Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribozymes are RNA molecules that cleave other RNA molecules. Thus, ribozymes offer a new way of inhibiting expression of specific genes whose nucleotide sequences are known. Intracellular stability of ribozymes is an important factor for their efficacy. We previously showed that hammerhead ribozyme directed against mRNA of tumour necrosis factor alpha (TNF alpha) slowly acquires resistance to degradation in cultured human cells. In order to explain this resistance, we now report on endogenous cellular protein(s) that bind to TNF alpha-ribozyme (TNF alpha-Rz) in solution to form stable complexes during native gel electrophoresis. Suppression of the effects of ribonucleases in the cytoplasmic extracts allowed approximately 80% of the input ribozyme RNA to be recovered in the form of complexes, indicating that complex formation protected the ribozyme from degradation. Treatment of the ribozyme-protein complexes with proteinase K prior to electrophoresis led to the recovery of full-length ribozyme. Interestingly, ribozyme-protein complexes retained cleavage activity, suggesting that the binding is in reversible equilibrium. Analysis of protein cytoplasmic extracts for binding to sub-fragments of TNF alpha-Rz demonstrated that protein binds to a conformational epitope formed by an interaction between the 5' end of TNF alpha-Rz and its catalytic domain. Competition of the ribozyme-protein binding with a ribozyme construct containing DNA instead of RNA at the 5' end, indicated that the ribose phosphate backbone of the 5' end is required for strong binding. The protein responsible for the formation of the complex with low electrophoretic mobility was found to be specific for the TNF alpha-Rz, since ribozyme for HIV-1 integrase gene (Int-Rz), or for human interleukin-2 (IL2-Rz) did not compete significantly with the TNF alpha-Rz binding. Covalent linkage of the IL2-Rz to the 3' end of TNF alpha-Rz, or to the proposed RNA protein binding site conferred protein binding and enhanced the stability and activity of the chimeric molecules. The RNA epitope identified in this study, through its endogenous protein binding, may serve as an oligonucleotide cassette for enhancing the in vivo stability and activity of other RNA molecules in general. This RNA epitope will also be useful in the study of RNA-protein interactions.
J Mol Biol 1994 Oct 07
PMID:Interaction between tumour necrosis factor alpha ribozyme and cellular proteins. Involvement in ribozyme stability and activity. 793 19

The detection limits of the polymerase chain reaction (PCR) for Mycoplasma pneumoniae were determined using specimens from persons known to have had M. pneumoniae pneumonia. Four primers were selected from the known sequence of the P1 gene. The primer pair (P1-178 and P1-809) which generates a 631 fragment gave the lowest detection limit. Nineteen of 21 throat swabs, which contained between 0.06 and 2 colony-forming units (CFU) per microlitre, from culture positive patients, were positive by PCR. The fact that M. pneumoniae grows in broth culture in spherules causes problems for determining the number of CFU detected in PCR. Filtering broth cultures through a 0.6 micron polycarbonate filter increased the number of CFUs two-to-ten-fold compared to unfiltered cultures. The lysis method needed to assay throat swabs differed from that necessary for broth cultures in that proteinase K treatment for 18 h increased the detection limit 10- to 100-fold when compared to NaOH digestion.
Mol Cell Probes 1994 Apr
PMID:Evaluation of the detection limits of PCR for identification of Mycoplasma pneumoniae in clinical samples. 793 10

The development of techniques allowing the unattended collection of RNA from cell samples at room temperature makes practical accurate and facile monitoring of circadian rhythms in Chlamydomonas reinhardtii. The utility of these methods was demonstrated by collecting RNA samples for three days from cells maintained in continuous darkness. Every hour, cells were automatically collected and lysed with buffer containing SDS and proteinase K. Samples were maintained at room temperature with little or no evidence of degradation of RNA. Strong, non-damping circadian rhythms of cab mRNA abundance were measured. Free-running rhythms of about 24 h were measured from cultures maintained at 16, 20, 25 and 30 degrees C, thus demonstrating temperature compensation of circadian period. Simultaneous collections from cultures previously entrained to 12 h light/12 h dark cycles of opposite phase displayed circadian rhythms of cab mRNA abundance that were in phase with their previous entraining light cycles. Thus, this result suggests that the measured circadian rhythms of cab mRNA abundance was not an artifact of the collection procedure.
Plant Mol Biol 1994 Oct
PMID:Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii. 794 76

Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The delta V17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB delta V17. Molecular modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonB delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the delta V17 mutation interrupted productive TonB-ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB delta V17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonB delta V17 export. Unlike the tonB delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.
Mol Microbiol 1994 Aug
PMID:Partial suppression of an Escherichia coli TonB transmembrane domain mutation (delta V17) by a missense mutation in ExbB. 799 75

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.
Mol Cell Biol 1994 May
PMID:An estrogen-inducible protein binds specifically to a sequence in the 3' untranslated region of estrogen-stabilized vitellogenin mRNA. 816 68

A PCR assay was developed to detect human rhinovirus (HRV) RNA in nasal washings from individuals experimentally infected with HRV-39 or HRV strain Hanks. Total RNA was purified from samples stored in the presence of vanadyl ribonucleoside complex (VRC) by one of two methods: proteinase K digestion followed by multiple extractions with phenol/chloroform (PK-PC); or denaturation with guanidinium thiocyanate followed by one phenol/chloroform extraction (GTC). The limit of detection of HRV in nasal washings spiked with HRV-39 was lower with the GTC method (1 TCID50) than with the PK-PC method. In a study of 31 nasal washings extracted by the PK-PC method, the sensitivity (93%) and negative predictive value (94%) were sub-optimal in comparison to cell culture. In a study of 60 nasal washings extracted by the GTC method, the number of samples positive by PCR (25) exceeded by two the number positive by isolation in cell culture. A GTC-based method for HRV RNA extraction in nasal washings was superior to a proteinase K-phenol/chloroform-based method in regard to sensitivity, consumption of reagents, material and time.
Mol Cell Probes 1993 Oct
PMID:Detection of human rhinovirus RNA in nasal washings by PCR. 826 71

As part of a retrospective study into the prevalence of the t(14;18) translocation in B-cell lymphomas, we assessed the suitability of the polymerase chain reaction (PCR) to amplify the t(14;18) major breakpoint region (MBR) in frozen and formalin-fixed tissue. Considering Southern blotting as a standard, the sensitivity of PCR was 81%. Of the various procedures used to extract DNA from paraffin-embedded tissue (PET), proteinase K digestion in the presence of nonionic detergents gave the highest yield and quality of DNA and the most efficient amplification rate. Using this method, excellent amplification rates (100%) were obtained for both the beta-globin control sequence and the MBR t(14;18) for fixed follicular lymphoma specimens collected in the previous 2 to 6 years (n = 27). Of nine older PETs, PCR on six gave inconsistent results, probably because of the poorer-quality substrate used for amplification. Specimens exposed to formol sublimate or formalin-acetic acid-alcohol were as suitable for amplification as tissues fixed in neutral-buffered formalin. The overall incidence of the MBR t(14;18) in all follicular lymphoma specimens as detected by both Southern blotting and PCR was 59% (23 of 39).
Diagn Mol Pathol 1993 Jun
PMID:Detection of the t(14;18) translocation in frozen and formalin-fixed tissue. 826 84

The oversynthesis of the secreted alkaline phosphatase (PhoA) in E.coli K12802 cells due to transformation with the PhoA+ plasmid pHI-7 leads to a change in its biogenesis--alternative localization and accumulation of the enzyme intermediate forms corresponding to different stages of the its post-translational modification. Instead of the soluble PhoA available in the parent strain mostly as a completely processed mature metazyme III localized in the periplasm, five enzyme forms were discovered in the PhoA overproducer: a cytoplasmic PhoA precursor (prePhoA) as insoluble aggregates; three soluble metazymes of a mature active form localized in the periplasm as in well as in culture medium; and a soluble high-molecular form in the periplasm. PrePhoA was isolated and purified by removal of soluble cell fractions using differential centrifugation, solubilization of membrane proteins with Triton X100, dissolution of the aggregates in the buffer with 8M urea and FPLC on MonoQ. Extracellular PhoA was purified by ultrafiltration, thermal treatment, and gel chromatography on Sepharose CL-4B. It was shown that the isolated prePhoA can be transformed into a mature form in the presence of a leader peptidase in 0.8 urea and is completely cleaved with proteinase K. Three forms of the mature PhoA vary in resistance to proteinase K and trypsin. Metazyme I, the unprocessed mature PhoA, is the most resistant to proteolysis.
Mol Biol (Mosk)
PMID:[Features of the biogenesis of Escherichia coli alkaline phosphatase during its supersynthesis]. 836 88

