to original sources
PubMed1999

Poliovirus/Hepatitis C virus (internal ribosomal entry site-core) chimeric viruses: improved growth properties through modification of a proteolytic cleavage site and requirement for core RNA sequences but not for core-related polypeptides.

H.-H. Lu and E. Wimmer (Proc. Natl. Acad. Sci. USA 93:1412-1417, 1996) have demonstrated that the internal ribosomal entry site (IRES) of poliovirus (PV) can be functionally replaced by the related genetic element from hepatitis C virus (HCV). One important finding of this study was that open reading frame sequences 3' of the initiating AUG, corresponding to the open reading frame of the HCV core polypeptide, are required to create a viable chimeric virus. This made necessary the inclusion of a PV 3C protease (3Cpro) cleavage site for proper polyprotein processing to create the authentic N terminus of the PV capsid precursor. Chimeric PV/HCV (P/H) viruses, however, grew poorly relative to PV. The goal of this study was to determine the molecular basis of impaired replication and enhance the growth properties of this chimeric virus. Genetic modifications leading to a different proteinase (PV 2Apro) cleavage site between the HCV core sequence and the PV polyprotein (P/H701-2A) proved far superior with respect to viral protein expression, core-PV fusion polyprotein processing, plaque phenotype, and viral titer than the original prototype PV/HCV chimera containing the PV 3Cpro-specific cleavage site (P/H701). We have used this new virus model to answer two questions concerning the role of the HCV core protein in P/H chimeric viral proliferation. First, a derivative of P/H701-2A with frameshifts in the core-encoding sequence was used to demonstrate that production of the core protein was not necessary for the translation and replication of the P/H chimera. Second, a viral construct with a C-terminal truncation of 23 amino acids of the core gene was used to show that a signal sequence for signal peptidase processing, when present in the viral construct, is detrimental to P/H virus growth. The novel P/H chimera described here are suitable models for analyzing the function(s) of the HCV elements by genetic analyses in vivo and for antiviral drug discovery.

3C Viral Proteases↗
PubMed2002

Full-length messenger RNA sequences greatly improve genome annotation.

BACKGROUND: Annotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism. RESULTS: Approximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation. CONCLUSIONS: Our results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.

Alternative Splicing↗
PubMed1990

Small ribosomal subunit RNA sequences, evolutionary relationships among different life forms, and mitochondrial origins.

A tree was constructed from a structurally conserved area in an alignment of 83 small ribosomal subunit sequences of eukaryotic, archaebacterial, eubacterial, plastidial, and mitochondrial origin. The algorithm involved computation and optimization of a dissimilarity matrix. According to the tree, only plant mitochondria belong to the eubacterial primary kingdom, whereas animal, fungal, algal, and ciliate mitochondria branch off from an internal node situated between the tree primary kingdoms. This result is at variance with a parsimony tree of similar size published by Cedergren et al. (J Mol Evol 28:98-112, 1988), which postulates the mitochondria to be monophyletic and to belong to the eubacterial primary kingdom. The discrepancy does not follow from the use of conflicting sequence alignments, hence it must be due to the use of different treeing algorithms. We tested our algorithm on a set of sequences resulting from a simulated evolution and found it capable of faithfully reconstructing a branching topology that involved very unequal evolutionary rates. The use of more limited or more extended areas of the complete sequence alignment, comprising only very conserved or also more variable portions of the small ribosomal subunit structure, does have some influence on the tree topology. In all cases, however, the nonplant mitochondria seem to branch off before the emergence of eubacteria, and the differences are limited to the branching pattern among different types of mitochondria.

Algorithms↗
PubMed1988

MP26 messenger RNA sequences in normal and cataractous lens. A molecular probe for abundance and distribution of a fiber cell-specific gene product.

