BavAI, a restriction endonuclease from Bacillus alvei.
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Bacillus↗
Biomedical subjects
I Fodor
Publications and source records attributed to I Fodor.
Overexpression and purification of enzymatically active recombinant integrase protein of Rous sarcoma virus.
The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control of lac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl-beta-D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity in Escherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn2+ over Mg2+, and showed substrate specificity at 1 mM MnCl2.
Avian Sarcoma Viruses↗
Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.
Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.
Animals↗
Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells.
Previous studies have shown that at least three polypeptides of 43, 39 and 38 kDa are translated from separate AUG codons of the thymidine kinase (TK) encoding mRNA of herpes simplex virus type 1. In addition, small tk-specific transcripts initiated within the tk coding region were observed. However, functional activity of these three proteins and their role in establishing of the TK+ cell phenotype is not yet clear. In order to locate the 5' boundary of the gene encoding functionally active TK, we constructed a set of deletion mutants with truncated 5' ends and examined their ability to provide a TK+ phenotype after microinjection into nuclei of LTK- cells. The results demonstrate that nucleotide sequences upstream from the second ATG codon can be removed without affecting the TK+ phenotype. Deletion of the second start codon and its downstream region inactivates the TK function. Those deletion mutants which contain only the third ATG codon are TK-. Thus, the 38-kDa polypeptide that initiates at the third start codon is not endowed with the TK+ activity. Constructs containing deletions up to nt +210 and lacking all 5'-end canonical and aberrant transcription control regions, as well as first start codon, can provide the TK+ function.
Animals↗
Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature.
Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.
Cell Fractionation↗
Nucleotide sequence of Citrus limon 26S rRNA gene and secondary structure model of its RNA.
The complete nucleotide sequence of Citrus limon 26S rDNA has been determined. The sequence has been aligned with large ribosomal RNA (L-rRNA) sequences of Escherichia coli, Saccharomyces cerevisiae and Oryza sativa. Nine extensive expansion segments in dicot 26S rRNA relative to E. coli 23S rRNA have been identified and compared with analogous segments of monocot, yeast, amphibian and human L-rRNAs. A secondary structure model for lemon 26S rRNA has been derived based on the refined model of E. coli 23S rRNA. It has been compared with other eukaryotic L-rRNAs models in terms of location of functionally important regions. Origin and evolution of L-rRNA expansion segments are discussed.
Base Sequence↗
[Recombinant RNA-dependent DNA-polymerase from Rous sarcoma virus. Isolation and properties].
Preparations of recombinant reverse transcriptase RSV were isolated from E. coli HB101/pMF14 cell cultures. The enzyme purified to homogeneity was shown to be made up of two subunits with molecular masses of 97 +/- 4 and 61 +/- 3 kDa. A comparison of enzymatic properties of recombinant transcriptase to those of the enzyme isolated from the RSV (Rauss sarcoma) virus demonstrated that in the preparations under study the recombinant reverse transcriptase exists in a subunit form, alpha beta, and may acquire a relatively stable configuration, alpha 2.
Avian Sarcoma Viruses↗
[Formation of an IS1-induced deletion in pNt6::IS1 plasmid].
The expression of oLpLN region of the plasmid pNT6 causes the high instability of the plasmid. Mutations in the promoter pL region and lesions in the structural part of the N gene result in the stable inheritance of the plasmid. The plasmids pNT6::IS1 containing the IS1-element inserted into the different loci of oLpLN region restore the high instability of the plasmid inheritance in the strain 4830 coding for oLpLN. The plasmids pIG3 and pIG4 of the series pNT6: :IS1 permit one to obtain the collection of random deletions in the cloned fragments induced by IS1-element.
Bacteriophage lambda↗
The distribution and aggregatability of intramembrane particles in phenylhydrazine-treated human erythrocytes.
Freeze-fracture analysis of phenylhydrazine-treated, unfixed human erythrocytes showed a random distribution of intramembrane particles both over membrane-bound Heinz-bodies and in the intervening areas when examined after fast freezing in liquid propane. The same results was obtained when unfixed, glycerinated red cells were frozen in liquid Freon. In contrast to previously published data (Low et al. (1985) Science 227, 531-533) these results indicate that binding of Heinz-bodies to the red cell membrane cannot cause morphologically detectable clustering of Band 3 in phenylhydrazine-treated red cells. Over numerous Heinz-bodies a decreased Acridine orange-induced particle aggregation was observed. The phenomenon of the oxidant-induced red cell fluorescence is described.
Erythrocyte Membrane↗
Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes.
Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.
Animals↗
[Cloning and expression, in Escherichia coli, of reverse transcriptase from Rous sarcoma virus].
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Avian Sarcoma Viruses↗
[Effective selection and analysis of IS1 element transposition using the oLpLN region of phage lambda].
The plasmid pNT6 permits selection of IS1-element insertions into the plasmid occurring with the frequency about 10%. The bacteriophage promoter pL cloned in pNT6 is a hot-spot region for IS1-element insertion. The frequency of IS1 transposition into pL depends on genotype. The plasmid pNT6 may be considered to be a useful target DNA for screening and analysis of IS1-element transposition.
Bacteriophage lambda↗
[Transducing properties of lambda phagemids].
