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W Arber

Publications and source records attributed to W Arber.

At least 55 records · Page 3Linked to original sources
PubMed1987

A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion.

A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions. The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK- mutants. Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not. In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation. Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration. The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.

Cloning, Molecular↗
PubMed1987

A transcriptional terminator sequence in the prokaryotic transposable element IS1.

The prokaryotic transposable element IS1 is known to exert a strong polar effect upon integration into an operon. To elucidate this polar effect, we constructed a plasmid which has an IS1 integrated between the 5' half of the tet gene for tetracycline resistance and the cat structural gene for chloramphenicol resistance. The cat gene is expressed by the tet promoter and the presence of IS1 in orientation I, in which the IS1 transposase genes insA and insB are in the same orientation as the cat gene, reduced the cat expression. By introducing deletions or insertions within the IS1 sequence, we were able to map a rho-dependent terminator TIS1A between the insA and insB genes. Translational interruption between these ins genes is important for TIS1A to be an active terminator.

Acetyltransferases↗
PubMed1987

Two DNA antirestriction systems of bacteriophage P1, darA, and darB: characterization of darA- phages.

Bacteriophage P1 is only weakly restricted when it infects cells carrying type I restriction and modification systems even though DNA purified from P1 phage particles is a good substrate for type I restriction enzymes in vitro. Here we show that this protection against restriction is due to the products of two phage genes which we call darA and darB (dar for defense against restriction). Each of the dar gene products provides protection against a different subset of type I restriction systems. The darA and darB gene products are found in the phage head and protect any DNA packaged into a phage head, including transduced chromosomal markers, from restriction. The proteins must, therefore, be injected into recipient cells along with the DNA. The proteins act strictly in cis. For example, upon double infection of restricting cells with dar+ and dar- P1 phages, the dar+ genomes are protected from restriction while the dar- genomes are efficiently restricted.

Coliphages↗
PubMed1987

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.

Chloramphenicol↗
PubMed1986

DNA inversion mediated by the r-determinant of plasmid NR1: evidence for the intramolecular replicative transposition of a 23 kb IS1-flanked transposon?

The r-determinant (r-det) of the R plasmid NR1-Basel is a 23 kb, IS1-flanked transposon, called Tn2671, which has been shown to transpose to the genome of bacteriophage P7. Among the derivatives of phage P7::r-det we found one which carried two copies of the r-det as inverted repeats and which also contained the P7 genome segment between them in inverted orientation. Its generation is best explained by assuming that the entire 23 kb Tn2671 transposon has undergone intramolecular replicative transposition.

Chromosome Inversion↗
PubMed1986

Sequence relations among the IncY plasmid p15B, P1, and P7 prophages.

Electron microscopic analysis of heteroduplex molecules between the 94-kb plasmid p15B and the 92-kb phage P1 genome revealed nine regions of nonhomology, eight substitutions, and two neighboring insertions. Overall, the homologous segments correspond to 83% of the P1 genome and 81% of p15B. Heteroduplex molecules between p15B and the 99-kb phage P7 genome showed nonhomology in eight of the same nine regions; in addition, two new nonhomologous segments are present and P7 carries a 5-kb insertion representing Tn902. The DNA homology between those two genomes amounts to 79% of P7 DNA and 83% of p15B. Plasmid p15B contains two stem-loop structures. One of them has no equivalent structure on P1 and P7 DNA. The other substitutes the invertible C segments of P1 and P7 and their flanking sequences including cin, the gene for the site-specific recombinase mediating inversion.

Coliphages↗
PubMed1986

The characterization of terminators of RNA transcription on IS30 and an analysis of their role in IS element-mediated polarity.

