Showing papers on "RNA published in 1995"
TL;DR: The Rev activation domain constitutes a nuclear export signal that redirects RRE-containing viral RNAs to a non-mRNA export pathway.
1,164 citations
TL;DR: It is suggested that an RNA encodes a soluble presynaptic protein that forms a predicted amphipathic alpha helix typical of the lipid-binding domain in apolipoproteins that may serve a novel function critical to the regulation of vertebrate neural plasticity.
834 citations
TL;DR: This system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative‐strand RNA genome.
Abstract: A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.
728 citations
TL;DR: This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway and indicates that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.
Abstract: The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.
692 citations
TL;DR: The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication and may be possible to genetically engineer recombinant VSVs displaying foreign antigens, which could be useful as vaccines conferring protection against other viruses.
Abstract: We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.
670 citations
TL;DR: An RNA chaperone hypothesis is presented, which fosters the view that constraints imposed by fundamental problems in the folding of RNA have profoundly influenced the nature of RNA/ protein interactions in biology, and provides a conceptual framework for RNA folding and RNA/protein interactions.
669 citations
TL;DR: The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes.
Abstract: The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest-neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single-stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blocker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors.
664 citations
TL;DR: In order to screen for brain region specific mRNAs which are transcriptionally regulated by acute cocaine and amphetamine, PCR differential display was employed and identified a previously uncharacterized mRNA whose relative levels in the striatum are induced four- to fivefold by acute psychomotor stimulant administration.
Abstract: involves alterations in specific patterns of gene expression. In order to screen for brain region specific mRNAs which are transcriptionally regulated by acute cocaine and amphetamine, PCR differential display was employed. This approach identified a previously uncharacterized mRNA whose relative levels in the striatum are induced four- to fivefold by acute psychomotor stimulant administration. Isolation and characterization of corresponding cDNA clones resulted in complete nucleotide sequence analysis, including prediction of the encoded protein product. Alternate polyA site utilization in the predicted 3′ noncoding region results in the appearance of an RNA doublet, approximately 700 and 900 bases in length, following Northern analysis. A presumed alternate splicing event further generates diversity within the transcripts, and results in the presence or absence of an in-frame 39 base insert within the putative protein coding region. As a result, the predicted translation products are either 129 or 116 amino acids in length. A common hydrophobic leader sequence at the amino terminus is present within each predicted polypeptide, suggesting that the protein product is targeted for entry into the secretory pathway. Basal expression of the RNA doublet is limited to neuroendocrine tissues, further implying that the protein product plays a functional role in both neuronal and endocrine tissues.
655 citations
Journal Article•
TL;DR: The modified precipitation does not prolong or increase the complexity of the TRI Reagent procedure and was tested by isolation of RNA from polysaccharide- and proteoglycan-rich tissues such as rat liver and aorta.
Abstract: A modification of the TRI Reagent procedure has been elaborated for isolation of RNA from polysaccharide- and proteoglycan-rich material. In the modified procedure, RNA is precipitated from the aqueous phase by the combined action of isopropanol and a high-salt concentration. Under these conditions, RNA is effectively precipitated while contaminating polysaccharides and proteoglycans remain in the soluble form. The modified precipitation does not prolong or increase the complexity of the TRI Reagent procedure. The new procedure was tested by isolation of RNA from polysaccharide- and proteoglycan-rich tissues such as rat liver and aorta.
620 citations
TL;DR: It is reported that eukaryotic ribosomes can initiate translation on circular RNAs, but only if the RNAs contain internal ribosome entry site elements.
Abstract: The ribosome scanning model predicts that eukaryotic ribosomal 40S subunits enter all messenger RNAs at their 5' ends. Here, it is reported that eukaryotic ribosomes can initiate translation on circular RNAs, but only if the RNAs contain internal ribosome entry site elements. Long-repeating polypeptide chains were synthesized from RNA circles with continuous open reading frames. These results indicate that ribosomes can translate such RNA circles for multiple consecutive rounds and that the free 5' end of a messenger RNA is not necessarily the entry point for 40S subunits.
