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At least 667 records · Page 37Linked to original sources
PubMed1998

In vitro processing of cucumber chloroplast tRNA(Leu)(CAA) precursor in a pea chloroplast soluble extract.

To study the sequential steps in the processing pathway of the chloroplast monocistronic intronless tRNA precursors, we examined cucumber chloroplast tRNA(Leu)(CAA) processing in a cucumber or pea chloroplast soluble extract. The tRNA(Leu)(CAA) precursor synthesized from SP6 RNA polymerase-directed transcription system, was used as a substrate. Incubation of the tRNA precursor with the pea extract resulted in processing of tRNA(Leu)(CAA) via 5'- and 3'-endonucleolytic cleavages followed by final trimming of extra 3' nucleotides by 3' exonuclease(s). No preferred order for endonucleolytic cleavages has been observed during the in vitro tRNA(Leu) processing and the simultaneous occurrence of the intermediates consisting of leader + tRNA(Leu) and tRNA(Leu) + trailer, indicate that either 5'- or 3'-endonucleolytic cleavage can occur as the first step in vitro.

Cell Extracts↗
PubMed1996

A purified Drosophila septin complex forms filaments and exhibits GTPase activity.

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.

Actin Cytoskeleton↗
PubMed1992

Cloning and characterization of the cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kD protein.

About 50% of patients with the polymyositis-scleroderma overlap syndrome are reported to have autoantibodies to a nucleolar particle termed PM/Scl. The particle consists of several polypeptides of which two proteins of 75 and 100 kD have been identified as the major antigenic components. Here we report on the cDNA cloning and partial epitope mapping of the 100-kD autoantigen from human placenta and HeLa lambda gt11 libraries. The deduced amino acid sequence encodes a protein of 885 amino acid residues with a molecular mass of 100.8 kD. Rabbit antibodies raised against a recombinant protein fragment reacted in immunofluorescence and immunoblotting in the same manner as human autoantibodies directed against the nucleolar 100-kD protein. Sequence analysis shows close homology to a consensus sequence of 12 amino acids from serine/threonine kinases, suggesting a possible function for this autoantigen. A major antigenic region is found to be located within the NH2-terminal third of the polypeptide.

Amino Acid Sequence↗
PubMed1993

Folding and assembly of major histocompatibility complex class I heterodimers in the endoplasmic reticulum of intact cells precedes the binding of peptide.

Major histocompatibility complex (MHC) class I molecules are heterotrimers consisting of a polymorphic H chain, beta 2-microglobulin (beta 2m) and peptide. Peptides are thought to associate early during biosynthesis but the order of assembly of class I molecules from their component subunits in intact cells is not settled. We have studied the assembly of MHC class I molecules in intact cells with or without peptide transporters. MHC class I H chain/beta 2m heterodimers can be efficiently recovered only 4 min after translation and are preceded by a folding intermediate. Approximately 2 min after their formation, the class I heterodimers are loaded with peptides resulting in stable class I heterotrimers. In these in vivo studies, no evidence was obtained that peptide binding to the H chain preceded the association with beta 2m. In contrast, nonassembled class I H chains could be recovered immediately after translation, but this pool did not participate in the formation of class I molecules.

Biological Transport↗
PubMed2003

Efficient lentiviral vectors for short hairpin RNA delivery into human cells.

RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.

Cell Line↗
PubMed2005

Frequent 14-3-3 sigma promoter methylation in benign and malignant prostate lesions.

14-3-3Sigma is a putative tumor suppressor gene involved in cell cycle regulation and apoptosis following DNA damage. 14-3-3Sigma loss of expression has been reported is several human cancers, including prostate adenocarcinoma and precursor lesions, and promoter hypermethylation has been proposed as the mechanism underlying gene silencing. Here, we investigate the frequency and extent of 14-3-3sigma promoter methylation in benign and cancerous prostate tissues. We examined tumor tissue from 121 patients with prostate carcinoma (PCa), 39 paired high-grade prostatic intraepithelial neoplasias (HGPIN), 29 patients with benign prostate hyperplasia (BPH), as well as four prostate cancer cell lines using quantitative methylation-specific PCR (QMSP). The percentage of methylated alleles (PMA) was calculated and correlated with clinical and pathological parameters. RT-PCR was performed in the cell lines to assess 14-3-3sigma mRNA expression. PCa, HGPIN, BPH, and cancer cell lines showed ubiquitous 14-3-3sigma promoter methylation. However, the PMA of HGPIN was significantly lower than that of PCa or BPH (P < 0.0001), while PCa and BPH did not significantly differ. The PMA did not correlate with any clinicopathological parameter. All prostate cancer cell lines expressed 14-3-3sigmamRNA. 14-3-3Sigma promoter methylation is a frequent event in prostate tissues and cancer cell lines. Furthermore, there is a progressive accumulation of neoplastic cells with 14-3-3sigma methylated alleles from HGPIN to PCa, suggesting a role for this epigenetic event in prostate carcinogenesis. However, other mechanisms besides promoter methylation might be required for effective 14-3-3sigma downregulation.

