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Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 199 records · Page 11Linked to original sources
PubMed1989

Characterization of a new prokaryotic transcriptional activator and its DNA recognition site.

The expression of the Bacillus subtilis phage phi 29 DNA is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter A3. Protein p4 binds specifically to a phi 29 DNA fragment containing the A3 promoter. DNase I footprinting analysis has shown that the DNA binding region for protein p4 is located between nucleotides -50 and -100 relative to the transcription start site. Methylation interference assays suggest that two eight base-pair long inverted repeats located within this binding region are the protein p4 recognition sequence. These results, together with the fact that the protein p4-dependent in vitro transcription requires the B. subtilis sigma 43-RNA polymerase, indicate that protein p4 is a transcriptional activator. The protein p4 DNA recognition region is statically bent as suggested by gel retardation and chemical cleavage assays. A model of protein p4 binding to its DNA target site is proposed.

Bacillus subtilis↗
PubMed1989

Regions at the carboxyl end of bacteriophage phi 29 protein p6 required for DNA binding and activity in phi 29 DNA replication.

Series of deletions corresponding to the carboxyl end of the phage phi 29 protein p6 have been constructed and their activity in the initiation of phi 29 DNA replication and their capacity to interact with the phi 29 DNA ends have been studied. Determination of the activity of the deletion mutants in phi 29 DNA replication indicated the dispensability of the 14 carboxy-terminal amino acids of the protein. The activity of protein p6 decreased with deletions from 23 to 39 amino acids and was undetectable when 44 amino acids were removed. A similar behaviour was obtained when the interaction of the mutant proteins with the phi 29 DNA ends was analyzed. These results indicate that the stimulation of phi 29 DNA replication by protein p6 requires a specific binding to the phi 29 DNA ends.

Amino Acid Sequence↗
PubMed1989

Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29 DNA-protein p3 replication.

The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off.

Amino Acids↗
PubMed1989

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication.

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.

Bacillus subtilis↗
PubMed1989

Characterization, overproduction and purification of the product of gene 1 of Bacillus subtilis phage phi 29.

Unit-length phi 29 DNA was not synthesized after restrictive infection of Bacillus subtilis with the phi 29 mutant sus1(629) indicating that the phage phi 29 protein p1 is needed for the viral DNA replication. Sequencing of the ORF-6 of mutant sus1(629) showed that a C in the wild-type (wt) phage had been changed to a T at nt position 19 of the ORF-6, giving rise to a TAA ochre codon, indicating that this ORF corresponds to gene 1. ORF-6 was cloned in plasmid pPLc28 under the control of the pL promoter of phage lambda and, after induction, a protein of about 10 kDa was overproduced, which was absent in the corresponding cells harbouring a recombinant plasmid with the sus1(629) mutation, indicating that the 10-kDa protein is the product of gene 1. In addition, a protein of lower Mr was synthesized after induction of the cells harbouring recombinant plasmids with the wt or the sus1(629) DNA. Both proteins were purified and characterized by N-terminal sequence determination and amino acid analysis. The low-Mr protein, named delta 1, has a size of 6 kDa and corresponds to an internal in-phase initiation event in ORF-6.

Amino Acid Sequence↗
PubMed1989

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.

Bacteriophages↗
PubMed1989

Replication of recombinant phi 29 DNA molecules in Bacillus subtilis protoplasts.

Recombinant phi 29 DNA molecules of different sizes and containing terminal protein at one or both ends, or without terminal protein, were prepared and their replication in Bacillus subtilis protoplasts was studied. Only phi 29 DNA molecules containing terminal protein at both ends replicate in vivo. The replication of symmetric DNA recombinant molecules (dimers) gives rise to displaced strands which by self-annealing create monomers with the two DNA strands covalently linked. Viral proteins p2, p3, and p6 are essential for replication of phi 29 DNA molecules in this system. Protein p17 is not essential, but stimulates the efficiency of replication. This stimulation depends on the host used.

Bacillus subtilis↗
PubMed1989

In vitro transcription of bacteriophage phi 29 DNA: inhibition of early promoters by the viral replication protein p6.

The effect of the phi 29 protein p6 on the in vitro initiation of transcription from the main phi 29 promoters was studied. Protein p6 interfered with the transcription from the early promoters C1 and C2, located at the right end of the phi 29 genome. Transcription initiated at the early promoters A1, A2b, and A2c and at the late promoter A3, located at the left region of the viral genome, was not affected by protein p6. These results suggest that protein p6 might play a dual role, acting as a negative modulator of some phi 29 early promoters apart from being a positive modulator of the viral DNA replication.

Bacteriophages↗
PubMed1989

[Correlation of etiology and severity in a series of 506 cases of acute pancreatitis].

We present a retrospective study on 506 patients with acute pancreatitis (AP), admitted in our hospital in the last five years (1984-1988). The goal of the paper is to establish a possible correlation between the severity and the etiology of the AP. Depending on the severity of the acute attack, and according to the Ranson's prognostic signs and the findings of the abdominal CAT, we have classified AP in three grades: mild, moderate and severe. 52% of AP were of biliary etiology, 25.7% alcoholic, and in 17.0% of the cases the responsible agent was not demonstrated. In relation with severity, the distribution was as follows: mild, 184 (36.4%), moderate, 254 (50.2%) and severe, 68 (13.4%). Among the cases of biliary and alcoholic etiology, 14.7% and 9.2%, respectively, were severe. Postoperative AP were severe in 71.4% of the cases. Systemic complications were more frequent in the severe forms, particularly of biliary etiology. Pancreatic abscesses and fistulas were also more frequent in biliary pancreatitis; on the other hand, pseudocysts and ascites were more common in alcoholic pancreatitis. Overall mortality was 2.8% (14 patients). Mortality was 19.1% in the severe forms. In relation to etiology the mortality was as follows: 3.7% in biliary AP; 0.8% in alcoholic AP; 14.3% in postoperative AP; and 2.3% in the idiopathic AP.

