PubMed Health⌕ Search

Biomedical subjects

W Arber

Publications and source records attributed to W Arber.

At least 19 recordsLinked to original sources
PubMed1992

Mutational analysis of the bacteriophage P1 late promoter sequence Ps.

The bacteriophage P1 late promoter sequence Ps controls the expression of the genes in the tail-fibre operon. Transcription from Ps only occurs during the second half of the P1 vegetative growth cycle and is positively regulated by the product of the phage gene 10. In this study degenerate oligonucleotides were used as primers in site-directed mutagenesis reactions in order to construct a large set of point mutations within the late promoter sequence Ps. A total of 35 independent single point mutations was isolated and the mutants were tested for promoter activity. Mutations in the Escherichia coli-like -10 region and in a late operator sequence, containing a symmetric sequence centred around position -22, resulted in significant reductions in promoter strength. Most of these mutations alter base-pairs that are highly conserved among the known late promoter sequences of the P1 family. In addition, insertion mutants that change the spacing between the -10 and the late operator indicate that a special topological arrangement between the two boxes is crucial for late promoter function. These results suggest that the product of gene 10 binds specifically to a late operator in order to activate transcription from P1 late promoter sequences.

Bacteriophage P1↗
PubMed1992

DNA inversion regions Min of plasmid p15B and Cin of bacteriophage P1: evolution of bacteriophage tail fiber genes.

Plasmid p15B and the genome of bacteriophage P1 are closely related, but their site-specific DNA inversion systems, Min and Cin, respectively, do not have strict structural homology. Rather, the complex Min system represents a substitution of a Cin-like system into an ancestral p15B genome. The substituting sequences of both the min recombinase gene and the multiple invertible DNA segments of p15B are, respectively, homologous to the pin recombinase gene and to part of the invertible DNA of the Pin system on the defective viral element e14 of Escherichia coli K-12. To map the sites of this substitution, the DNA sequence of a segment adjacent to the invertible segment in the P1 genome was determined. This, together with already available sequence data, indicated that both P1 and p15B had suffered various sequence acquisitions or deletions and sequence amplifications giving rise to mosaics of partially related repeated elements. Data base searches revealed segments of homology in the DNA inversion regions of p15B, e14, and P1 and in tail fiber genes of phages Mu, T4, P2, and lambda. This result suggest that the evolution of phage tail fiber genes involves horizontal gene transfer and that the Min and Pin regions encode tail fiber genes. A functional test proved that the p15B Min region carries a tail fiber operon and suggests that the alternative expression of six different gene variants by Min inversion offers extensive host range variation.

Amino Acid Sequence↗
PubMed1992

Bacteriophage P1 gene 10 is expressed from a promoter-operator sequence controlled by C1 and Bof proteins.

Gene 10 of bacteriophage P1 encodes a regulatory function required for the activation of P1 late promoter sequences. In this report cis and trans regulatory functions involved in the transcriptional control of gene 10 are identified. Plasmid-borne fusions of gene 10 to the indicator gene lacZ were constructed to monitor expression from the gene 10 promoter. Production of gp10-LacZ fusion protein became measurable at about 15 min after prophage induction, whereas no expression was observed during lysogenic growth. The activity of an Escherichia coli-like promoter, Pr94, upstream of gene 10, was confirmed by mapping the initiation site of transcription in primer extension reactions. Two phage-encoded proteins cooperate in the trans regulation of transcription from Pr94: C1 repressor and Bof modulator. Both proteins are necessary for complete repression of gene 10 expression during lysogeny. Under conditions that did not ensure repression by C1 and Bof, the expression of gp10-LacZ fusion proteins from Pr94 interfered with transformation efficiency and cell viability. Results of in vitro DNA-binding studies confirmed that C1 binds specifically to an operator sequence, Op94, which overlaps the -35 region of Pr94. Although Bof alone does not bind to DNA, together with C1 it increases the efficiency of the repressor-operator interaction. These results are in line with the idea that gp10 plays the role of mediator between early and late gene transcription during lytic growth of bacteriophage P1.

Base Sequence↗
PubMed1991

Gene organization in the multiple DNA inversion region min of plasmid p15B of E.coli 15T-: assemblage of a variable gene.

