Construction of a DNA-polymerase I overproducing plasmid and isolation of the enzyme

The polA gene of Escherichia coli coding for DNA polymerase I was cloned under the control of bacteriophage lambda promoter pL and gene N in a high copy number plasmid vector. The chromosomally located lambda cIts repressor gene kept the synthesis of the polA gene product at 28 degrees C at a low level. Raising the temperature to 43 degrees C resulted in inactivation of the repressor and overproduction of DNA polymerase I, which could easily be purified to homogeneity.     

Cloning the polB gene of Escherichia coli and identification of its product

Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDa protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.     

Streptococcus pneumoniae polA gene is expressed in Escherichia coli and can functionally substitute for the E. coli polA gene.

The Streptococcus pneumoniae polA+ gene was introduced into Escherichia coli on the recombinant plasmid pSM31, which is based on the pSC101 replicon. Extracts of E. coli polA5 mutants containing pSM31 showed DNA polymerase activity, indicating that the pneumococcal DNA polymerase I was expressed in the heterospecific host. Complete complementation of the E. coli polA5 mutation by the pneumococcal polA+ gene was detected in excision repair of DNA damage.     

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.     

A new in vivo termination function for DNA polymerase I of Escherichia coli K12

Escherichia coli deleted for the tus gene are viable. Thus there must be at least one other mechanism for terminating chromosome synthesis. The tus deletion strain yielded a small fraction of cells that overproduce DNA, as determined by flow cytometry after run-out chromosome replication in the presence of rifampicin and cephalexin. A plasmid, paraBAD tus+, prevented the excess DNA replication only when arabinose was added to the medium to induce the synthesis of the Tus protein. Transduction studies were done to test whether or not additional chromosomal deletions could enhance the excess chromosome replication in the tus deletion strain. A strain containing a second deletion in metE udp overproduced DNA at a high level during run-out replication. Further studies demonstrated that a spontaneous unknown mutation had occurred during the transduction. This mutation was mapped and sequenced. It is polA(G544D). Transduction of polA(G544D) alone into the tus deletion strain produced the high DNA overproduction phenotype. The polA(G544D) and six other polA alleles were then tested in wild-type and in tus deletion backgrounds. The two alleles with low levels of 5'-->3' exonuclease (exo) overproduced DNA while those with either high or normal exo overproduce much less DNA in run-out assays in the wild-type background. In contrast, all seven mutant polA alleles caused the high DNA overproduction phenotype in a tus deletion background. To explain these results we propose a new in vivo function for wild-type DNA polymerase I in chromosome termination at site(s) not yet identified.     

Cloning of the DNA BglII fragments of bacteriophage T4 in the vector plasmid pSCC31

An attempt has been made to clone six BglII fragments of T4 DNA in the range of 3.3-8.1 kb in the vector plasmid pSCC31 containing a single BglII site within the gene for endonuclease EcoRI and pL promoter of phage lambda. DNA fragments were extracted from the corresponding bands of agarose gel. The following BglII fragments were cloned: the 3.3 kb fragment No. 9 containing a portion of gene 20, the gene 21 and a portion of gene 22; the 4.2 kb fragment No. 8.1 with genes 17, 18, 19 and a portion of gene 20; the 5.2 kb fragment No. 7.1 with genes 25-29 and a portion of gene 48. In the case of the fragment No. 7.1, the recombinant plasmids pRL705 and pRL707 with different orientation of phage DNA fragment were obtained. An attempt to clone the fragments No. 8.2 (4.2 kb), No. 7.2 (5.45 kb) and No. 6 (8.1 kb) was unsuccessful and this probably indicates the presence of the genes, whose products are deleterious to the growth of bacterial cell.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.     

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.     

In vitro stability and expression of green fluorescent protein under high pressure conditions

AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions. METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.     

Overproduction of transcription termination factor Rho in Escherichia coli

A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the pL promoter and the N-antitermination regulatory system of bacteriophage lambda. The pL-directed synthesis of Rho crucially depends on the lambda N gene product and is promoted most effectively when this product is supplied from the N gene cloned on a separate compatible plasmid with a moderate copy number. The requirement for N can be circumvented partly, but not completely, by deletion of the region preceding the rho structural gene. Attempts were also made to optimize the construction of rho-expression plasmids by adjusting the orientation and location of pL and rho inserts on the pBR322 vector. With optimal conditions, Rho protein is overexpressed 100-fold and can become as much as 10% of the total cellular protein. Using this plasmid system, Rho can be purified with a yield of more than 20 mg from 10 g of induced cells.     

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.     

Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins.

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.     

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.     

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.     

Overproduction of Escherichia coli NusA protein

The nusA gene of Escherichia coli has been cloned into the plasmid vector pKC30 under the control of the inducible lambda pL promoter. When a strain carrying this plasmid is induced, NusA protein is overproduced more than 100-fold and constitutes 20-30% of the total cellular protein. The NusA protein purified from this strain appears identical to authentic NusA protein in its migration on SDS polyacrylamide gels and on isoelectric focusing gels. It is also able to function properly in in vitro termination and antitermination assays and in its ability to bind to E. coli core RNA polymerase.