609 citations
Patent•
TL;DR: In this article, purine-based compounds for inclusion into oligonucleotides are presented, which are especially useful as "antisense" agents, agents that are capable of specific hybridization with a nucleotide sequence of an RNA.
Abstract: This invention presents novel purine-based compounds for inclusion into oligonucleotides. The compounds of the invention, when incorporated into oligonucleotides are especially useful as "antisense" agents--agents that are capable of specific hybridization with a nucleotide sequence of an RNA. Oligonucleotides are used for a variety of therapeutic and diagnostic purposes, such as treating diseases, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins, and cleaving RNA in site specific fashions. The compounds of the invention include novel heterocyclic bases, nucleosides, and nucleotides. When incorporated into oligonucleotides, the compounds of the invention can be useful for modulating the activity of RNA.
Patent•
01 Feb 1995TL;DR: In this article, purine-based compounds for inclusion into oligonucleotides were proposed, which are especially useful as "antisense" agents, agents that are capable of specific hybridization with a nucleotide sequence of an RNA.
Abstract: This invention is directed to novel purine-based compounds for inclusion into oligonucleotides. The compounds of the invention, when incorporated into oligonucleotides are especially useful as 'antisense' agents -- agents that are capable of specific hybridization with a nucleotide sequence of an RNA. The compounds of the invention may also be used for cross-linking oligonucleotides. Oligonucleotides are used for a variety of therapeutic and diagnostic purposes, such as treating deseases, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins, and cleaving RNA in site specific fashions. The compounds of the invention include novel heterocyclic bases, nucleosides, and nucleotides. When incorporated into oligonucleotides, the compounds of the invention can be useful for modulating the activity of RNA.
TL;DR: The present review summarizes key progress made in characterizing the small nucleolar RNAs (snoRNAs) of eukaryotic cells.
Abstract: The present review summarizes key progress made in characterizing the small nucleolar RNAs (snoRNAs) of eukaryotic cells. Recent studies have shown snoRNA populations to be substantially more complex than anticipated initially. Many newly discovered snoRNAs are synthesized by an intron-processing pathway, which provides a potential mechanism for coordinating nuclear RNA synthesis. Several snoRNAs and snoRNP proteins are known to be needed for processing of ribosomal RNA, but precise functions remain to be defined. In principle, snoRNAs could have several roles in ribosome synthesis including: folding of pre-rRNA, formation of rRNP substrates, catalyzing RNA cleavages, base modification, assembly of pre-ribosomal subunits, and export of product rRNP particles.
TL;DR: This analysis of intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls indicated that this concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.
Abstract: We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different fluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.
TL;DR: Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.
Abstract: A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.
TL;DR: Analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the Higgs boson-like AU-rich motif targets mRNA for rapid deadenYLation as an early step in the mRNA decay process.
Abstract: Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process.
TL;DR: Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome, rendering the biology of VSV fully accessible to genetic manipulation of theiral genome.
Abstract: Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.
TL;DR: It would be surprising if some of the variety of poly P functions observed in microorganisms did not apply to aspects of human growth and development, such as aging and the aberrations of disease.