Using expression vectors carrying the lacUV5 or Pgal promoters and the galK gene, we have studied terminators of transcription on the prokaryotic mobile genetic element IS30. The long open reading frame, ORF-A, of IS30 contains a relatively Rho-independent terminator, T30A, within its coding sequence. T30A terminates the majority of transcripts initiated at either an external promoter or the IS30-borne promoter P30A. No other terminator functions on this strand of IS30 (orientation left to right). In the orientation right to left, the previously identified terminator T30C, which follows ORF-C, is Rho-independent. T30C together with T30D, a newly identified, strong, partially Rho-dependent terminator near the left end of IS30, permits less than 2% read-through from external promoters. Neither ORF-A nor ORF-C appears to be protected from transcription by external promoters. As a consequence of the internal terminators, the insertion of IS30 into an operon can be expected to reduce the expression of genes downstream of the site of insertion weakly for one orientation of IS30 and strongly for the other orientation.

Base Sequence↗
PubMed1986

Terminal inverted repeats of prokaryotic transposable element IS186 which can generate duplications of variable length at an identical target sequence.

The insertion element IS186, which resides in the chromosome of Escherichia coli K-12, is 1338 bp long. Its termini represent 23-bp perfectly inverted repeats, but a variant carries a mismatch at position 23. IS186 transposes preferentially into G + C-rich sequences and generates target duplications of variable length, even at the same integration site.

Base Composition↗
PubMed1985

Promotion of RNA transcription on the insertion element IS30 of E. coli K12.

Two promoters of RNA transcription have been identified on IS30 by an in vivo assay, in which various DNA fragments with IS30 sequences were inserted in front of the promoterless galK gene of plasmid pFD51. Both promoters have a similar activity of approximately 10% of the activity of the lacUV5 promoter. Promoter P30A precedes the long open reading frame (ORFA), and its proposed -35 region lies within the left-hand terminal inverted repeat of IS30. However, the apparent activity of promoter P30A is significantly reduced when measured in the 3' region of ORFA. Thus, either the activity of promoter P30A is controlled by an IS30-encoded product from the same element, or some termination of transcription from P30A occurs within the coding region of ORFA. Promoter P30C precedes a short open reading frame (ORFC) in-frame with ORFA, but in the opposite strand. Reading frame ORFC is closely followed by a terminator of RNA transcription, T30C. None of the other potential open reading frames predicted from the DNA sequence, with one possible exception, are preceded by a promoter of RNA transcription active in the assay. No significant transcription was detected out of the left-hand end of the complete element. However, a small amount, probably due to read-through from promoter P30A, was detected out of the right-hand end of a complete copy of IS30. In addition the right-hand end of IS30 has been shown to have the potential to create promoters by insertion.

Base Sequence↗
PubMed1985

An active variant of the prokaryotic transposable element IS903 carries an amber stop codon in the middle of an open reading frame.

The prokaryotic mobile genetic element IS903.B is an active variant of IS903. It differs from IS903 and IS102 by 34 and 61 nucleotide substitutions, respectively. The large open reading frame (ORFI) which probably encodes the transposase is conserved in all three IS elements, whereas the smaller open reading frame (ORFII), which codes on the opposite DNA strand and entirely overlaps ORFI, contains an amber stop codon past the middle of ORFII in IS903.B. Experiments using Escherichia coli K12 strains permissive or non-permissive for amber mutations revealed no difference in the cointegration frequency mediated by IS903.B. Therefore, a possible peptide encoded by ORFII on the IS903-related element is unlikely to be necessary for transposition.

Base Sequence↗
PubMed1985

Reversion of a truncated gene for ampicillin resistance by genetic rearrangements in Escherichia coli K12.

The composite transposon Tn2672 is a derivative of the Tn3-related transposon Tn902 whose bla gene providing ampicillin resistance had been inactivated by the insertion of the IS1-flanked multiple drug resistance transposon Tn2671. Most ampicillin resistant revertants of Tn2672 are due to precise excision of Tn2671. However, a rare Bla+ revertant which still retains all the previously acquired drug resistance markers was isolated. On this revertant, the 5' part of the split bla gene on Tn2672 has converted to an intact, active bla gene, and the entire Tn902 is structurally restored. In contrast, the adjacent IS1b element belonging to Tn2671 has its terminal 142 base pairs deleted. Despite of this rearrangement, the split 3' part of bla and its adjacent sequences have remained unchanged. Models are presented to explain the observed DNA rearrangements, and their similarity with gene conversion events is discussed.

