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

Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.
Mol Gen Genet 1990 Jan
PMID:Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures. 232 20

This report describes a new assay for detecting filarial parasites of the genus Brugia in blood samples using labeled DNA probes. The sequences of these DNA probes are based on the HhaI repeat DNA family found in the genus Brugia. These DNA probes are species-specific and can detect the DNA from a single microfilaria in hybridization assays. To adapt this test for use on blood samples collected in the field, complex steps to separate microfilariae from blood cells and to purify parasite DNA were eliminated. We found that the most effective method was to filter blood samples through 5.0 microns pore nitrocellulose membranes, lyse the microfilariae on the membranes by proteinase K digestion, denature the parasite DNA with sodium hydroxide, and hybridize with the DNA probe. With this method, individual microfilariae can be visualized and counted on autoradiograms. The assay was evaluated in a mock field study using Brugia malayi-infected jirds and was found to be an efficient and accurate method for quantifying microfilariae in blood samples.
Mol Biochem Parasitol 1990 Apr
PMID:A rapid DNA assay for the species-specific detection and quantification of Brugia in blood samples. 234 29

Modification of the alkaline lysis at elevated temperature technique is proposed isolation of plasmid DNA from lactobacilli. Modification consists of colorimetric control of culture phase during the biomass growth, pH control at the probes treatment with lysozyme and alkaline solution of natrium dodecylsulfate by adding the indicator bromcrezolpurple into the medium for biomass growth. The high concentration of lysozyme is used (10 mkg.ml-1). Lactobacilli are lysed at 2 min incubations of the probes with the lytic solution in the boiling water bath. The treatment of the probes by proteinase K, by the mixture of chloroform:phenol:isoamyl spirit (25:24:1 vol/vol/vol) and by diethylpirocarbonate increased considerably the quality of the obtained DNA preparations. The modified technique is suitable for isolation of the plasmid DNA from lactobacilli of different species, enterococci, streptococci and other lactic bacteria. The connection of antibiotic resistance marker and the plasmid profile of lactobacilli under different conditions with the presence of the plasmid DNA- protein complex is discussed.
Mol Gen Mikrobiol Virusol 1990 Mar
PMID:[Optimization of the method of isolation of microamounts of plasmid DNA from lactobacilli]. 236

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.
Mol Cell Biol 1990 Sep
PMID:Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei. 238 17

Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross-links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen-induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA-interstrand and DNA-protein associated cross-links in cultured epithelial cells. Cells were exposed to three known DNA cross-linking agents, nitrogen mustard (HN2), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN2 at 0.25, 1.0, and 4.0 microM or of MMC at 20, 40, and 60 microM produced a dose-dependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN2 and MMC induced both DNA-protein cross-links and DNA-interstrand cross-links. Ultraviolet irradiation induced both DNA cross-links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity-flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross-linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents.
Environ Mol Mutagen 1989
PMID:Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution. 249 39

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.
Mol Cell Biol 1989 Nov
PMID:Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells. 251 81

Restriction fragments of bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) were packaged in a completely defined in vitro system that included purified proheads, the DNA packaging protein gp16 and ATP. Both left and right end DNA-gp3 fragments were packaged in this system, in contrast to the oriented and selective packaging of left end DNA-gp3 fragments in extracts; left ends could be packaged quantitatively in the defined system, while the packaging efficiency of right ends was generally about threefold lower. In addition, certain internal (non-end) DNA fragments were packaged at efficiencies of about 10% to 15%. Digestion of the gp3 with trypsin or proteinase K reduced the packaging of whole-length DNA by a factor of 2 or 4, respectively, and removal of the gp3 from whole-length DNA or end fragments with piperidine reduced packaging to the level of internal fragments. Though the terminal protein gp3 was non-essential for DNA translocation in the defined system, it stimulated packaging of left and right end fragments, and stabilized packaging of the left end. The packaging of end and internal DNA fragments of the related phage M2Y into phi 29 proheads was similar to that of phi 29 DNA fragments, and certain fragments of lambda DNA were packaged at the efficiency of the internal phi 29 DNA fragments. Selective packaging of DNA-gp3 left ends was restored by the addition of bacterial cell extracts or glycerol to the defined system, and these packaging conditions discriminated between phi 29 and M2Y DNAs that have distinct terminal proteins.
J Mol Biol 1989 Sep 05
PMID:In vitro packaging of bacteriophage phi 29 DNA restriction fragments and the role of the terminal protein gp3. 253 Mar 57

The application of DNA probes to detect foreign DNA in whole arthropods has been limited by the inability of the probes to distinguish between small quantities of target DNA and the background signal generated by non-specific hybridization of mosquito material. We report that treatment of nitrocellulose filters upon which mosquitoes have been squashed with chitinase and proteinase K eliminates non-specific hybridization of DNA probes to mosquito components. Using this technique we have been able to detect a single larva of Brugia malayi, sporozoites of Plasmodium falciparum and Plasmodium berghei, and a single-copy gene in directly squashed vector mosquitoes. Use of this simple, rapid technique should facilitate the successful use of DNA probes in field studies.
Mol Biochem Parasitol 1989 May 01
PMID:Use of chitinase to facilitate detection of protozoan, helminth and single copy genes in squashed whole mosquitoes. 265 22

Several novel hybridization techniques are described. Cells or specimens are treated to release nucleic acids and a liquid phase hybridization is carried out with a dA-tailed capture probe and a reporter probe in chaotropic salts or in salts containing SDS/proteinase K. In another format the tailed capture probe is preimmobilized on polystyrene and used to capture target nucleic acids from the solution. No phenol extraction or centrifugation is required to prepare the nucleic acids. Capture of the target on the poly (dT)-solid supports is used to remove excess labelled probe and sample impurities prior to non-radioisotopic or radioisotopic detection. This paper shows the advantage of a single round of capture on polystyrene, including the ability to assay large numbers of samples manually, the ability to analyse each sample for many analytes simultaneously, the use of rapid non-radioisotopic detection, and the ability to readily adapt the assay for automation.
Mol Cell Probes 1989 Jun
PMID:Nucleic acid hybridization assays employing dA-tailed capture probes. Single capture methods. 267 81

The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.
J Mol Biol 1989 Nov 05
PMID:Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli. Implications for protein function. 268 32


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