Abstract: Pursuit of the enzymes that make and degrade poly P has provided analytic reagents which confirm the ubiquity of poly P in microbes and animals and provide reliable means for measuring very low concentrations. Many distinctive functions appear likely for poly P, depending on its abundance, chain length, biologic source, and subcellular location. These include being an energy supply and ATP substitute, a reservoir for Pi, a chelator of metals, a buffer against alkali, a channel for DNA entry, a cell capsule and, of major interest, a regulator of responses to stresses and adjustments for survival in the stationary phase of culture growth and development. Whether microbe or human, we depend on adaptations in the stationary phase, which is really a dynamic phase of life. Much attention has been focused on the early and reproductive phases of organisms, which are rather brief intervals of rapid growth, but more concern needs to be given to the extensive period of maturity. Survival of microbial species depends on being able to manage in the stationary phase. In view of the universality and complexity of basic biochemical mechanisms, it would be surprising if some of the variety of poly P functions observed in microorganisms did not apply to aspects of human growth and development, such as aging and the aberrations of disease. Of theoretical interest regarding poly P is its antiquity in prebiotic evolution, which along with its high energy and phosphate content make it a plausible precursor to RNA, DNA, and proteins. Practical interest in poly P includes many industrial applications, among which is its use in the microbial depollution of P1 in marine environments.
TL;DR: The results establish that K88 encodes an IFN-inducible ds RNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specificAdenosineDeaminase occur in human cells.
Abstract: A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.
Journal Article•
TL;DR: It appears that no organism is immune to the effects of nonsense codons on mRNA abundance, and challenges for the future include identifying the gene products and RNA sequences that function in nonsense mediated RNA loss, and resolving the cause and consequences of there apparently being more than one cellular site and mechanism for nonsense-mediated RNA loss.
Abstract: It appears that no organism is immune to the effects of nonsense codons on mRNA abundance. The study of how nonsense codons alter RNA metabolism is still at an early stage, and our current understanding derives more from incidental vignettes than from experimental undertakings that address molecular mechanisms. Challenges for the future include identifying the gene products and RNA sequences that function in nonsense mediated RNA loss, resolving the cause and consequences of there apparently being more than one cellular site and mechanism for nonsense-mediated RNA loss, and understanding how these sites and mechanisms are related to both constitutive and specialized pathways of pre-mRNA processing and mRNA decay.
TL;DR: It is shown that the rapid appearance of drug-resistant virus in serum is followed by an increase in viral RNA load, which is similar to that seen in patients treated with the reverse transcriptase inhibitor lamivudine.
Abstract: The effect of the appearance of drug-resistant human immunodeficiency virus type 1 (HIV-1) on viral RNA load was studied in patients treated with the reverse transcriptase inhibitor lamivudine. During the first 12 weeks of treatment, HIV-1 RNA concentrations and amino acid changes in codon 184, causing high-level resistance to lamivudine, were determined in longitudinal serum samples from HIV-1 p24 antigen-positive and -negative patients. A marked decline in the amount of HIV-1 RNA (approximately 95% below baseline) and HIV-1 p24 antigen was observed within 2 weeks, followed by a rise that coincided with the appearance of lamivudine-resistant viruses in serum (isoleucine mutants initially, which were subsequently replaced by valine variants). After 12 weeks, a partial antiviral effect was observed despite the presence of a complete codon 184 mutant virus population in serum. This study shows that the rapid appearance of drug-resistant virus in serum is followed by an increase in viral RNA load.
TL;DR: In db/db and lesioned mice, quantitative differences in expression level of ob RNA correlated with adipocyte lipid content, suggesting that both the db gene and the hypothalamus are downstream of the ob gene in the pathway that regulates adipose tissue mass.
Abstract: The gene product of the recently cloned mouse obese gene (ob) is important in regulating adipose tissue mass. ob RNA is expressed specifically by mouse adipocytes in vivo in each of several different fat cell depots, including brown fat. ob RNA is also expressed in cultured 3T3-442A preadipocyte cells that have been induced to differentiate. Mice with lesions of the hypothalamus, as well as mice mutant at the db locus, express a 20-fold higher level of ob RNA in adipose tissue. These data suggest that both the db gene and the hypothalamus are downstream of the ob gene in the pathway that regulates adipose tissue mass and are consistent with previous experiments suggesting that the db locus encodes the ob receptor. In db/db and lesioned mice, quantitative differences in expression level of ob RNA correlated with adipocyte lipid content. The molecules that regulate expression level of the ob gene in adipocytes probably are important in determining body weight, as are the molecules that mediate the effects of ob at its site of action.