Ampicillin↗
PubMed1985

Gene organization and target specificity of the prokaryotic mobile genetic element IS26.

The 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII. The 702-bp ORFI is thus likely to code for the IS26 transposase. An 82-bp long sequence from the left end of IS26 contains a promoter-like structure in front of the start of ORFI at coordinate 64. In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene. The observation provides additional evidence for the functional relevance of ORFI. Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter. Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content.

Base Sequence↗
PubMed1985

Crossover sites cix for inversion of the invertible DNA segment C on the bacteriophage P7 genome.

The bacteriophage P7 genome contains an invertible DNA segment called C which determines its host range. P7 C(+) phages produce plaques on Escherichia coli K12. The C segment consists of a 3-kb unique sequence and 0.62-kb inverted repeats of which one carries an internal 0.2-kb deletion. This deletion has been mapped within the right inverted repeat in the C(+) orientation. The crossover sites cix for inversion of the C segment do not map at the inside boundaries of the inverted repeats, as had been proposed. They are localized at the external ends of these repeats. Thus organization of the C segment in phage P7 is analogous to that in the related phage P1.

Base Sequence↗
PubMed1985

Site-specific DNA inversion is enhanced by a DNA sequence element in cis.

A segment of the bacteriophage P1 genome, called the C segment, can be inverted by site-specific recombination; the two different orientations of the invertible segment confer different host ranges to the phage. Inversion is catalyzed by the product of the cin gene which is adjacent to one of the crossover sites flanking the C segment. The Cin-catalyzed recombination can be measured in trans by using tester plasmids in which inversion switches on antibiotic-resistance genes. We show here that an additional sequence, distinct from the two crossover sites, is needed in cis for efficient inversion. This sequence is part of the cin structural gene and stimulates recombination more than 100-fold. We have localized the major enhancer sequence on a 72-base-pair fragment and found its activity to be largely independent of the orientation or position of the sequence with respect to the crossover sites.

Journal Article↗
PubMed1985

Transposable element IS1 intrinsically generates target duplications of variable length.

Target duplication during transposition is one of the characteristics of mobile genetic elements. IS1, a resident insertion element of Escherichia coli K-12, was known to generate a 9-base-pair target duplication, while an IS1 variant, characterized by a nucleotide substitution in one of its terminal inverted repeats, was reported to duplicate 8 base pairs of its target during cointegration. We have constructed a series of transposons flanked by copies of either the normal or the variant IS1. The analysis of their transposition products revealed that transposons with normal termini as well as those with variant termini can intrinsically generate either 9- or 8-base-pair target duplications. We also observed that a normal IS1 from the host chromosome generated an 8-base-pair repeat. The possible relevance of the observation for the understanding of transposition processes and models to explain the variable length of target duplications are discussed.

Base Sequence↗
PubMed1985

Bacteriophage P1 derivatives unaffected in their growth by a large inversion or by IS insertions at various locations.

Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described. They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation. One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1. The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment. This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation. These results are discussed with regard to the genome organization of phage P1.

Coliphages↗
PubMed1984

Nucleotide sequence of the prokaryotic mobile genetic element IS30.

The complete nucleotide sequence of the mobile genetic element IS30, a resident of Escherichia coli K12, is 1221 bp long. A large open reading frame, preceded by possible transcription and translation control signals, could encode a basic protein of 383 amino acids which might presumably function as transposase. No large in-frame open reading frame is present on the opposite strand. The 26 bp long terminal inverted repeats have some sequence homology with the c-end of the phage Mu genome and with the terminal inverted repeats of the Halobacterium halobium insertion element ISH50. The IS30 sequence has no significant homology with any other sequenced prokaryotic insertion sequences.

Base Composition↗