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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Various extracellular signals (i.e. transmitters, hormones, growth factors, etc.), together with their respective second-messenger pathways, regulate transmitter biosynthesis and neuronal function by altering gene expression. In this study we validated a protocol for isolating rat striatum and adrenal medullary nuclei for the purpose of extracting, identifying, and characterizing, nuclear regulatory factors which may serve a functional role in signal-transduction processes. Through gel retardation studies using a 299 base pair (bp) XmnI-SacI 32P-labeled probe (derived from the 5' untranslated region of the rat preproenkephalin gene), we show that different patterns of retained bands result from nuclear extracts derived from rat adrenal medulla and striatum (as well as from other tissue). These tissue differences may have biological significance since rat adrenal medullae have low basal enkephalin levels while the striatum has high levels of this peptide and its respective mRNA. Additionally, certain retained bands were common to both cytosolic and nuclear compartments, suggesting binding factors may be located in either cell space. An initial biochemical characterization of these factors was also undertaken. Generally, salt levels of 100 mM or more reduced factor binding while 10-50 mM sodium ion levels showed preferentially enhanced bands. Binding activity appeared optimal at pH 6.8. As all retained bands were abrogated by proteinase K treatment, these factors appear to have a significant protein component. Finally, of particular interest is that this 299 bp region contains many sequences showing over 80% sequence identity with several previously characterized transcriptional control elements (i.e. cAMP and phorbol ester inducible enhancers, GCN4, AP1, Sp1, CCAAT binding factor, ATF, and AP2). If binding is confirmed (footprint analysis) and function validated (transfection studies), the evolutionary significance of the apparent presence of gene regulatory sequences and functional element divergence of the DNA region between different species can be evaluated.
Brain Res Mol Brain Res 1989 Mar
PMID:Preproenkephalin DNA-binding proteins in the rat: 5' flanking region. 271 96

The requirement for TonB protein in a variety of membrane-related processes suggests that TonB is an envelope protein. Consistent with this suggestion, the deduced TonB amino acid sequence (Postle, K., and Good, R. F., (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5235-5239) contains an amino-terminal region similar to leader (signal) sequences of exported proteins, although its charged region falls outside the rules which characterize these sequences (von Heijne, G. (1985) J. Mol. Biol. 184, 99-105). The deduced TonB amino acid sequence contains three potential methionine start codons in the first six codons of the open reading frame. In this report, we show, by Edman degradation of [35S]methionine-labeled protein, that TonB protein synthesized in vitro initiates at the third of these methionine codons. A method for detecting TonB synthesized in vivo has been developed that involves expression of TonB from the lambda PL promoter and pulse labeling with [35S]methionine. TonB synthesized in vivo has a chemical half-life of 10 min at 42 degrees C. It is exported from the cytoplasm, as determined by proteinase K accessibility experiments. It fractionates with spheroplasts under conditions where maltose-binding protein fractionates with the periplasm. It has the same mobility in three different polyacrylamide gel systems as TonB synthesized in vitro. We concluded that the amino terminus of TonB is uncleaved following its export from the cytoplasm and that TonB is a membrane-associated protein. Characterization of a tonB-phoA gene fusion suggests that the amino-terminal 41 amino acids of TonB are sufficient to promote export of the fusion protein and presumably TonB as well. Models for TonB orientation within the cell envelope are presented.
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PMID:Escherichia coli TonB protein is exported from the cytoplasm without proteolytic cleavage of its amino terminus. 283 13

A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.
Mol Biol (Mosk)
PMID:[Rapid isolation of phage lambda DNA]. 285 52

ICR 2A frog cells and two solar ultraviolet (UV)-sensitive cell lines, DRP 36 and DRP 153, were irradiated with 150 kJ/m2 of the UV radiation produced by a fluorescent sun lamp, the radiation from which was passed through a sheet of 48A Mylar (DuPont, Wilmington, DE) to eliminate wavelengths shorter than approximately 315 nm. The irradiated cultures were also exposed to photoreactivating light (PRL), resulting in the removal of most of the pyrimidine dimers induced by the sun lamp UV irradiation, and then incubated 0-4 hr. At the end of the incubations, the cells were subjected to the alkaline elution assay. In these elutions, the cell lysates were either treated with proteinase K (proK) to eliminate any DNA-protein crosslinks (DPC) that may be present in the cells, or left untreated with proK. For the ICR 2A cells, the level of apparent DNA single-strand breaks (ssb) detected in elutions using proK increased with the incubation time after irradiation and remained high. However, when the DNA was eluted without proK pretreatment, the number of ssb fell rapidly. In contrast, the levels of ssb decreased in the DRP 36 and DRP 153 cells regardless of the use of proK in the elutions. Hence, this differential response in ssb induction may be indicative of a system involved with recovery following irradiation with solar UV wavelengths.
Environ Mol Mutagen 1989
PMID:DNA strand breakage in normal and solar ultraviolet-sensitive ICR 2A frog cell lines exposed to solar ultraviolet wavelengths. 291 Jul 4

