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A I Bukhari

Publications and source records attributed to A I Bukhari.

At least 19 recordsLinked to original sources
PubMed1986

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu.

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

Bacteriophage mu↗
PubMed1985

Comparison of left-end DNA sequences of bacteriophages Mu and D108.

The nucleotide sequences of the left ends of bacteriophage Mu DNA and that of its close relative D108 have been determined. The first 100 bp of phages Mu and D108 are substantially the same except for an octanucleotide change from bp 53 to 61 and other small interspersed base-pair changes from bp 61 to 200. The first five host nucleotides preceding the host-phage junction are generally, but not always, G + C-rich and these five nucleotides display no obvious consensus sequence. Both phages Mu and D108 share striking similarity in their end DNA sequences to the end sequences of the newly described Escherichia coli movable genetic element IS30.

Bacteriophage mu↗
PubMed1985

Regulation of bacteriophage mu and mini-mu DNA replication in vivo.

To study the regulation of bacteriophage Mu DNA's integrative-replication (transposition) during lytic growth in a cell containing both a Mu and a helper-dependent Mini-Mu (short, internally-deleted Mu genome), we placed "marker" genes (bla, lacZ) within either genome and then measured their encoded enzymes as indicators of the gene dosage. These results, corroborated using DNA-DNA hybridization, show that Mu and Mini-Mu DNA transposition is well regulated, requires both the Mu A and B gene products, and can be readily monitored by measuring beta-galactosidase and beta-lactamase expressed from the lacZ and bla genes, respectively.

Coliphages↗
PubMed1984

Methylation dependent expression of the mom gene of bacteriophage Mu: deletions downstream from the methylation sites affect expression.

The expression of the DNA modification gene (mom) of bacteriophage Mu requires the cellular deoxyadenosine methylase (dam) and a transactivation factor from the phage. By hypothesis, the transcription of mom is activated by methylation of three GATC sequences upstream from the mom gene. We have introduced small deletions at a fourth GATC site located about 140 base pairs downstream from the primary methylation region. Some of the deletions severely affect the mom gene expression. We propose from this analysis that (1) some important elements, possibly the promoter, concerned with the expression of mom are located between nucleotides 840 and 880 from the right end of Mu and (2) the mom protein starts with the codon GTG located at position 810. We favor the hypothesis that methylation turns off transcription upstream, thereby allowing the main mom promoter to function.

Base Sequence↗
PubMed1984

Expression of the gin and mom genes of bacteriophage Mu.

The gin and mom genes are located in the rightmost 1.6-kb segment, designated the beta segment, of bacteriophage Mu DNA. The gin gene is responsible for the inversion of the G segment of Mu, whereas the mom gene is involved in an unusual modification of the DNA. We have analyzed recombinant plasmids carrying one or both ends of Mu DNA for the expression of the Gin and Mom functions. The Gin protein and the presumptive Mom protein are not always detected in minicells, even though the plasmids being tested have the gin- and mom-containing segment of Mu DNA. However, some plasmids, in which the right end segment of Mu DNA is confined to the 1.6-kb beta segment, do give rise to these gene products in minicells. It seems that synthesis of the Gin and Mom proteins is inhibited in minicells, but this inhibition is lifted if most of the DNA to the left of the beta segment is eliminated from the plasmids. The most prominent Mu product detected in minicells is a 23-25-kDal polypeptide, termed here the zeta (zeta) protein. The function of the zeta protein remains unknown. In vitro transcription of Mu DNA with purified Escherichia coli RNA polymerase is limited to only two regions of the genome. The early region of Mu DNA is transcribed at a relatively high efficiency, whereas the beta region is transcribed at a low efficiency. This low-efficiency transcription appears to be specific for the gin gene; the mom gene transcript cannot be detected.

Bacteriophage mu↗
PubMed1983

Infecting bacteriophage mu DNA forms a circular DNA-protein complex.

Upon superinfection of immune (lysogenic) cells with bacteriophage Mu, a form of Mu DNA accumulates that sediments about twice as fast as the linear phage DNA marker in neutral sucrose gradients. This form is also detected upon infection of sensitive cells with Mu. We have purified it and examined its physical nature. Under the electron microscope it appears circular and supertwisted. Upon treatment with Pronase, phenol or sodium dodecyl sulfate, however, it is converted to a linear Mu-length form, indicating that the circle is not covalently closed. The linear DNA still has heterogeneous host sequences at its termini. The circular DNA is resistant to the action of Escherichia coli exonuclease III and T7 exonuclease, but becomes sensitive to these nucleases after treatment with Pronase showing the presence of a protein that binds non-covalently to the ends of the DNA to circularize it as well as protect it from digestion with exonucleases. The complex is resistant to high salt (up to 6 M-NaCl) but can undergo transitions between forms that are partially open, open circular, linear and circular dimers and trimers. Examination of DNA from mature phage particles reveals that a circular DNA species is present in at least 0.1 to 1% of the population. The purified complex is extremely efficient in transfection of E. coli spheroplasts. We estimate the molecular weight of the protein in this DNA-protein complex to be approximately 64,000, and suggest that this complex might represent the integrative precursor of infecting Mu DNA.

