The effect of growth rate and culturing conditions on the stability of plasmid pCJ55 and on the level of expression of a large fragment of DNA-polymerase I, cloned in Escherichia coli

Plasmid pCJ55 with a cloned gene for the large fragment of Escherichia coli DNA polymerase I is stable in the population of a recombinant strain under the conditions of batch and continuous cultivation at different dilution rates in the presence of ampicillin. The level of Klenow fragment expression is determined by at least two factors: the stability of the recombinant strain and its specific growth rate. The maximal activity of the Klenow fragment was found after thermoinduction of the culture growing at a rate of mu = 0.6 h-1 in a synthetic medium with bactopeptone and glucose as a carbon source.     

RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro

To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro. Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes. Escherichia coli SSB, causes no such improvement in Klenow fidelity. RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP. Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime. These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.     

Mechanism of Inhibition of DNA Synthesis by 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)uracil

The mechanism of the inhibitory action of 1-β-D -arabinofuranosyl-E-5-(2-bromovinyl) uracil triphosphate (BV-araUTP) on DNA synthesis by Escherichia coli DNA polymerase I Klenow fragment was studied. Acting as a chain terminator, BV-araUTP inhibited DNA synthesis by Klenow fragment more effectively than 2′, 3′-dideoxythymidine triphosphate (ddTTP). However, the incorporation sites of BV-araU monophosphate were restricted at consecutive dTMP sequence whereas ddTMP was incorporated at every dTMP site.     

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.     

Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons

Partially single-stranded amplicons, formed during PCR amplification of single and mixed templates, are a potential source of bias in genetic diversity studies. The analysis of 16S rRNA gene diversity in mixed template samples by the fingerprinting technique terminal restriction fragment length polymorphism (T-RFLP) analysis can be biased by the occurrence of pseudo-T-RFs, i.e., restriction fragments occurring in addition to the expected terminal restriction fragments of single amplicons. This bias originates from PCR products, which are single-stranded at their terminal restriction site. Here we show that treatment of PCR amplicons with Klenow fragment prior to restriction digest and T-RFLP analysis minimized effectively the occurrence of pseudo-T-RFs. Klenow fragment activity filled in bases into the partially single-stranded amplicons and thereby restored the affected amplicons to complete double strands. Our method allowed to improve the assessment of genetic diversity and gene ratios from T-RFLP analysis of an original environmental sample. Since partially single-stranded amplicons might influence many PCR-based techniques, post-amplification treatment with Klenow fragment may be useful for a wide range of applications, which assess the composition of amplicon pools, e.g., the analysis of marker gene diversity in mixed template samples by fingerprinting techniques or the analysis of sequence diversity by cloning.     

Temperature-inducible amber suppressor: construction of plasmids containing the Escherichia coli serU- (supD-) gene under control of the bacteriophage lambda pL promoter.

An Escherichia coli DNA fragment containing the structural gene serU132 for the nonsense suppressor tRNASer2am was identified and purified by being cloned into a plasmid vector. Information obtained from DNA sequence analysis was used to select a serU132 fragment for insertion downstream from the bacteriophage lambda pL promoter in two pBR322-lambda derivatives. In nonsense mutant strains bearing the resulting serU132 hybrid plasmids, the presence of the lambda cI857 repressor gene carried on the same plasmid or in a prophage genome permits thermal regulation of suppressor synthesis.     

DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.     

An alternative approach in gene synthesis: use of long selfpriming oligodeoxynucleotides for the construction of double-stranded DNA

A novel approach for the synthesis of double-stranded DNA fragments from only one long oligodeoxynucleotide (oligo) is presented. The basic strategy is to use oligos which possess a short inverted repeat at their 3' end resulting in the formation of a hairpin structure. The 3' end of this hairpin then serves as a primer in the Klenow (large) fragment of E. coli DNA polymerase I-mediated synthesis of the second DNA strand. Removal of the loop structure as well as generation of sticky ends for subsequent cloning is achieved by digestion with restriction enzymes. Several oligos ranging in size from 130 to 147 nt were synthesized and successfully used in the cloning of gene fragments of up to 120 bp in length. Furthermore, a strategy for the simultaneous cloning of two synthetic DNA fragments is outlined yielding even larger gene fragments. By sequential cloning of these gene fragments the methodology presented here will allow the synthesis of genes of any size. The proposed methodology should also be useful for site-directed mutagenesis as well as saturation mutagenesis.     

8-Hydroxyadenine (7,8-dihydro-8-oxoadenine) induces misincorporation in in vitro DNA synthesis and mutations in NIH 3T3 cells.

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.     

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.     

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.     

DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases

A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapable of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.     

C4-methyldeoxythymidine replacing deoxythymidine in poly[d(A-T)] renders the polymer resistant to the 3''----5'' exonuclease activity of the Klenow and T4 DNA polymerases.

We previously reported that O4-alkyl dTTPs could replace, for short times, dTTP in polymer synthesis [Singer et al., PNAS 83, 26-32, 1986]. The reasons for such early termination of synthesis could be either proofreading or the eventual formation of weakly paired primer termini. Utilizing the known 3'----5' exonucleolytic activity of polymerases, in the absence of dNTPs, enabled us to conclude that, in contrast to the digestibility of poly[d(A-T)] which yielded the expected 3'-mononucleotides, the polymerizing enzymes did not digest O4-methyl dT or its neighbors. The presence of the resistant alpha-phosphorothionate linkage did not prevent measurable digestion of poly[d(A-T)] by the Klenow fragment. This, together with evidence that polymerization of O4-methyl dTTP is favored at low temperatures, supports the model proposed by Ollis et al. [Nature 313, 762-766, 1985] showing independent domains for the two activities in the Klenow fragment.     

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.