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G Chaconas

Publications and source records attributed to G Chaconas.

53 records · Page 3Linked to original sources
PubMed1985

A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative transposition, of Mu DNA.

The phage-encoded proteins required for conservative integration of infecting bacteriophage Mu DNA were investigated. Our findings show that functional gpA, an essential component of the phage transposition system, is required for integration. The Mu B protein, which greatly enhances replicative transposition of Mu DNA, is also required. Furthermore, a truncated form of gpB lacking 18 amino acids from the carboxy terminus is blocked in replicative transposition, but not conservative integration. Our results point to a more prominent role for gpB than simply a replication enhancer in Mu DNA transposition. The ability of a truncated form of B to function in conservative integration, but not replicative transposition, also suggests a key role for the carboxy-terminal domain of the protein in the replicative reaction. The existence of a shortened form of gpB, which uncouples conservative integration from replicative transposition, should be invaluable for future dissection of Mu DNA transposition.

Bacteriophage mu↗
PubMed1985

Primary structure of phage mu transposase: homology to mu repressor.

The phage Mu transposase is essential for integration, replication-transposition, and excision of Mu DNA. We present the complete nucleotide and derived amino acid sequence of the transposase and analyze implications for transposase/DNA interaction. The NH2 terminus of the Mu transposase has considerable sequence homology with the Mu repressor and with the NH2 terminus of the transposase of the Mu-like phage D108. These three proteins are known to share binding sites on DNA. The protein sequence and predicted secondary structural similarities at the NH2 termini of the three proteins suggest a common DNA-binding region similar to the regions found in proteins of known structure. An internal sequence in the Mu A protein also shares these features. We anticipate that these regions will be involved in DNA recognition during transposition.

Amino Acid Sequence↗
PubMed1984

The nucleotide sequence of the B gene of bacteriophage Mu.

Bacteriophage Mu is a highly efficient transposon which requires the products of the Mu A and B genes in order to transpose at a normal frequency. We have determined the nucleotide sequence of the B gene as well as that of the A-B intergenic region upstream of B. The protein product of the gene contains 312 amino acids and has a predicted molecular weight of 35,061. As expected, there do not appear to be any potential promoter sequences in the intergenic region prior to the gene, but it is preceded by a strong Shine-Dalgarno sequence. The intergenic region does not contain any obvious transcription termination sequences. The frequency of optimal codon usage is similar to that for other transposon and phage genes, and the amino acid composition is comparable to that of an "average" E. coli protein. A region near the amino terminus of the protein resembles the highly conserved bihelical fold which is involved in DNA contact and sequence specific recognition in a number of DNA binding proteins.

Amino Acid Sequence↗
PubMed1983

Predominant integration end products of infecting bacteriophage Mu DNA are simple insertions with no preference for integration of either Mu DNA strand.

The integration of 32P-labeled infecting Mu DNA into the Escherichia coli chromosome was investigated. Cleavage of the integrated Mu DNA with restriction endonuclease EcoRI, which cuts twice in the Mu genome, liberated the internal EcoRI fragment but not the left and right end fragments. The ends of the Mu genome became fused with host DNA at a variety of locations generating a smear of radioactive DNA fragments following cleavage with EcoRI. The predominant integration end products of infecting Mu DNA molecules are therefore generated by a mechanism which results in simple insertions and not cointegrates. Since predominantly simple insertions are found after infection (during lysogenization or lytic growth) but not after prophage induction, the transposition mode which is utilized appears to be a function of the source of the transposing DNA. Use of the integrated, 32P-labeled Mu DNA as a hybridization probe with the separated strands of Mu DNA or lambda phages carrying various regions of Mu showed no strand preference in the integration process. Both labeled DNA strands at both ends of the Mu genome were integrated. These results suggest the lack of a site-specific recombination site in the genome; the simple insertions which are the end products of Mu DNA integration following infection appear to be generated by a separate pathway rather than by the resolution of cointegrate structures.

Bacteriophage mu↗
PubMed1981

Studies on the heterogeneity of the 5' ends of the protamine mRNAs from rainbow trout testis.

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m7-GMP), an analogue of 'cap' structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T4 polynucleotide kinase and [gamma-32P]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding for protamine components CI and CIII have at least a cap 1 structure while the mRNAs coding for CII do not appear to be capped or methylated.

Animals↗
PubMed1980

Association of Mu-containing plasmids with the Escherichia coli chromosome upon prophage induction.

To determine the structure of a prophage-containing plasmid during Mu transposition, we have monitored the physical state of pSC101[unk]Mucts after thermoinduction. We have also examined the fate of a mini Mu plasmid constructed in vitro by deleting 27 kilobases from the center of the Mu prophage in pSC101[unk]Mucts. At various times after prophage induction, DNA was extracted from Mu or mini Mu plasmid-containing strains and subjected to electrophoresis in low concentration agarose gels followed by transfer of the DNA to nitrocellulose paper. Separate hybridization with (32)P-labeled pSC101 and Mu DNA revealed the position of the plasmids and the replication of Mu DNA. At times after induction when Mu replication was clearly visible, Mu and mini Mu plasmids were found to migrate with Escherchia coli DNA. This Mu-specific association requires the phage coded A and B proteins. Electron microscopy has shown that some of the associated DNA is comprised of circular plasmid molecules which appear to be in contact with the chromosomal DNA. These structures may represent intermediates or end products of the replication-integration process. The finding that Mu and mini Mu plasmids do not give rise to any detectable excision products and apparently remain intact during Mu transposition supports our proposal that the predominant event after Mu induction is the replication of Mu DNA in situ to generate integrative intermediates.

Bacteriophage mu↗
PubMed1979

Antibiotic induced electrophoretic mobility shifts of DNA restriction fragments.

Several antibiotics, netropsin, distamycin A, actinomycin D, Hoechst 33258 and olivomycin, which demonstrate base specificity in their DNA binding properties have been found to alter the electrophoretic mobility of DNA restriction fragments in native polyacrylamide gels. The antibiotics mostly reduced the migration of larger DNA fragments, but netropsin and Hoechst 33258 were observed to increase the migration rate of several DNA fragments of intermediate size. DNA fragments of similar molecular weight which comigrate as a single gel band can at times be separated as the result of differential mobility shifts promoted by antibiotic DNA complex formations.

Anti-Bacterial Agents↗
PubMed1978

Polyadenylic acid sequences in the RNA of Hyphomicrobium.

Heterogeneous RNA containing polyadenylic acid [poly(A)] sequence has been isolated from Hyphomicrobium by affinity chromatography on oligothymidylic acid cellulose and polyuridylic acid Sepharose columns. About 0.1 to 0.3% of [3H]adenine-labeled RNA over a 60-min period is associated with poly(A) sequences. This percentage decreases to about 0.03 in a 20-h labeling period. The poly(A) tracts recovered after digestion with ribonuclease A and T1 are composed of greater than 95% adenine residues and are up to 200 nucleotides in length with a predominant range of 15 to 40 nucleotides. Adenosine and AMP are present in the ratio of 1:36 in alkaline digests of Hyphomicrobium poly(A) tracts. This is compatible with nucleotide lengths determined on acrylamide gels and location at the 3'-OH terminus of the RNA molecule.

Bacteria↗
PubMed1977

End labeling of enzymatically decapped mRNA.

A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.

Adenosine Triphosphatases↗
PubMedDate not supplied

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers.

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.

Bacteriophage mu↗