Acute Disease↗
PubMed1988

Protein-primed replication of bacteriophage phi 29 DNA.

The replication of phi 29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication. The phi 29 DNA polymerase involved both in the initiation and elongation steps of phi 29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins. To correlate functional and structural domains in the phi 29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed. Most of the mutations had a decreased initiation and elongation activity, but normal 3'----5' exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation. Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions. The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both phi 29 DNA ends. A good correlation of binding and stimulation of replication has been obtained by using fragments containing phi 29 DNA-terminal sequences and deletion mutants of protein p6. The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate phi 29 DNA-protein p3 replication in vitro.

Bacteriophages↗
PubMed1988

Characterization and cloning of gene 5 of Bacillus subtilis phage phi 29.

Sequencing of the phi 29 DNA region [open reading frames (ORFs) 12, 11 and 10] between genes 6 and 4 of the mutant ts5(219) showed that a G in the wild-type phage had been changed to an A in the mutant at position 218 of ORF 10 indicating that this ORF corresponds to gene 5. ORF 10 was cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda and, after heat induction of the Escherichia coli cells carrying the recombinant plasmid pGM26, a 12-kDa protein was overproduced, accounting for about 5% of the de novo synthesized protein. Introduction of a nonsense mutation in ORF 10 indicated that the latter codes for the 12-kDa protein. The predicted secondary structure, the hydrophilicity values and the antigenic regions of protein p5 are discussed.

Bacillus subtilis↗
PubMed1988

Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant.

By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue. The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA. The results obtained indicate a high specificity in the linking site of the terminal protein.

Bacteriophages↗
PubMed1988

Characterization of the origins of replication of bacteriophage phi 29 DNA.

The origins of replication of phi 29 DNA have been studied by analyzing the activity as templates in the phi 29 in vitro replication system of E. coli recombinant plasmids and M13 derivatives containing phi 29 DNA terminal sequences. Plasmid pITR, containing the 6 bp long inverted terminal repeat of phi 29 DNA, was shown to be essentially inactive. The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right phi 29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the mutagenesis at these sequences was carried out. Changes of the second or third A into a C completely abolished the template activity. In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat. The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.

Bacteriophages↗
PubMed1988

[Vesico-ureteral-renal reflux in the adult. Preliminary results of endoscopic treatment].

Vesico-ureteric reflux in adult patient may now be treated endoscopically by injecting teflon under the ureteral orifices. Twenty-six ureteral units were treated by this technique in 18 patients. Correction of the reflux was obtained in 22 units (85 p. 100); the technique was ineffective in 4 units (15 p. 100). No complication was observed in this series; in particular, there was no stenosis of the intramural ureter. These encouraging preliminary results have to be confirmed on a long-term basis in large series of patients.

Endoscopy↗
PubMed1988

Symmetrical transcription in bacteriophage phi 29 DNA.

Transcription of some early genes occurring during phi 29 infection in the absence but not in the presence of chloramphenicol has been shown to depend upon the synthesis of the viral protein p4, the positive regulator of late transcription. In addition, the early promoter B1, responsible for early transcription on the late region of the phi 29 genome, has been accurately mapped by nuclease S1 protection experiments. The deduced promoter sequence shares homology with that of the other early phi 29 promoters previously described and with the consensus sequence of the promoters recognized by the Bacillus subtilis sigma 43-RNA polymerase.

Bacillus subtilis↗
PubMed1988

Initiation of phage phi 29 DNA replication by mutants with deletions at the amino end of the terminal protein.

Series of deletions at the amino end of protein p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized. Measurements of the activity of the deletion mutants in the formation of the protein p3-dAMP initiation complex in vitro indicate the dispensability of the first 13 amino acids (aa) of the protein. The activity of protein p3 decreased considerably when 17 or more aa were deleted. The results on the in vitro phi 29 DNA replication primed by the p3 deletion mutants correlated very well with those obtained in the formation of the TP-dAMP initiation complex.

Adamantane↗
PubMed1988

Interaction of the bacteriophage phi 29 protein p6 with double-stranded DNA.

The Bacillus subtilis bacteriophage phi 29 protein p6 binds to double-stranded DNA, but not to single-stranded DNA, as determined by a gel retardation assay. The nature of the interaction was further studied by DNase I "footprinting" experiments. Protein p6 binds to fragments containing the right or left terminal sequences of phi 29 DNA, producing a characteristic pattern of hypersensitive bands spaced about 24 nucleotides apart along most of the fragment, flanking protected regions. Binding of protein p6 to an internal phi 29 DNA fragment was also observed, but the footprint pattern was more salt sensitive than that obtained with the terminal phi 29 DNA fragments. By electron microscopy, protein p6 was shown to cover the DNA, totally or partially, from one end. In addition, binding of protein p6 to relaxed circular DNA induced positive supercoiling, indicating that a topological change in the DNA occurred.

Bacillus subtilis↗