Previous work from this laboratory has suggested that swollen nucleated fiber cells can survive in mature galactose-cataracts. Evidence for this observation was derived from analysis on the in vitro translation products of mRNA isolated from normal lens and lens undergoing development of galactose-cataracts. Therefore, studies on the fate of a fiber cell-specific gene product (MP26 mRNA) in both normal and cataractous lens should map out gene response to: (1) differentiation of epithelial cells to fiber cells; (2) levels of this differential gene activity and its anatomical location in initiation and maturation of galactose-cataracts; and (3) distribution of MP26 and mRNA in fibers of normal and cataractous lens. The MP26 probe was isolated by methods of cDNA cloning into expression vectors, then subcloned into a transcription vector, and the recombinant plasmid was transcribed into sense and anti-sense [35S]-UTP-labeled RNA. The [35S]-labeled RNA products were used to localize MP26 mRNA in tissue sections by methods of hybridization in situ. The results on normal lens show that gene transcription for MP26 mRNA is initiated immediately in elongating fibers, where new fiber cell nuclei begin to migrate to within the cortex. The MP26-positive grains are absent from the epithelium, highest at the bow, with a lower number in fibers below the posterior capsule, and lowest in the lens nucleus. The cataractous lens exhibits continued manifestation of MP26 mRNA at the bow, but at significantly lower concentrations than found in the controls, and this low level persisted in viable areas of the cortex. Absence of significant grains in areas containing cell debris is evident. The emerging picture for MP26 mapping in lens suggests that: (1) in cataractous as well as in normal lens MP26 mRNA first develops in elongating fibers; and (2) that MP26 mRNA localization gives an exact measure of point of cell specialization, and levels or "storage" of a specific gene product in fiber cells undergoing maturation, aging and cataractogenesis.

Animals↗
PubMed1997

Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5' to cleavage sites.

The endoribonuclease RNase E is believed to initiate the degradation of many mRNAs in Escherichia coli, yet the mechanism by which it recognizes cleavage sites is poorly understood. We have prepared derivatives of the mRNA encoding ribosomal protein S20 which contain a single major RNase E cleavage site at residues 300/301 preceded by variable 5' extensions. Three of these RNAs are cleaved in vitro with significantly reduced efficiencies relative to the intact S20 mRNA by both crude RNase E and pure Rne protein (endonuclease component of RNase E). In all three substrates as well as in the full-length mRNA the major cleavage site itself remains single-stranded. One such substrate (t84D) contains a 5' stem-loop structure characterized by three noncanonical A-G pairs. Removal or denaturation of the stem restores efficient cleavage at the major RNase E site. The other two contain single-stranded 5'-termini but apparently lack cleavage sites near the termini. Our data show that sensitivity to RNase E can be influenced by distant structural motifs in the RNA and also suggest a model in which the initial recognition and cleavage of a substrate near its 5' end facilitates sequential cleavages at more distal sites. The model implies that RNase E contains at least a dimer of the Rne subunit and that the products of the first cleavage are retained by Rne prior to the second cleavage.

Bacterial Proteins↗
PubMed1991

Polyadenylated RNA sequences produced in vaccinia virus-infected cells under aberrant conditions inhibit protein synthesis in vitro.

We have previously demonstrated that small nontranslated polyadenylated RNAs (POLADS) produced in vaccinia virus (VV)-infected cells inhibit the translation of cellular mRNAs, but minimally affect the translation of VV mRNAs in a cell-free protein synthesizing system. Infection of HeLa cells with ultraviolet-irradiated vaccinia virus or infection in the presence of actinomycin D (ACD) amplifies the synthesis of POLADS compared to the amount produced in cells infected under normal conditions. The effect of these POLADS on translation was studied in the reticulocyte lysate system. Polyadenylated RNAs isolated from cells infected with wild-type virus (V-POLADS) had a greater inhibitory effect on HeLa cell protein synthesis than on VV protein synthesis. Polyadenylated sequences obtained from cells infected with ultraviolet-irradiated virus (UV-POLADS) or from cells infected in the presence of ACD (ACD-POLADS), however, inhibited translation of both HeLa and viral mRNAs. Ultraviolet-POLADS and ACD-POLADS were found to possess, on average, longer poly(A) tails than V-POLADS. The inhibition of translation of both host and viral mRNAs effected by V-POLADS, UV-POLADS, and ACD-POLADS was reversed by poly(A) binding protein.

