Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The location of the protein bound to bacteriophage phi29 DNA has been studied with restriction endonucleases, exonucleases, and polynucleotide kinase. The protein is invariably associated with the two terminal DNA fragments generated by restriction endonucleases. The phi29 DNA prepared with or without proteinase K treatment is resistant to the action of the 5'-terminal-specific exonucleases, lambda-exonuclease and T7 exonuclease. The phi29 DNA is also inaccessible to phosphorylation by polynucleotide kinase even after treatment with alkaline phosphatase. On the other hand, phi29 DNA is sensitive to exonuclease III, and the 3' termini of the DNA can be labeled by incubating with alpha-[32P]ATP and terminal deoxynucleotidyl transferase. The protein remains associated with the phi29 DNA after treatment with various chaotropic agents, including 8 M urea, 6 M guanidine-hydrochloride, 4 M sodium perchlorate, 2 M sodium thiocyanate, and 2 M LiCl. These results are consistent with the notion that the protein is linked covalently to the 5' termini of the phi29 DNA.
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PMID:Bacteriophage phi29 terminal protein: its association with the 5' termini of the phi29 genome. 73 97

We describe a whole mount in situ hybridization procedure to detect and localize low abundance transcripts (qmf1) and myosin heavy chain proteins in quail embryos. The critical factor in transcript detection was the extent of proteinase K digestion. Optimal digestion increased probe accessibility and reduced background. The use of digoxigenin-labeled probes and immunological detection with anti-digoxigenin-alkaline phosphatase conjugated antibody allowed signal development in 6-10 h, unlike the 7 days required using 35S-labeled probes. Myosin heavy chain proteins were detected using immunofluorescence and confocal laser scanning microscopy to allow precise localization of signal within developing embryos.
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PMID:Whole mount in situ detection of low abundance transcripts of the myogenic factor qmf1 and myosin heavy chain protein in quail embryos. 141 72

An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non-radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde-fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis.
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PMID:In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs. 171 91

A dot blot hybridization protocol was developed for the direct detection of molluscum contagiosum virus (MCV) DNA in clinical specimens submitted for virus isolation. Samples were concentrated by high-speed centrifugation and treated with proteinase K; this was followed by a single phenol-chloroform extraction step. The DNA was denatured, and the entire volume was spotted onto a nitrocellulose membrane. A biotinylated DNA probe specific for the BamHI-C region of MCV type 1 was used for hybridization. Evidence of MCV DNA was visualized by using streptavidin alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as the substrate. Results showed that nonspecific hybridization does not occur with herpes simplex virus- or orf virus-infected clinical specimens and that dot blotting is more sensitive and reproducible than electron microscopy.
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PMID:Direct detection of molluscum contagiosum virus in clinical specimens by dot blot hybridization. 177 21

Antigens extracted from Cryptosporidium oocysts, which had been purified from faeces or chick egg culture, were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels, and blotted onto nitrocellulose membranes. A Cryptosporidium genus-specific monoclonal antibody MAb-C1 bound to multiple bands using several detection techniques, and these corresponded to bands detected using immune rabbit antisera. Using a detection system with fluorescein isothiocyanate (FITC)-labelled MAb-C1 and alkaline phosphatase-labelled anti-FITC, bands were detected between 50 and 300 kDa. Blots were examined directly and by using a laser scanner. The system was shown to be specific for Cryptosporidium spp., giving no staining with a variety of other pathogens, and with negative samples. The oocyst antigen which bound MAb-C1 was stable, and banding patterns were not significantly affected by pretreatment of oocysts with proteinase K, trypsin, formalin, or sodium hypochlorite, methods commonly used during preparation and storage of C. parvum oocysts. However, banding was reduced with potassium dichromate. Of 76 samples containing Cryptosporidium oocysts, 53 showed one or more MAb-C1 staining bands. Cryptosporidium baileyi and C. parvum could be clearly differentiated by their banding patterns, indicating that the system will distinguish between species. Some isolates, including a single isolate of C. muris, produced weak bands which made interpretation difficult. The technique showed differences between isolates of C. parvum, with two different banding types found in human isolates, and other banding types seen in calf and lamb isolates. This method provides a useful way of characterising isolates which may be new species.
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PMID:A technique for typing Cryptosporidium isolates. 181 85

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.
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PMID:A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees. 186 4

The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4'/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).
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PMID:Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. 190 21

Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40-45 degrees C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4 degrees C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9 degrees C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1 x SSC, 45 degrees C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4-11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions. 196 56

Sequences of hepatitis B virus DNA were detected specifically in the sera of infected individuals by a non-radioactive riboprobe nucleic acid hybridization assay. The nucleic acids contained in serum specimens were prepared by an overnight proteinase K-/SDS-digest, phenol/chloroform-extraction and ethanol-precipitation, and immobilized on nylon membranes by the dot-blot technique. Digoxigenin-labelled 1.4 kb RNA-probes were generated by the phage sp 6 RNA-polymerase from a linearized HBV DNA transcription template containing the appropriate promoter. Hybridized probes were detected by alkaline phosphatase-conjugated sheep anti-digoxigenin Fab-fragments, and 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium salt (NBT) as chromogenic substrates for the subsequent ELISA procedure. With a detection limit of 0.5-1.0 pg HBV DNA and a high specificity, the results were comparable to those obtained by the standard assay employing radioactively labeled DNA-probes.
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PMID:A nonradioactive riboprobe assay for the detection of hepatitis B virus DNA in human sera. 208

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.
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PMID:In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes. 212 70


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