14-3-3 Proteins↗
PubMed1985

Comparative studies on (2'-5')oligoadenylate-related enzyme systems and the antiviral effect of interferon in two mouse cell lines which differ in (2'-5')oligoadenylate sensitivity of their protein synthesizing system.

Effect of (2'-5')oligoadenylate (2-5A) on cellular and viral protein and RNA syntheses was investigated with two mouse cell lines, L929 and Lz (a subclone of L929). The oligonucleotide was introduced into the cells either by using calcium phosphate coprecipitation technique or by microinjection method. In L929 cells protein and viral RNA syntheses were severely inhibited by 2-5A, whereas in Lz cells, both were only slightly inhibited. The activities of 2-5A synthetase and double-stranded (ds)RNA-dependent protein kinase were enhanced by interferon (IFN) treatment roughly to the same extent and there was no significant difference in the level of 2'-5' phosphodiesterase activity either. On the other hand, 2-5A-dependent RNase (RNase L) activity in Lz cells was low, being about 10-20% of that of L929 cells. It was increased twofold after IFN treatment, but protein synthesis of Lz cells was not as sensitive to 2-5A as that of L929 cells even after IFN treatment. L929 and Lz cells were sensitive to the antiviral effect of mouse IFN against vesicular stomatitis virus (VSV) and Mengovirus. In contrast, however, Lz cells were relatively insensitive to the antiviral effect of IFN on vaccinia virus, whereas L929 cells were sensitive.

2',5'-Oligoadenylate Synthetase↗
PubMed1999

tRNAs and proteins are imported into mitochondria of Trypanosoma brucei by two distinct mechanisms.

Import of tRNA into the mitochondrial matrix of Trypanosoma brucei was reconstituted in vitro. Efficient import required the hydrolysis of externally added ATP and was shown to be a carrier-mediated process depending on proteinaceous receptors on the surface of mitochondria. A partly synthetic tRNA(Tyr) as well as a physiological tRNA(Lys) were imported along the same pathway. Contrary to import of all matrix-localized proteins, tRNA import does not require a membrane potential. Furthermore, addition of an excess of import-competent tRNA had no effect on import of a mitochondrial matrix protein. In summary, these results show that tRNAs and proteins in T. brucei are imported by fundamentally different mechanisms.

Adenosine Triphosphate↗
PubMed1995

Localization and possible functions of Drosophila septins.

The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface. We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins. The predicted Sep1 amino acid sequence is 35-42% identical to the known S. cerevisiae septins; 52% identical to Pnut, a second D. melanogaster septin; and 53-73% identical to the known mammalian septins. Sep1-specific antibodies have been used to characterize its expression and localization. The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation. Other aspects of Sep1 localization suggest roles not directly related to cytokinesis. For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells. The distribution of Sep1 typically overlaps, but is distinct from, that of actin. Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex.

Amino Acid Sequence↗
PubMed2004

Enhanced expression of 14-3-3sigma in pancreatic cancer and its role in cell cycle regulation and apoptosis.

14-3-3sigma belongs to the 14-3-3 family of proteins, which are involved in the modulation of diverse signal transduction pathways. Loss of 14-3-3sigma expression has been observed in a number of human cancers, suggesting that it may have a role as a tumor suppressor gene. The aim of the study was to investigate the expression and the functional role of 14-3-3sigma in pancreatic ductal adenocarcinoma (PDAC). Expression of 14-3-3sigma was analyzed using laser capture microdissection (LCM), quantitative real-time-PCR (QRT-PCR), DNA arrays, immunohistochemistry and western blot analysis. The role of 14-3-3sigma in apoptosis and cell cycle regulation was evaluated by western blotting, immunoprecipitation and FACS analysis. By QRT-PCR, 14-3-3sigma mRNA levels were 54-fold increased in pancreatic adenocarcinoma in comparison with normal pancreatic samples and localized in pancreatic cancer cells as determined by LCM. In pancreatic cancer cells, the degree of 14-3-3sigma expression was not decisive for the maintenance of G(2)/M cell cycle checkpoint or induction of apoptosis. Responses to radiation or apoptosis-inducing agents were neither accompanied by a significant 14-3-3sigma accumulation nor by a change in association of 14-3-3sigma with cdc2, bad and bax. In conclusion, the marked over-expression of 14-3-3sigma in PADC together with multiple known genetic and epigenetic alterations of potential 14-3-3sigma interacting partners suggests an important role of aberrant 14-3-3sigma downstream signaling in pancreatic cancer.

14-3-3 Proteins↗
PubMed1998

Cloning and characterization of human Sep1 (hSEP1) gene and cytoplasmic localization of its product.

We isolated and sequenced a human cDNA (designated as hSEP1) encoding both a homologue of mouse Dhm2 and budding yeast SEP1. The gene was shown to be located on the long arm of chromosome 3 (3q25-26.1). The putative hSEP1 product (hSEP1p) consisted of 1694 amino acid residues with a molecular mass of about 190 kDa. Northern blot analysis showed a major 10-kb mRNA expressed ubiquitously in various organs as well as a minor 5.5-kb mRNA expressed relatively highly in the testis and placenta. hSEP1p is localized in the cytoplasm as examined by cytochemical and Western blot analyses of fractionated cellular extracts. The biological function of hSEP1p was discussed in correlation with its cytoplasmic localization.