TL;DR: Pretreatment serum HCV RNA levels and HCV genotype are the main and independent factors associated with sustained response to IFN therapy and these factors are significantly different in the three response groups.
TL;DR: Using this highly sensitive RT-PCR during the follow-up of CML patients, a signal was unexpectedly found in healthy controls and showed the presence of bcr-abl transcript in the blood of 22 of 73 healthy adults and in theBlood of 1 of 22 children but not in 22 samples of umbilical cord blood.
TL;DR: Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses.
TL;DR: These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.
Abstract: Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.
TL;DR: In this paper, the relative abundance of each individual tRNA isoacceptor in Escherichia coli can be altered by varying its cognate codon concentration, which results in a loss of ribosomes and protein synthetic capacity.
Abstract: We attempted to test the idea that the relative abundance of each individual tRNA isoacceptor in Escherichia coli can be altered by varying its cognate codon concentration. In order to change the overall codon composition of the messenger pool, we have expressed in E. coli lacZ with the aid of T7 RNA polymerase so that their respective gene products individually accounted for 30% of the total bacterial protein. Unexpectedly, the maximum expression of either test gene has no specific effect on the relative rates of synthesis of the tRNA species that we studied. Instead, we find that there is a cumulative breakdown of rRNAs, which results in a loss of ribosomes and protein synthetic capacity. After either of the test genes is maximally induced, there is a growing fraction of protein synthesis invested in beta-galactosidase or delta tufB that is matched by a comparable decrease of the fraction of normal protein synthesis. We have also observed enhanced accumulation of two heat shock proteins during overexpression. Finally, after several hours of overexpression of either test protein, the bacteria are no longer viable. These results are relevant to the practical problems of obtaining high expression levels for cloned proteins.
TL;DR: The 3-D structures of HIV-1 and MoMuLV NC proteins, deduced from NMR studies, are characterized by a central globular domain with one (MoMuLV) or two (HIV-1) zinc fingers, which should facilitate a rational approach of new anti-HIV therapies based on inhibition of NC protein functions.
TL;DR: A Mg(2+)-dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approximately 10(5)-fold greater than that of the uncatalyzed reaction, raising the possibility that DNA enzymes might be made to operate in vivo.
TL;DR: It is established that H19 and Igf2 utilize the same endoderm enhancers, but on different parental chromosomes, as shown in mice inheriting the enhancer deletion from fathers.
Abstract: The distal end of mouse Chromosome 7 contains four tightly linked genes whose expression is dependent on their parental inheritance. Mash-2 and H19 are expressed exclusively from the maternal chromosome, whereas Insulin-2 (Ins-2) and Insulin-like growth factor 2 (Igf2) are paternally expressed. The identical expression during development of the 3'-most genes in the cluster, Igf2 and H19, led to the proposal that their imprinting was mechanistically linked through a common set of transcriptional regulatory elements. To test this hypothesis, a targeted deletion of two endoderm-specific enhancers that lie 3' of H19 was generated by homologous recombination in embryonic stem cells. Inheritance of the enhancer deletion through the maternal lineage led to a loss of H19 gene expression in cells of endodermal origin, including cells in the liver, gut, kidney, and lung. Paternal inheritance led to a very similar loss in the expression of Igf2 RNA in the same tissues. These results establish that H19 and Igf2 utilize the same endoderm enhancers, but on different parental chromosomes. Mice inheriting the enhancer deletion from fathers were 80% of normal size, reflecting a partial loss-of-function of Igf2. The reduction was uniformly observed in a number of internal organs, indicating that insulin-like growth factor II (IGFII), the product of Igf2, acts systemically in mice to affect prenatal growth. A modest decline in Ins-2 RNA was observed in the yolk sac. In contrast Mash-2, which is expressed in spongiotrophoblast cells of the placenta, was unaffected by the enhancer deletion.