PubMed Health⌕ Search

Biomedical subjects

F Heffron

Publications and source records attributed to F Heffron.

90 records · Page 5Linked to original sources
PubMed1981

Transposon Tn3 encodes a site-specific recombination system: identification of essential sequences, genes, and actual site of recombination.

The bacterial transposon Tn3 encodes a site-specific recombination system. The recombination requires the product of tnpR, a gene previously identified as a repressor of the transposase. This recombination is site specific and takes place somewhere within the sequence C-G-A-A-A-T-A-T-T-A-T-A-A-A-T-T-A-T-C but requires at least one additional sequence outside this. The phenotype of mutations in this recombination system suggests that transposition proceeds by a mechanism in which cointegrates are intermediates.

Base Sequence↗
PubMed1979

The E. coli gene encoding heat stable toxin is a bacterial transposon flanked by inverted repeats of IS1.

Restriction endonuclease subclones of the Escherichia coli gene encoding the heat stable (ST) toxin exhibit a stem and loop structure similar to those seen in many procaryotic transposons. An EcoRI DNA fragment encoding tetracycline (Tc) resistance but no transposition functions was spliced into the ST gene in one of these subclones. By monitoring Tcr, we were able to show that the ST gene transposes. Restriction and DNA sequence data strongly suggest that the ST transposon, Tn 1681, is flanked by inverted repeats of IS1.

Bacterial Toxins↗
PubMed1979

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

Base Sequence↗
PubMed1978

In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites.

A method for mutagenizing circular DNA molecules has been developed that uses synthetic oligodeoxynucleotide restriction sites as mutagens. A single synthetic restriction site is introduced at random by cleaving circular DNA with a nonspecific double-strand endonuclease. The restriction site is then ligated to the ends and the molecule is subsequently recircularized. These small additions to the genome are mapped by digestion with the appropriate restriction enzyme. Rearrangements such as duplications and deletions can be engineered at will by using the added restriction sites. This technique has been used to produce a fine-structure map of RSF1050, a ColE1 derivative, 60% of which is a transposable DNA sequence encoding the TEM beta-lactamase (Tn3). A subset of the mutations, mapping within a narrow region of Tn3, result in an increased frequency of Tn3 transposition; mutations in other regions abolish transposition entirely.

Bacteriocin Plasmids↗
PubMed1978

Method for the genetic labeling of cryptic plasmids.

A recently developed method for detecting transposition was employed to genetically "label" conjugative plasmids such as F and Ent P307, which do not normally exhibit a readily identifiable phenotype.

Conjugation, Genetic↗
PubMed1978

Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12.

Replicating DNA molecules of the nonconjugative R plasmid RSF1010 (Smr Sur) were cleaved with the EcoRI restriction endonuclease and examined with the electron microscope. Results of this analysis indicated that replication is initiated from an origin located at about 19% of total genome size from one of the EcoRI ends. Replication proceeded either unidirectionally or bidirectionally with equal frequency. Results of the analysis of replicative intermediates of RSF1010 containing the Apr-transposable sequence (Tn) are also presented.

DNA Replication↗
PubMed1978

Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3.

The Tn1 and Tn3 elements are closely related transposons which carry the structural gene for ampicillin resistance. Two classes of deletion mutants of the plasmid pMB8::Tn3 (RSF1050) are unable to transpose ampicillin resistance but can be complemented in trans by a coresident Tn1 or Tn3 element. The analysis of the sequences transposed upon complementation of one class of mutants (type I) showed that the mutant element had undergone bona fide transposition. Complementation of the type II mutants led to the transposition of a sequence analogous to bacteriophage mu-promoted integration of non-mu DNA. The transposed sequence consisted of two Tn3 elements which flanked a single copy of the pMB8 portion of the RSF1050 genome. Complementation data indicated that the type II mutants are defective in at least one trans-acting function which must be supplied for transposition to occur. The nature of sequence transposed from the type II mutant is the consequence of a defective cis-acting function (or site). In addition, the type II mutants were defective in a trans-acting function which regulated the frequency of transposition.