Gene Expression Regulation, Viral↗
PubMed1987

Identification of renin and renin messenger RNA sequence in rat ovary and uterus.

An increase in plasma prorenin during pregnancy suggests that prorenin might be synthesized in the ovary and the secretion of renin or prorenin may be stimulated by an ovarian steroid-mediated process. Recently, renin and angiotensinogen have been identified in human ovarian follicular fluid. However, there is considerable controversy over whether renin is synthesized in the ovary or derived from circulation. In the present study, we confirmed the presence of renin and renin mRNA in rat ovary and uterus by Northern blot analysis with rat renin cRNA as a hybridization probe. Our data show that ovarian or uterine renin is synthesized in the same cells. This suggests that the function of renin might be closely linked to the reproductive process.

Animals↗
PubMed1993

Assessment of the viral RNA sequence heterogeneity for control of OPV neurovirulence.

By using sensitive mutant analysis by PCR and restriction enzyme cleavage we have found that among several positions that differ between the wild-type and attenuated type 3 poliovirus genomes, only two positions, 472 and 2493, showed variability in vaccine lots. Of these two, only position 472 correlates with neurovirulence in monkeys, while the abundance of revertants at position 2493 indicated the type of seed virus and the passage level. Conditions of cell culture influence the rate of mutant selection, suggesting that some cellular factor(s) may be involved in selection of 472-C. Determination of 472-C content predicts which vaccine lots would fail the monkey neurovirulence test. These results imply that the PCR method can be used for optimization of manufacturing conditions.

Animals↗
PubMed2000

Detection of Isospora belli by polymerase chain reaction using primers based on small-subunit ribosomal RNA sequences.

The aim of the present study was to use small-subunit (SSU)-rRNA sequences of Isospora belli to design specific primer pairs and a hybridization probe for the detection of Isospora belli in human samples by PCR and Southern blot hybridization. PCR amplification with the primer pairs produced correct DNA fragments with target DNA from samples of Isospora belli-infected patients and from cloned SSU-rRNA of Isospora belli. The nature of the PCR products was confirmed by Southern blot hybridization. No amplification was seen with template DNA extracted from other parasites. Although Isospora belli infections can be easily diagnosed using light microscopy, molecular-based techniques may prove useful as an additional diagnostic tool.

Animals↗
PubMed1993

Role of polyadenylated RNA sequences (POLADS) in vaccinia virus infection: correlation between accumulation of POLADS and extent of shut-off in infected cells.

The selective inhibition of host-cell protein synthesis was studied in cells infected with vaccinia virus (VV) under aberrant conditions of transcription. Previous studies in our laboratory have correlated this selective inhibition with a class of short polyadenylated virus-directed RNAs (POLADS) which are synthesized in VV-infected cells during the early phase of transcription. Moreover, it was shown that infection of HeLa cells with UV-irradiated VV or infection in the presence of actinomycin D (ACD) amplifies the synthesis of POLADS compared to the amount produced in cells infected under normal conditions. To further study the role of POLADS in shut-off, we utilized a temperature sensitive mutant of VV which induces only marginal host shut-off at the restrictive temperature. POLADS were isolated from cells infected with either unirradiated or UV-irradiated VV ts mutant at the permissive and restrictive temperatures and their inhibitory activity on translation in vitro was assayed. The study yields further evidence associating excess of POLADS with greater inhibitory potential and supports the speculation that the increased production of POLADS is correlated with the inhibition of translation of both host-cell and viral polypeptides, albeit to different degrees. The results also demonstrate that a lack of POLADS accumulation is related to a corresponding reduction of shut-off.

Adenosine↗
PubMed1997

Detection of rare RNA sequences by single-enzyme in situ reverse transcription-polymerase chain reaction. High-resolution analyses of interleukin-6 mRNA in paraffin sections of lymph nodes.