A sample of 171 natural isolates of Neurospora crassa and Neurospora intermedia was tested for senescence. Of these, 28 strains senesced within the duration of the experiment. These senescent strains, together with a selection of nonsenescent strains, were examined for the presence of mitochondrial plasmids. This was done by digesting mitochondrial DNA preparations with proteinase K, and running these samples on agarose gels. Most of the strains examined, both senescent and nonsenescent, contained plasmids, many of them new. Some new plasmids were linear, as inferred from their resistance to 5' exonuclease and sensitivity to 3' exonuclease. New circular plasmids were also found. Some strains carry several plasmids, and mixtures of circular and linear elements were common. A cross-homology study was performed on a sample of plasmid-bearing strains, and several cases of apparent relatedness were found, some between strains from distant geographical locations. Linear plasmids homologous to the maranhar linear senescence plasmid were quite common. A new member of the LaBelle circular plasmid homology group was found. In the sample tested for homology, no strains contained elements related to the kalilo linear senescence plasmid. The relationship of the new plasmids to senescence is not known. In addition to plasmid monomers, several different types of derivatives were found. The kalilo linear plasmid was found to occur in linear and circular forms of low mobility, presumed to be giant concatamers, and, in some strains, variant sibling structures and ladders of short derivatives were found. Circular plasmids also gave rise to extensive ladders on electrophoresis, probably representing different relaxation states and head-to-tail concatameric series. Some such forms migrated more slowly than mitochondrial DNA. One unique type of plasmid modification observed was a pair of linear elements that had apparently arisen de novo which showed homology to a circular plasmid.
Mol Gen Genet 1993 Feb
PMID:Plasmid diversity in senescent and nonsenescent strains of Neurospora. 845 55

Four temperature-sensitive mutations in the divIB gene of Bacillus subtilis have been localized to the region corresponding to the C-terminal half of the 263-residue DivIB protein. Antiserum was raised to the 80% C-terminal portion lying on one side of a putative transmembrane (hydrophobic) segment, and used to examine aspects of the nature and localization of the DivIB protein in the cell. A single DivIB species of a size equal to the full-length protein encoded by the divIB gene was detected in wild-type cells. Cell fractionation studies established that DivIB is associated preferentially with the cell envelope (membrane plus cell wall), with approximately 50% being released into solution upon treatment of cells with lysozyme under conditions that yield protoplasts. Of the remaining 50%, approximately half remained firmly associated with the membrane fraction. On the basis of the 'positive-inside rule' of von Heijne (1986) it is suggested that the topology of membrane-bound DivIB is such that the long C-terminal portion is directed to the outside and the smaller N-terminal portion to the inside of the cell. DivIB in protoplasts was rapidly degraded by proteinase K under conditions where there was no general proteolysis of the cytoplasmic proteins. This is consistent with its absence from the cytoplasm, and with the predicted membrane topology. Septum positioning in a divIB null mutant, which grows as filaments at temperatures of 30 degrees C and below, was found to be normal. It appears that DivIB is needed for achieving the appropriate rate of initiation of septum formation at normal division sites. It is proposed that the C-terminal portion of DivIB, localized on the exterior surface of the membrane and in juxtaposition to the peptidoglycan, normally interacts with another protein (or proteins) to initiate septum formation.
Mol Microbiol 1993 Feb
PMID:Characterization of mutations in divIB of Bacillus subtilis and cellular localization of the DivIB protein. 845 77


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