The bacteriophage P1-related plasmid p15B of E. coli 15T- contains a 3.5 kb long region which frequently undergoes complex rearrangements by DNA inversion. Site-specific recombination mediated by the Min DNA invertase occurs at six crossover sites and it eventually results in a population of 240 isomeric configurations of this region. We have determined 8.3-kb sequences of the invertible DNA and its flanking regions. The result explains how DNA inversion fuses variable 3' parts to a constant 5' part, thereby alternatively assembling one out of six different open reading frames (ORF). The resulting variable gene has a coding capacity of between 739 and 762 amino acids. A large portion of its constant part is composed of repeated sequences. The p15B sequences in front of the variable fusion gene encode a small ORF and a phage-specific late promoter and are highly homologous to P1 DNA. Adjacent to the DNA invertase gene min, we have found a truncated 5' region of a DNA invertase gene termed psi cin which is highly homologous to the phage P1 cin gene. Its recombinational enhancer segment is inactive, but it can be activated by the substitution of two nucleotides.

Amino Acid Sequence↗
PubMed1991

Bleomycin-resistance gene derived from the transposon Tn5 confers selective advantage to Escherichia coli K-12.

The plasmid pRAB2 contains a silent operon derived from the transposon Tn5 and carrying the gene neo for neomycin-kanamycin resistance and a truncated ble gene (ble333) for bleomycin resistance. Spontaneous mutants that express the two resistances provide Escherichia coli cells an improved fitness during the phase of decline in the absence of the antibiotics. It is shown that the ble333 gene product is responsible for this better fitness. These results can explain a previously described selective advantage attributed to the presence of Tn5. The improved fitness of bleomycin-resistant bacteria is proposed to relate to DNA repair by the ble gene product. The consequences of the presence of an accessory gene improving fitness are discussed in terms of evolutionary stable strategy of a transposon in populations of E. coli.

Bleomycin↗
PubMed1991

Elements in microbial evolution.

Spontaneous mutation, selection, and isolation are key elements in biological evolution. Molecular genetic approaches reveal a multitude of different mechanisms by which spontaneous mutants arise. Many of these mechanisms depend on enzymes, which often do not act fully at random on the DNA, although a large number of sites of action can be observed. Of particular interest in this respect are DNA rearrangement processes, e.g., by transposition and by site-specific recombination systems. The development of gene functions has thus to be seen as the result of both DNA rearrangement processes and sequence alterations brought about by nucleotide substitutions and small local deletions, insertions, and duplications. Prokaryotic microorganisms are particularly appropriate for studying the effects of spontaneous mutation and thus microbial evolution, as they have haploid genomes, so that genetic alterations become rapidly apparent phenotypically. In addition, bacteria and their viruses and plasmids have relatively small genomes and short generation times, which also facilitate research on evolutionary processes. Besides the strategy of development of gene functions in the vertical transmission of genomes from generation to generation, the acquisition of short DNA segments from other organisms appears to be an important strategy in microbial evolution. In this process of horizontal evolution natural vector DNA molecules are often involved. Because of acquisition barriers, the acquisition strategy works best for relatively small DNA segments, hence at the level of domains, single genes, or at most operons. Among the many enzymes and functional systems involved in vertical and horizontal microbial evolution, some may serve primarily for essential life functions in each individual and only secondarily contribute to evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression.

Amber mutants of bacteriophage P1 were used to identify functions involved in late regulation of the P1 lytic growth cycle. A single function has been genetically identified to be involved in activation of the phage-specific late promoter sequence Ps. In vivo, P1 gene 10 amber mutants fail to trans activate a lacZ operon fusion under the transcriptional control of promoter Ps. Several P1 segments, mapping around position 95 on the P1 chromosome, were cloned into multicopy plasmid vectors. Some of the cloned DNA segments had a deleterious effect on host cells unless they were propagated in a P1 lysogenic background. By deletion and sequence analysis, the harmful effect could be delimited to a 869-bp P1 fragment, containing a 453-bp open reading frame. This open reading frame was shown to be gene 10 by sequencing the amber mutation am10.1 and by marker rescue experiments with a number of other gene 10 amber mutants. Gene 10 codes for an 18.1-kDa protein showing an unusually high density of charged amino acid residues. No significant homology to sequences present in the EMBL/GenBank data base was found, and the protein contained none of the currently known DNA-binding motifs. An in vivo trans activation assay system, consisting of gene 10 under the transcriptional control of an inducible promoter and a gene S/lacZ fusion transcribed from Ps, was used to show that gene 10 is the only phage-encoded function required for late promoter activation.

Amino Acid Sequence↗
PubMed1990

The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element.

The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction. The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions. In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element. In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion. It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase. Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu.

Amino Acid Sequence↗
PubMed1990

Site-specific DNA recombination system Min of plasmid p15B: a cluster of overlapping invertible DNA segments.