Amino Acid Sequence↗
PubMed2002

A complex prediction: three-dimensional model of the yeast exosome.

We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase). Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome. We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one. The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.

Amino Acid Sequence↗
PubMed1997

A ribonuclease specific for inosine-containing RNA: a potential role in antiviral defence?

RNA transcripts in which all guanosine residues are replaced by inosine are degraded at a highly accelerated rate when incubated in extracts from HeLa cells, sheep uterus or pig brain. We report here the partial purification and characterization of a novel ribonuclease, referred to as I-RNase, that is responsible for the degradation of inosine-containing RNA (I-RNA). I-RNase is Mg2+ dependent and specifically degrades single-stranded I-RNA. Comparison of the Km of the enzyme for I-RNA with the Ki for inhibition by normal RNA suggests a approximately 300-fold preferential binding to I-RNA, which can account for the specificity of degradation. The site of cleavage by I-RNase is non-specific; I-RNase acts as a 3'-->5' exonuclease generating 5'-NMPs as products. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. We show that I-RNase is able to degrade RNAs that previously have been modified by the RED-1 double-stranded RNA adenosine deaminase (dsRAD). dsRADs destabilize dsRNA by converting adenosine to inosine, and some of these enzymes are interferon inducible. We therefore speculate that I-RNase in concert with dsRAD may form part of a novel cellular antiviral defence mechanism that acts to degrade dsRNA.

Adenosine Deaminase↗
PubMed1998

Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3' end formation of 5.8S rRNA in Saccharomyces cerevisiae.

The temperature-sensitive mutation, dob1-1, was identified in a screen for dependence on overexpression of the yeast translation initiation factor eIF4B (Tif3p). Dob1p is an essential putative ATP-dependent RNA helicase. Polysome analyses revealed an under accumulation of 60S ribosomal subunits in the dob1-1 mutant. Pulse-chase labelling of pre-rRNA showed that this was due to a defect in the synthesis of the 5.8S and 25S rRNAs. Northern and primer extension analyses in the dob1-1 mutant, or in a strain genetically depleted of Dob1p, revealed a specific inhibition of the 3' processing of the 5.8S rRNA from its 7S precursor. This processing recently has been attributed to the activity of the exosome, a complex of 3'-->5' exonucleases that includes Rrp4p. In vivo depletion of Dob1p also inhibits degradation of the 5' external transcribed spacer region of the pre-rRNA. A similar phenotype was observed in rrp4 mutant strains and, moreover, the dob1-1 and rrp4-1 mutations show a strong synergistic growth inhibition. We propose that Dob1p functions as a cofactor for the exosome complex that unwinds secondary structures in the pre-rRNA that otherwise block the progression of the 3'-->5' exonucleases.

Cloning, Molecular↗
PubMed2001

The exosome of Trypanosoma brucei.

The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species. An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.

Alleles↗
PubMed2002

CCR4, a 3'-5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase.

The CCR4-NOT complex from Saccharomyces cerevisiae is a general transcriptional regulatory complex. The proteins of this complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation. The evolutionarily conserved CCR4 protein, which is part of the cytoplasmic deadenylase, contains a C-terminal domain that displays homology to an Mg2+-dependent DNase/phosphatase family of proteins. We have analyzed the putative enzymatic properties of CCR4 and have found that it contains both RNA and single-stranded DNA 3'-5' exonuclease activities. CCR4 displays a preference for RNA and for 3' poly(A) substrates, implicating it as the catalytic component of the cytoplasmic deadenylase. Mutations in the key, conserved catalytic residues in the CCR4 exonuclease domain abolished both its in vitro activities and its in vivo functions. Importantly, CCR4 was active as a monomer and remained active in the absence of CAF1, which links CCR4 to the remainder of the CCR4-NOT complex components. These results establish that CCR4 and most probably other members of a widely distributed CCR4-like family of proteins constitute a novel class of RNA-DNA exonucleases. The various regulatory effects of the CCR4-NOT complex on gene expression may be executed in part through these CCR4 exonuclease activities.

Amino Acid Sequence↗
PubMed2002

Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae.

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis. In addition, we demonstrate that at least some of the Ccr4p/Pop2p-associated Not proteins are cytoplasmic, and lesions in some of the NOT genes can lead to defects in mRNA deadenylation rates. The Ccr4p deadenylase is inhibited in vitro by addition of the poly(A) binding protein (Pab1p), suggesting that dissociation of Pab1p from the poly(A) tail may be rate limiting for deadenylation in vivo. In addition, the rapid deadenylation of the COX17 mRNA, which is controlled by a member of the Pumilio family of deadenylation activators Puf3p, requires an active Ccr4p/Pop2p/Not deadenylase. These results define the Ccr4p/Pop2p/Not complex as the cytoplasmic deadenylase in yeast and identify positive and negative regulators of this enzyme complex.

Binding Sites↗
PubMed2003

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase.

The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.

14-3-3 Proteins↗