To study the distribution pattern of interleukin-6 (IL-6)-producing cells in normal human lymph nodes, we applied the in situ reverse transcription-polymerase chain reaction technique. We describe a new modification of this technique for monitoring small amounts of specific nucleotide sequences in conventional paraffin sections. This technique differs in at least two respects from those described earlier. The two decisive steps are: 1) the reverse transcription of mRNA and the subsequent amplification of cDNA by polymerase chain reaction are performed by a new single enzyme capable of both reaction types in one and the same medium without buffer exchange; and 2) for the specific detection of the amplified cDNA, a modified version of the primed in situ labeling technique was used. The technique, carried out on normal human lymph nodes, traces a low load of IL-6 mRNA in fibroblasts, endothelial cells, and a minor population of T lymphocytes in the pulp region. High levels of expression were encountered in about 20% of perisinusoidal pulp macrophages. In addition, moderate activity was detectable in sinus lining cells. Because no major activity was found in the germinal centers of the lymphoid B follicles and in the T zone, it is suggested that the plasma cell differentiation ensuing from primary and secondary B-cell immunization is mainly effected by the sinus lining cells as well as perifollicular and perisinusoidal pulp macrophages capable of producing high amounts of IL-6.

Humans↗
PubMed1992

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

Animals↗
PubMed1990

Polyadenylated RNA sequences from vaccinia virus-infected cells selectively inhibit translation in a cell-free system: structural properties and mechanism of inhibition.

The mechanism of vaccinia virus-induced selective inhibition of host cell protein synthesis was studied in a nonpermissive (Chinese hamster ovary, CHO) and in a permissive mouse cell line ( L cells). Small polyadenylated RNAs obtained from uninfected and infected cells were fractionated into six size classes by polyacrylamide gel electrophoresis. The RNAs from the first two largest fractions (greater than 500 nucleotide, nt) were translated into some low-molecular-weight polypeptides, whereas, the RNAs from the remaining fractions (400-500, 300-400, 200-300, and 100-200 nt) had no translational activity in reticulocyte lysates. When these nontranslating polyadenylated short sequences (POLADS) were added to the cell-free system together with HeLa cell mRNAs, translation was inhibited from 70%, by the 400- to 500-nt fraction, to about 20%, by the 100- to 200-nt fraction. The degree of inhibition of protein synthesis was clearly dependent on the size of POLADS. The translation of vaccinia virus mRNAs in the cell-free system was inhibited by about 25% with the 400- to 500-nt fraction, by 5% with the 300- to 400-nt fraction, while the smaller size POLADS had no inhibitory effect. The inhibition of HeLa cell and vaccinia virus mRNA translation by POLADS was reversed by the simultaneous addition of oligo(dT) to the cell-free system. POLADS were also obtained from uninfected cells, but they inhibited the translation of HeLa cell and vaccinia virus mRNAs to a much lesser extent. The removal of the poly(A) moiety from POLADS by treatment with ribonuclease H and oligo(dT) abolished their inhibitory effect on HeLa cell mRNA translation. The average length of the poly(A) tails of POLADS obtained from infected cells was longer than that of POLADS from normal cells. Inhibition of HeLa cell mRNA translation mediated by POLADS in the cell-free system was reversed (approximately 70%) by addition of crude initiation factors (ribosomal salt wash, RSW). Significantly, inhibition of translation of POLADS was reversed (greater than 90%) by addition of purified poly(A) binding protein (PAB). Purified initiation factor 4A (eIF-4A) also reversed this inhibition, but to a lesser extent than RSW and PAB. Our results show that the translation of vaccinia virus mRNAs is resistant to POLADS, suggesting that POLADS, by virtue of their long poly(A) tails, may sequester PAB and thus, play a role in selective inhibition.

Animals↗
PubMed1993

Amplification of polyadenylated nontranslated small RNA sequences (POLADS) during superinfection correlates with the inhibition of viral and cellular protein synthesis.