Plasmid p15B of Escherichia coli 15T- carries a 3.5-kilobase segment that undergoes frequent DNA inversion mediated by the DNA inversion enzyme Min, a member of the Din family of site-specific recombinases. While the previously described Din inversion systems invert a DNA segment between two crossover sites in inverted orientation, the Min system produces more complex DNA rearrangements. These have been physically characterized by electron microscopy and by restriction cleavage analysis. The results can best be explained by a model that involves six crossover sites (called mix) and predicts 240 isomeric forms of the invertible region. The model was confirmed by sequencing the six mix sites in plasmids that contain the invertible DNA segments in a frozen configuration. All mix sites fit the dix consensus sequence, and they are all good substrates for DNA inversion when carried in inverted orientation. Recombination between two mix sites in direct orientation was rare, in line with the notion that Din inversion systems are topologically biased to the inversion reaction. Another recently described multiple inversion system, the shufflon of the E. coli plasmid R64, is neither functionally nor structurally related to the Min system of p15B.

Chromosome Deletion↗
PubMed1990

The Min DNA inversion enzyme of plasmid p15B of Escherichia coli 15T-: a new member of the Din family of site-specific recombinases.

Plasmid p15B is a bacteriophage P1-related resident of Escherichia coli 15T-. Both genomes contain a segment in which DNA inversion occurs, although this part of their genomes is not identical. This DNA segment of p15B was cloned in a multicopy vector plasmid. Like its parent, the resulting plasmid, pAW800, undergoes complex multiple DNA inversions: this DNA inversion system is therefore called Min. The min gene, which codes for the p15B Min DNA invertase, can complement the P1 cin recombinase gene. The Min inversion system is thus a new member of the Din family of site-specific recombinases to which Cin belongs. The DNA sequence of the min gene revealed that Min is most closely related to the Pin recombinase of the e14 defective viral element on the E. coli K12 chromosome. Like other members of the Din family, the min gene contains a recombinational enhancer element which stimulates site-specific DNA inversion 300-fold.

Amino Acid Sequence↗
PubMed1989

Bacteriophage P1 tail-fibre and dar operons are expressed from homologous phage-specific late promoter sequences.

Two plasmid systems, containing the easily assayable galK and lacZ functions, were employed to study the regulation of the bacteriophage P1 tail-fibre and dar operons. Various P1 DNA fragments carrying either the 5' end of lydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the lacZ gene. In the presence of an induced P1 prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters. The corresponding DNA operons are expressed from positively regulated, late promoters. The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties. Deletion analysis mapped the promoter of a gene 19-lacZ fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19. The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U). Sequence comparison between this promoter region, upstream sequences of the lydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a P1-specific late promoter. This was confirmed by 5' mapping of P1 mRNA. Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence. The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22.

Base Sequence↗
PubMed1989

Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein.

A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.

Bacteriophages↗
PubMed1989

Mutational analysis of a prokaryotic recombinational enhancer element with two functions.

The site-specific DNA inversion system Cin encoded by the bacteriophage P1 consists of a recombinase, two inverted crossing-over sites and a recombinational enhancer. The latter approximately 75 bp long genetic element is bifunctional due to its location within the 5' part of the cin gene encoding the recombinase. In order to determine the essential nucleotides for each of its two biological functions we randomly mutated the recombinational enhancer sequence sis(P1) and analysed both functions of the mutants obtained. Three distinct regions of this sequence were found to be important for the enhancer activity. One of them occupies the middle third of the enhancer sequence and it can suffer a number of functionally neutral base substitutions, while others are detrimental. The other two regions occupy the two flanking thirds of the enhancer. They coincide with binding sites of the host-coded protein FIS (Factor for Inversion Stimulation) needed for efficient DNA inversion in vitro. These sequences appear to be highly evolved allowing only a few mutations without affecting either of the biological functions. Taking the effect of mutations within these FIS binding sites into account a consensus sequence for the interaction with FIS was compiled. This FIS consensus implies a palindromic structure for the recombinational enhancer. This is in line with the orientation independence of enhancer action with respect to the crossing-over sites.

Amino Acid Sequence↗
PubMed1989

Characterization of in vitro constructed IS30-flanked transposons.

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.

Bacteriophages↗
PubMed1989

Organization of the bacteriophage P1 tail-fibre operon.

The revised sequence of a bacteriophage P1 DNA fragment containing the 5' end of the tail-fibre gene, gene 19, revealed that this gene is closely preceded by another open reading frame (ORF) of 432 bp. We have designated this ORF as gene R. The tail-fibre gene and gene R are transcriptionally and translationally coupled. Thus, the tail-fibre operon of bacteriophage P1 consists of three genes: gene R, gene 19 (or gene S) and gene U.

Bacteriophages↗
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↗
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↗