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.
Mol Cell Biol 1989 Jan
PMID:Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. 292 99

In vitro transcription of the rat rRNA gene led to the identification of a region within a 3.4-kilobase fragment of the nontranscribed spacer (NTS) which significantly increased the transcription of rat ribosomal DNA. Promoter constructs containing this region were transcribed up to 17-fold more efficiently in vitro than templates with only 167 or 286 base pairs of NTS. This effect was also observed when the 3.4-kb fragment of the NTS was subcloned in the opposite orientation and 4 kb upstream of the promoter. The region responsible for the enhanced level of transcription was found between -286 and -1018. The results of order-of-addition experiments suggested that the enhanced level of transcription was the result of the formation of a stable complex between a trans-acting factor and the nontranscribed spacer. DNA-protein binding assays demonstrated that the same region of the NTS determined to have enhancer activity also specifically bound a proteinase K-sensitive factor present in nuclear extracts. The sequence of this region was not found to have any significant homology with the promoter of the rat rRNA gene. This is the first report to assign a transcriptional role to the NTS of a mammalian rRNA gene.
Mol Cell Biol 1986 Aug
PMID:Transcriptional role for the nontranscribed spacer of rat ribosomal DNA. 302 48

The membrane orientation of the NB protein of influenza B virus, a small (Mr, approximately 18,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.
Mol Cell Biol 1986 Dec
PMID:Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region. 302 52

Potassium chromate induced the formation of DNA-protein complexes in cultured Chinese hamster ovary cells. The DNA-protein complexes were isolated by ultracentrifugal sedimentation in the presence of 2% sodium dodecyl sulfate (SDS) and 5 M urea. Two-dimensional SDS-polyacrylamide gel electrophoresis analysis of the chromate-induced DNA-protein complexes revealed that two acidic proteins of 53 and 45 kDa and a basic protein of 54 kDa were selectively complexed to the DNA. Numerous other proteins also became associated with the DNA to a lesser degree as the chromate concentration was increased. Nuclease digestion was not a prerequisite for the resolution of the protein component of the DNA-protein complexes using two-dimensional gel electrophoresis. Ultracentrifugal analysis of the DNA-protein complexes in the presence of proteinase K, nucleases, or a chelating agent demonstrated that protein aggregation was not responsible for the increased protein recovery in chromate-treated samples and that the complexes were disrupted by EDTA. These data suggest that the selectively complexed proteins were associated with the DNA through strong interactions that may be mediated by the trivalent form of chromium.
Mol Carcinog 1988
PMID:Characterization of DNA-protein complexes induced in intact cells by the carcinogen chromate. 315 Dec 60

cDNA, synthesized on bovine coronavirus (BCV) genomic RNA templates, could be used to detect very small quantities (i.e. 1 pg) of viral RNA by hybridization with either radioisotopic-labelled or biotinylated recombinant plasmids. Virus was optimally attached to nitrocellulose membranes when spotted in 1 x SSC, whereas 20 x SSC was superior for viral RNA. Denaturation and RNA fixation of both RNA, still encapsidated in virus particles and isolated genomic RNA, was achieved by baking of the blots in vacuum. Virus detection in the supernatant of infected HRT-18 cells was feasible, but improved significantly after proteinase K treatment. No homology was observed between virus cDNA with either plasmid DNA or nucleic acid isolated from non-infected HRT-18 cells. Hybridization with radioisotopic-labelled probes in higher formamide concentrations (up to 60%) increased the detection signals, possibly by reducing reassociation of the probe. Significant detection amplification (30-50 times) was achieved in the case of biotinylated probes by stimulation of hyperpolymer formation on already hybridized target sequences, by additional hybridization with biotinylated pUC-19. A detection amplification was also obtained when hybridization was done with two probes (pBC-52 and pBC-247), containing non-overlapping viral sequences. Although the detectability was surpassed by biotinylated probes, sensitivity was superior in radioisotopic virus detection.
Mol Cell Probes 1988 Sep
PMID:Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus. 322 84

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.
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PMID:T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K. 327 1


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