The production and the role of POLADS in cells infected with vaccinia virus (VV) was studied in doubly infected HeLa and L cells. In cells first infected with VV followed by superinfection with UV-irradiated virus (VVuv), referred to as VV + VVuv, the course of viral polypeptide synthesis was not significantly altered. However, if cells were first infected with VVuv, followed by superinfection with VV, referred to as VVuv + VV, both host and viral polypeptide synthesis was compromised. Labeling of such infected cells with 3H-adenosine revealed that infection with VVuv or VVuv + VV, caused an amplification of incorporation of label both in the large and small size class RNAs compared to RNAs obtained from a normal infection. On the other hand, labeling of cells with 3H-adenosine which were infected with VV + VVuv, caused no significant change in the labeling pattern of large and small size class RNAs compared to labeled RNAs from normal infection. The large size class RNAs isolated from infection with VVuv or VVuv + VV, were translated in the reticulocyte lysate cell-free system much more productively compared to the same size RNAs obtained from normal infection. When these large size class RNAs were added together with HeLa cell mRNAs to the cell-free translational system, competition between the two types of mRNAs ensued. The small size class RNAs (POLADS) isolated from infection with VVuv or VVuv + VV, had little translational activity, but when added together with HeLa cell mRNAs, caused a striking inhibition of HeLa cell mRNA translation which was more pronounced than the inhibition caused by the small size class RNAs obtained from normal infections. POLADS obtained from infection with VV or VV + VVuv inhibited HeLa cell protein synthesis to the same extent and were about two times less active than the POLADS obtained from infection with VVuv or VVuv + VV. These results demonstrate that if cells are first infected with normal virus, the superinfecting UV-irradiated virus has little or no effect on the course of VV replication, suggesting that the production of POLADS under these conditions is regulated. However, when cells are first infected with VVuv, POLADS production is amplified to an "abnormal" level, thus, both viral polypeptide synthesis and replication of the superinfecting VV is compromised resulting in lower yields of virus.

Animals↗
PubMed2026

Metabolism pathway-based subtyping in pancreatic adenocarcinoma: an integrated study by bulk RNA-sequence and machine learning algorithms.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is highly aggressive, and its tumor microenvironment has significant metabolic and immune microenvironment complexity and genomic instability. In this study, by integrating the metabolic pathway activity score and clinical data, we constructed a novel risk assessment model to reveal the unique biological behavior and clinical significance behind different PAAD subtypes. METHODS: In this study, the transcriptome and clinical data of TCGA and GSE57495 databases were integrated to explore the interaction between metabolic pathways. Based on unsupervised clustering analysis of pathway activity and survival prognosis, patients with PAAD were classified into metabolic subtypes with significant prognostic differences. Subsequently, we assessed the heterogeneity of these subtypes in terms of clinical outcomes, genomic characteristics, and immune microenvironment composition. Based on the differentially expressed genes (DEGs) among metabolic subtypes, a clinical prognostic risk model and nomogram were constructed, which were double-validated by GSE57495-independent cohort and GSE57495 + TCGA-PAAD combined cohort. Finally, the correlations between risk scores (RSs) and signaling pathway activity and tumor immune microenvironment characteristics were evaluated. RESULTS: Based on metabolic pathway correlation and prognostic information, 240 patients in the TCGA-PAAD and GSE57495 datasets were divided into three subgroups. There were significant differences between subgroups in gene expression, pathway activity, clinical prognosis, and immune infiltration characteristics among the subtypes. Using machine learning algorithms, an RS model was constructed from DEGs among the subgroups, with the random forest method showing the best performance. A nomogram integrating the RS and clinical indicators demonstrated excellent predictive accuracy for 1-, 3-, and 5-year survival rates, confirming the RS as an independent prognostic factor. High- and low-risk groups exhibited significant differences in immune infiltration, pathway activity, and gene mutations. Drug sensitivity analysis showed that the high-risk group was more sensitive to AZD6244, ABT737, and other drugs. CONCLUSION: This study stratified patients with PAAD into three subgroups based on metabolic pathways and prognostic information, revealing significant differences in clinical outcomes, immune characteristics, and genetic mutations. The robust RS model developed from these findings demonstrated strong predictive power for patient survival and identified promising therapeutic strategies, providing valuable insights for advancing precision medicine in PAAD.

immune microenvironment↗