Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNase protection-gel retention studies show human host cell-specific ribonucleoprotein complexes with human immunodeficiency virus type 1 Rev-responsive element (RRE) RNA. Nuclear proteins from rodent or murine cells appear to lack the ability to form these complexes. Human-mouse somatic cell hybrids retaining a single human chromosome, either 6 or 12, form the RRE-nuclear-protein complexes. One of the complexes requires the entire RRE RNA, while the other needs RRE RNA stem-loops 1 and 2 only. Two major proteins with molecular masses of 120 and 62 kDa specifically bind to RRE RNA. Rodent cells (CHO) either lack or contain small amounts of these RRE-binding proteins.
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PMID:Human immunodeficiency virus type 1 Rev-responsive element RNA binds to host cell-specific proteins. 813 7

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.
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PMID:Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. 818 16

The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.
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PMID:Tat-mediated delivery of heterologous proteins into cells. 829 May 79

In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional structures were determined by x-ray crystallography. Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site. The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges. These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases.
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PMID:Crystal structures of ribonuclease HI active site mutants from Escherichia coli. 840 67

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes.
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PMID:Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture. 847 82

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.
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PMID:Isolation of virus-neutralizing RNAs from a large pool of random sequences. 852 93

We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.
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PMID:Three functional classes of transcriptional activation domain. 862 70

Specific binding of the human immunodeficiency virus Tat protein to its RNA site (TAR) is mediated largely by a single arginine residue located within a basic region of the protein. Many essential features of the interaction can be mimicked by the free amino acid arginine, and an NMR model has been proposed in which the arginine guanidinium group binds to a guanine base in the major groove and to two phosphates adjacent to a bulge, with the RNA structure stabilized by a base triple between a U in the bulge and an adjacent A:U base pair. To compare the TAR structure to other arginine-binding RNAs, we performed in vitro selection experiments and identified RNAs with arginine-binding affinities similar to TAR. About 40% of the selected RNAs contained the same motif found in TAR: two stems separated by a bulge of at least two nucleotides, a U at the 5' position of the bulge, and G:C and A:U base pairs above the bulge. In many cases, the upper stems contained only the G:C and A:U pairs, located next to small loops. Chemical modification experiments demonstrated that these "TAR-like" RNAs bound arginine in a manner similar to TAR, and in some cases identified nucleotides outside the binding site that contributed to binding. To explore how small loops might help stabilize the structures of adjacent arginine-binding sites, we measured arginine-binding affinities of TAR-like RNAs having all possible three-nucleotide loops. An RNA with a UAG loop bound with highest affinity, and chemical modification and RNase mapping experiments suggested that the RNA changes conformation upon arginine binding, converting a large unstructured loop into a bulge conformation related to that of TAR. The results suggest that the arginine-binding site in TAR is structurally versatile and demonstrate how binding can be modulated by the surrounding RNA context.
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PMID:Arginine-binding RNAs resembling TAR identified by in vitro selection. 865 64

To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3' terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozyme cleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to serve export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a long stretch of poly(A) near the 3' end of a ribozyme-cleaved transcript is not sufficient for directing mRNA export. Taken together, these results suggest that polyadenylation is required for the nucleocytoplasmic transport of mRNA and that Rev interaction with the Rev-responsive element cannot bypass this requirement.
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PMID:Role of polyadenylation in nucleocytoplasmic transport of mRNA. 865 27

A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding domain allowing target RNA/DNA recognition was fused to a (deoxy)ribonucleolytic domain allowing target RNA/ DNA cleavage. The design and construction of such a chimeric enzyme could be of value for both basic research involving structure-function relationships and applied research requiring inactivation of harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of this designer nuclease approach for inactivating specific RNA/DNA molecules was assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model. Trans-activator of transcription (Tat) protein is one of the key regulatory proteins encoded by HIV-1. It binds to the trans-activation-responsive (TAR) RNA element located within the 5' non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA in vitro. Cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA.
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PMID:Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro. 865 73


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