Bacteriophage mu↗
PubMed1982

Low level and high level DNA rearrangements in Escherichia coli.

It can be argued that all organisms exhibit two levels of DNA rearrangements. At a low level they may occur sporadically in cells, perhaps largely because of spontaneous activity of transposable genetic elements. A high level may be induced in special circumstances if functions that cause rearrangements are hyperactive. As an example of low level genetic rearrangements, we have studied the occurrence of spontaneous polar mutations in the early regions of prophage Mu. We isolated 49 independent prophage mutants, which are defective in replication ad expression of late genes; 44 were in the B region and 5 were in the A region. In the B region, 68% were IS1 insertions, 9% were IS5 insertions and 9% were IS2 insertions; 14% showed no insertion. In the A region, all 5 were IS5 insertions. Thus most spontaneous polar mutations in Escherichia coli appear to the insertions. IS1 is the most common insertion; however, certain DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.

Bacteriophage mu↗
PubMed1982

DNA intermediates in transposition of phage Mu.

Transposable genetic elements can insert into DNA sites that have no homology to themselves. Evidence that there is a physical linkage between a transposable element and its target DNA sequence during transposition comes from studies on bacteriophage Mu DNA transposition in which plasmids containing Mu DNA have been shown to attach to host DNA. We report the isolation of key structures, seen after induction of Mu DNA replication, after cloning lac operator into Mu DNA and using the lac repressor-operator interaction to trap Mu DNA on nitrocellulose filters. We have localized Mu sequences within these structures in the electron microscope by visualizing the lac operator-repressor interaction after binding with ferritin-conjugated antibody. This analysis shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu.

Bacteriophage mu↗
PubMed1981

A mechanism of DNA transposition.

Bacteriophage Mu and many other transposable elements undergo transposition by a process that involves replication of the element. We describe here a mechanism by which such integrative replication may take place. We hve examined electron microscopically the DNA structures generated in host cells after Mu induction and have deduced the following steps in the transposition process, (i) Association. A protein-mediated association is brought about between the transposable element and the target DNA. (ii) Attachment. One end of the element is nicked and attached to a site that undergoes a double-stranded cleavage. (iii) Roll-in replication. While one strand of the target DNA is linked to the nicked strand of the element, the complementary strand of the target DNA is used as a primer for replication into the element such that the replicating DNA is threaded through the replication complex. (iv) Roll-in termination. When the distal end of the element arrives at the replication complex, replication is terminated. The roll-in replication mechanism can also explain laying down of tandem repeats--i.e., amplification of circular DNA sequences.

Coliphages↗
PubMed1981

DNA rearrangements associated with reversion of bacteriophage Mu-induced mutations.

Excision of transposable genetic elements from host DNA is different from the classical prophage lambda type of excision in that it occurs at low frequency and is mostly imprecise; only a minority of excision events restores the wild-type host sequences. In bacteriophage Mu, a highly efficient transposon, imprecise excision is 10-100 times more frequent than precise excision. We have examined a large number of these excision events by starting with mucts X mutants located in the Z gene of the lac operon of Escherichia coli. Mucts X mutants are defective prophages whose excision occurs at a measurable frequency. Imprecise excision was monitored by selecting for melibiose+ (Mel+) phenotype, which requires only a functioning lacY gene. Mel+ revertants exhibit an array of DNA rearrangements and fall in four main classes, the predominant one being comprised of revertants that have no detectable Mu DNA. Most of these revertants can further revert to Lac+. Perhaps 5 base-pair duplications, originally present at prophage-host junctions, are left in these lacZ-Y+ revertants, and they can be further repaired to lacZ+. Another class has, in addition to the loss of Mu DNA, deletions that extend generally, but not always, to only one side of the prophage. The other two classes of revertants, surprisingly, still have Mu DNA in the lacZ gene. One class has deletions in the Z gene, whereas, no deletions can be detected in the other. Many of the revertants in the last class can further revert to lacZ+, indicating that the lacY gene must have been turned on by a rearrangement within Mu DNA. Apparently, all of the detectable precise and most of the imprecise excision events require functioning of the Mu A gene. We suggest that a block in large-scale Mu replication allows the excision process to proceed.

Bacteriophage mu↗
PubMed1980

Effects of prophage Mu induction on expression of adjacent host genes.

The extent of induction and approximate amount of DNA replication of a Mu prophage carrying a gene for ampicillin resistance can be monitored by assaying the level of beta-lactamase. The expression of the lacZ gene adjacent to either end of an induced Mu prophage remains virtually unaffected, until late in the Mu lytic cycle, while Mu DNA is replicating and transposing.

Bacteriophage mu↗