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Query: HUMANGGP:012675 (S100)
 6,012 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Northern blot analysis of HeLa cell nuclear extract following electrophoresis in nondenaturing gels revealed that a small proportion of U2 small nuclear ribonucleoprotein (snRNP) displays a low mobility, in confirmation of previous reports. This low mobility form of U2 snRNP (termed LMC, for low mobility complex) also formed in vitro when U2 snRNP present in HeLa cytoplasmic S100 was added to a micrococcal nuclease-treated nuclear extract. Of greater experimental value, we found that the LMC also formed when a T7 U2 RNA transcript was assembled into U2 snRNP in a HeLa cytoplasmic S100 system, followed by its incubation in micrococcal nuclease-treated nuclear extract. LMC formation was ATP-dependent and was specific for U2 snRNP since it was not observed with S100-assembled U1 or U4 snRNPs. RNase H cleavage of U2 snRNP in the nuclear extract with an oligonucleotide complementary to nucleotides 28-42 of U2 RNA, as opposed to micrococcal nuclease treatment, rendered the extract competent to form the LMC, indicating that the nuclear factors responsible for LMC formation reside on endogenous U2 snRNP. LMC formation was not competed by excess U2 RNA but was competed by partially purified native U2 snRNP, providing further evidence that the LMC represents an interaction of nuclear factors with already assembled U2 snRNP. LMC formation did not take place on a mutant U2 snRNP lacking the binding site for the two U2-specific proteins, A' and B", nor on mutant U2 snRNPs lacking nucleotides 34-37 or nucleotides 46-49. Further results revealed that nucleotides 35 and 36 of U2 RNA, but not 34 and 37, are required for LMC formation. These experiments demonstrate a nucleotide sequence-specific interaction of U2 snRNP with nuclear factors in the absence of pre-mRNA. Among the U2 RNA nucleotides involved in the formation of this complex are ones previously implicated in base pairing between U2 RNA and the pre-mRNA lariat branch site. These findings are discussed in the context of the possibility that the LMC is on the spliceosome assembly pathway.
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PMID:The U2 small nuclear ribonucleoprotein particle associates with nuclear factors in a pre-mRNA independent reaction. 165 22

DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.
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PMID:Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor. 167 Oct 44

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.
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PMID:Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro. 171 77

An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 degrees C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5' cap analogue 7-methylguanosine 5'-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 degrees C.
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PMID:Efficient translation of synthetic and natural mRNAs in an mRNA-dependent cell-free system from the dimorphic fungus Candida albicans. 185 80

We identified a Mg2+ dependent 5' exo-ribonuclease and an RNA ligase in cell-free extracts of Trypanosome brucei. The exo-ribonuclease in S100 or nuclear extracts, removes about 20 nts from the 5' end of SP6 derived capped as well as uncapped RNA and then stops. In contrast to the activity of the exo-ribonuclease on capped SP6 mini-exon transcripts, the exonuclease cannot degrade trypanosome-derived mini-exon transcripts or the mini-exon located at hsp 70 mRNAs. We therefore assume that the four secondary base modifications adjacent to the mini-exon cap, generated in vivo, confer resistance to the exo-ribonuclease. After exonuclease shortening of SP6 transcripts, an RNA ligase catalizes intramolecular ligation, generating a 3'-5' phosphodiester bond in a Mg2+ and ATP dependent reaction.
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PMID:A 5' exo-ribonuclease and RNA ligase of T. brucei. 246 Aug 26

Incubation of a SP6-transcribed human U2 RNA precursor molecule in a HeLa cell S100 fraction resulted in the formation of ribonucleoprotein complexes. In the presence of ATP, the particles that assembled had several properties of native U2 snRNP, including resistance to dissociation in Cs2SO4 gradients, their buoyant density, and pattern of digestion by micrococcal nuclease. These particles also reacted with Sm monoclonal antibody and a human autoantibody with specificity for the U2 snRNP-specific proteins A' and B", but not with antibodies for U1 snRNP-specific proteins. In contrast, the particles that formed in the absence of ATP did not have these properties. ATP analogs with non-hydrolyzable beta-gamma bonds did not substitute for ATP in U2 snRNP assembly. Additional experiments with a mutant U2 RNA confirmed that nucleotides 154-167 of U2 RNA are required for binding of the U2 snRNP-specific proteins but not of the "Sm" core proteins. Pseudouridine formation, a major post-transcriptional modification of U2 RNA, was enhanced under assembly permissive conditions.
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PMID:U2 small nuclear RNP assembly in vitro. 274 38

We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.
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PMID:Autonomous splicing and complementation of in vivo-assembled spliceosomes. 292 Dec 83

Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5' cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.
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PMID:Identification of a functional mammalian spliceosome containing unspliced pre-mRNA. 297 71

Pro-OmpA that is synthesized in vitro can assemble into bacterial inner membrane vesicles in the presence of ATP and NADH. We have purified pro-OmpA to determine which additional soluble proteins are necessary for its membrane assembly. [35S]Pro-OmpA was bound to Sepharose-linked antibody to OmpA, then eluted with 8 M urea and chromatographed on an anion-exchange resin in 8 M urea. This pro-OmpA is purified 2000-fold and is radiochemically pure. After dialysis, it is soluble but incompetent for membrane assembly. Addition of an Escherichia coli cytoplasmic fraction (S100) to the assembly reaction does not allow translocation. However, when S100 is added to pro-OmpA prior to dialysis, full assembly competence is restored, suggesting that a soluble factor, termed "trigger factor," triggers the folding of pro-OmpA into an assembly-competent form as the urea is removed. We noted that, prior to the last purification step, the immunoaffinity-purified pro-OmpA was partially competent for membrane assembly without addition of trigger factor. To test whether trigger factor had bound to the antibody column by means of its association with pro-OmpA, the crude pro-OmpA was acid-denatured prior to immunoadsorption. In this experiment, the trigger factor did not bind to the anti-OmpA column, and S100 was required for renaturation of this [35S]pro-OmpA. As suggested by this experiment, the crude [35S]pro-OmpA was in a complex with other proteins. Sedimentation velocity studies showed that the trigger factor has an apparent molecular weight of approximately 60,000. We propose that it is required for translocation-competent folding of pro-OmpA and other precursor proteins.
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PMID:Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form. 329 81

Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.
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PMID:Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. 619 2


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