pBR322-Red在大肠杆菌lac操纵子基因敲除和敲入中的应用

pBR322-Red是一种新型重组工程系统,它携带了λ-噬菌体Red重组酶基因和一系列调控元件.对pBR322-Red最优重组条件进行探索后应用该质粒提供的体内同源重组功能,在菌株W3110体内,对染色体上的lac操纵子进行了基因修饰,包括:①运用kan/sacB选择反选择方法和重叠引物方法敲除了阻遏基因lacⅠ,②运用kan/sacB选择反选择方法和线性双链DNA介导的DNA重组方法将报告基因lacZ敲入lacA和lacY的位置,并且首次测定了报告基因lacZ在这三个结构基因位置的组成性表达情况.结果表明运用不同的重组策略,pBR322-Red系统都能方便有效地对大肠杆菌W3110染色体进行基因敲除和敲入修饰.     

Correlation between umuC induction and Salmonella mutagenicity assay for quinolone antimicrobial agents

Abstract A novel genetic procedure is described to identify stress genes in Bacillus subtilis by insertion mutagenesis. In addition, this method allows the rapid mapping of the mutation and the establishment of the DNA sequence of the gene impaired by the mutation. Small restriction fragments of chromosomal DNA of B. subtilis are inserted into a pBR322-based vector, the recombinant plasmids are transformed into B. subtilis , and integrants are selected which arise by recombination between the insert and its homologous region within the bacterial chromosome. About two dozen heat-, cold- and salt-sensitive mutants were isolated. Four mutations were mapped using PBS1 transduction, and the physiology of one salt-sensitive mutant was analysed.     

n' Protein activator sites of plasmid pBR322 are not essential for its DNA replication

The lagging strand DNA synthesis of the Escherichia coli bacterial chromosome and plasmids is thought to be initiated by the mobile promotor, the primosome. This primosome is assembled at a specific site on single-stranded DNA. This process is initiated by the interaction of one of the at least seven components, the n' protein, with this site. Indeed n' protein activator sites are found in the plasmids Col E1 and pBR322. To investigate the in vivo function of these n' protein sites, deletion derivates of pBR322 were constructed in which the n' protein sites are removed. The deletion plasmids show no change in stability and only threefold reduction in copy number compared to pBR322. Using a transduction system for single-stranded plasmid DNA it was shown that no other specific initiation signals for lagging strand DNA synthesis were present in the deletion plasmids. It was concluded that the n' protein activator sites in pBR322 are not essential for its DNA replication in vivo.     

Hfr-mediated conjugative transfer of pBR322 vector carrying the chromosomal DNA of Escherichia coli

Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and a segment of chromosomal DNA of Escherichia coli could be transferred from an Hfr donor to recipient cells by a bacterial mating. When the chromosomal DNA in the plasmid corresponded to the early transfer region of the Hfr, the frequency of the transfer was high. The recA function of both donor and recipient cells was required in the transfer. The physical association of the hybrid plasmid with the transferring Hfr chromosome through the homologous sequences may mediate the transfer of the non-mobilized plasmid. This phenomenon is useful for the determination of the chromosomal location of an unidentified fragment cloned in a non-mobilized plasmid.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Site-directed mutagenesis of the Escherichia coli chromosome near oriC: identification and characterization of asnC, a regulatory element in E. coli asparagine metabolism

We developed a new method for the specific mutagenization of the E. coli chromosome. This method takes advantage of the fact that a pBR322 plasmid containing chromosomal sequences is mobilizable during an Hfr-mediated conjugational transfer, due to an homologous recombination between the E. coli Hfr chromosome and the pBR322 derivative. Transconjugants are screened with a simple selection procedure for integration of mutant sequences in the chromosome and loss of pBR322 sequences. Using this method we specifically inactivated several genes near the E. coli replication origin oriC. We found that a gene coding for asparagine synthetase A. This regulatory mechanism was investigated in detail by determining in vivo regulation of asnA promoter activity by the 17kD protein under different growth conditions. Results obtained also suggest a general regulatory role of the 17kD protein in E. coli asparagine metabolism. Therefore the 17kD gene is proposed to be renamed asnC.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA

We describe here simple techniques for increasing the frequency of UV-induced mutations in a DNA fragment cloned in plasmid pBR322. Irradiation of both the host and the plasmid DNA before transformation is necessary to produce new mutations in the plasmid DNA, presumably because the UV-damaged pBR322 replicon cannot efficiently induce the error-prone repair pathway of Escherichia coli. In contrast, U V irradiation of the plasmid DNA alone before transformation primarily causes the transfer of preexisting mutations from the host chromosome to homologous DNA present in the plasmid. The only other kind of mutants obtained were large deletions of the plasmid DNA. Two chromosomal mutations from the host galK gene and one from the lacZ gene have been transferred to the plasmid by UV irradiation of the plasmid DNA alone. The technique can thus be of general use.     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.     

Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes

Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density. The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322. Thus topoisomerase I is involved in determining the level of supercoiling in bacteria. Three of the topoisomerase I-defective strains we studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli.

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [32P]-pBR322 at moderately high stringency. Moreover, nick-translated [32-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the 32P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.