Base Sequence↗
PubMed1977

Deletions affecting the transposition of an antibiotic resistance gene.

The structural gene for plasmid-mediated ampicillin resistance resides upon a 3.2 X 10(6) dalton transposable sequence (TnA) flanked by short inverted repeated sequences that accompany its insertion. TnA was transposed to pMB8, a 1.8 X 10(6) dalton derivative of the colicingenic plasmid ColE1. Random deletions were introduced in the resultant 5 X 10(6) dalton recombinant plasmid by a combination of nuclease treatments in vitro. From this set of deletions a subset was isolated that contained deletions affecting the transposition of TnA. The deletions were mapped by digestion with restriction nucleases and electron microscopic analysis of DNA hetero-duplexes and were found to include one of the inverted repeated sequences or lie in the central portion of TnA. Complementation experiments were attempted between these plasmids and another compatible plasmid carrying a deletion in TnA that abolished its ampicillin resistance. The results of the deletion data indicate that approximately 2 X 10(6) daltons of TnA is required for transposition; the complementation experiments suggest that the terminal inverted repetition and the central region of TnA play different essential roles in TnA transposition.

Crosses, Genetic↗
PubMed1977

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.

Ampicillin↗
PubMed1976

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion.

Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010. There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells. The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases. Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations. Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar. In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number.

Ampicillin↗
PubMed1975

Two replication initiation sites on R-plasmid DNA.

Replicating DNA molecules of a deletion mutant of the conjugative R-plasmid R 6 K are cleaved at a single site by the EcoRI restriction endonuclease. Electron microscope examination and measurements of the EcoRI treated replicative intermediate molecules indicate that replication can be initiated at two sites on the plasmid DNA molecule. The two sites are located at about 23 and 39% of total length, respectively, from the EcoRI cleavage site. About 5% of the replicating molecules use both replication initiation sites simultaneously.

Conjugation, Genetic↗
PubMed1975

Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion.

A series of recombinant plasmids was generated in Escherichia coli in which the TEM beta-lactamase translocon (TnA) was inserted into the small plasmid RSF1010. RSF1010 is a 5.5 X 10(6) dalton nonconjugative plasmid which confers resistance to streptomycin and sulfonamide. The recombinant plasmids can be classified into three clearly defined phenotypic groups. Group I is ampicillin-, streptomycin- and sulfonamide-resistant. Group II is ampicillin- and sulfonamide-resistant but has lost streptomycin resistance. Group III is ampicillin-resistant but is sensitive to sulfonamide and shows a simultaneous 30-fold reduction in the minimal inhibitory concentration of streptomycin. It was possible to map the site of insertion of TnA within RSF1010 by electron microscope studies of DNA heteroduplexes formed between RSF1010 and recombinant plasmids. Insertions of TnA occur at, at least, 12 distinct sites in a region corresponding to one-third of the RSF1010 DNA molecule. Those insertions giving rise to particular phenotypes are clustered. Insertions of TnA-like insertion sequences (IS) appear to give rise to strongly polar mutations.

Ampicillin↗
PubMed1975

Origin of the TEM-beta-lactamase gene found on plasmids.

A sequence of deoxyribonucleic acid of 2.7 times 10-6 to 3.3 times 10-6 daltons which includes the TEM beta-lactamase gene is present on the small plasmid RSF 1030 (R-Amp). This same sequence is present on plasmid derivatives that have received a translocation of deoxyribonucleic acid specifying the TEM beta-lactamase and is also present on naturally occurring plasmids of the F1, F11, N, X, O, I, C, and W incompatibility groups that do not specify ampicillin resistance or specify O-type beta-lactamases.

Ampicillin↗
PubMedDate not supplied

Identification of the protein encoded by the transposable element Tn3 which is required for its transposition.

Protein products have now been identified which account for the entire coding capacity of the transposable element Tn3. Mutations in Tn3 have allowed us to map the genes encoding each of these peptides and to identify their role in transposition. We have found that only a single Tn3-encoded peptide is required for transposition. Expression of this peptide is repressed by the product of a second gene, which is itself autogenously regulated.

Bacterial Proteins↗