Transduction of genetic markers conferring drug-resistance with lambda phagemids has been studied in terms of the influence of various genetic conditions on the level of transduction. For this purpose, we have constructed and analysed a new series of phagemids - lambda gt::pBR322 and lambda gt::pUC19, in addition to earlier published phagemids. Experimental data indicate that the transduction frequency depends on the function of cI repressor and bacterial recA system, the plasmid orientation in the phagemid DNA, the selective marker used in an experiment and the concentration of the antibiotic in the medium.
Bacteriophage lambda↗
Genetic selection of lambda phage clones from a gene clonotheque.
A genetic procedure for selection of specific lambda clones, by homologous recombination between lambda clones from a gene clonotheque and sequences cloned into a plasmid, was developed. Resulting clones are isolated in transduction experiments by plating infected Escherichia coli cells under conditions selecting for the antibiotic resistance marker carried by the plasmid. The feasibility of the method was demonstrated in a model test system as well as by isolation of alpha-interferon-specific sequences from the human gene clonotheque.
Bacteriophage lambda↗
Coated pit formation: a membrane function involved in the regulation of cellular iron uptake.
Diferric-transferrin induces a marked increase in the number of coated pits on the reticulocyte membrane. This increase is followed by a decline, often to below the initial number. Since a good correlation was found between the rate of iron uptake and the number of coated pits, but not between the rate of transferrin recycling and the coated pit count, it is likely that coated pit formation is necessary for the removal of iron from transferrin. The decline in the number of transferrin-induced coated pits was observed only when haem synthesis was undisturbed, indicating that the accumulation of intracellular haem inhibits coated pit formation. Based on these results we suggest that haem regulates the rate of iron uptake by inhibiting iron removal rather than receptor recycling.
Animals↗
The mobility of intramembrane particles in non-haemolysed human erythrocytes. Factors affecting acridine-orange-induced particle aggregation.
It has previously been shown that reversible intramembrane particle aggregation can be induced in non-haemolysed human erythrocytes. This phenomenon, which can be induced by the cationic dye Acridine Orange, has been further investigated using different experimental conditions that are expected to influence the rate of aggregation of the particles. In addition to the concentration of the dye, the rate of aggregation was also found to be dependent on the extracellular and intracellular pH, as well as on the type of buffer used. While lowering the pH of the Acridine Orange solutions resulted in decreased particle clustering, low intracellular pH increased and elevated intracellular pH decreased particle aggregation. Furthermore, at a given dye concentration and a given pH, Acridine Orange caused more intense aggregation in Tris-buffered saline than in isotonic phosphate buffer or phosphate-buffered saline. Under appropriate conditions Acridine Orange caused significant particle aggregation at concentrations as low as 0.25 mM within 30 s. During this period only discocyte-stomatocyte transformation occurred; neither agglutination nor vesiculation of the erythrocytes could be detected. Treatment of the erythrocytes with Diamide (Serva), which cross-links spectrin via disulphide bridges and thereby reduces lateral diffusion of integral membrane proteins over large distances, had no inhibitory effect on Acridine-Orange-induced particle aggregation. Heating the erythrocytes to 50 degrees C, at which temperature denaturation of spectrin and fragmentation of the erythrocytes occur, and subsequently incubating them in Acridine Orange at room temperature, caused an almost maximal rate of particle aggregation within 10-30 s, without haemolysis. The possible mechanism and significance of the particle aggregation phenomenon are discussed.
Acridine Orange↗
A comparative study on the cellular processing of free and gold-conjugated transferrin.
The kinetics of the transferrin-reticulocyte interaction was studied using free and colloidal gold-conjugated double labelled transferrin (Tf and AuTf, respectively). Simultaneous biochemical and morphological experiments provided the following information: 1. The cellular recycling of Tf is significantly faster than that of AuTf. 2. AuTf induces a marked increase in the number of multivesicular elements (MVE) in rabbit reticulocytes. 3. The release of AuTf from the cells is very slow and accumulation of gold particles in MVEs can be observed during the process. The results suggest that the two postulated pathways of the transferrin-cell cycle (a fast, iron-donating and a slow, receptor-shedding cycle) are not similarly involved in the cellular processing of Tf and AuTf. While it has been suggested that in the Tf-cell interaction the fast recycling mechanism is dominating, it is likely that, probably due to steric effects, the majority of AuTfs are forced into the slower receptor-shedding pathway. These observations call attention to the possible limitations of the colloidal gold labelling technique in the determination of the kinetics and pathway of intracellular processing of free ligands.
Animals↗
A site-specific endonuclease from Pseudomonas aeruginosa.
PaeI, a new restriction endonuclease from Pseudomonas aeruginosa clinical strain was isolated and characterized. It recognizes and cleaves the sequence 5'-GCATG reduced C-3' generating DNA fragments with 3'-tetranucleotide sticky ends. DNAs of pBR322, SV40 and bacteriophage lambda have one, two and six PaeI recognition sites, respectively. Seventy-two strains of Pseudomonas, Clostridium, Escherichia coli, Shigella, Proteus and Saccharomyces were screened for the presence of site-specific endonucleases. Here we describe the PaeI restriction enzyme found in Pseudomonas aeruginosa; other data will be published elsewhere. Earlier Hinkle and Miller isolated from P. aeruginosa a PaeR7 restriction endonuclease recognizing and cleaving a sequence 5'-C reduced TCGAG-3' (1). Sequence analysis of DNAs cleaved by PaeI shows that the enzyme is the isoschizomer of SphI (2).
Base Sequence↗