Non-random binding of N-acetoxy-N-2-acetylaminofluorene to chromatin subunits as visualized by immunoelectron microscopy

The influence of chromatin structure on the accessibility of DNA to the model ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) was investigated by means of an immunoelectron microscopic technique developed recently. An homogeneous population of core particles or trinucleosomes from chicken erythrocytes, was submitted to electrophilic attack by N-Aco-AAF. After DNA isolation, N-2-acetylaminofluorene (AAF) binding sites were mapped upon the DNA fragments using specific antibodies as a probe. Our results indicate a non-random binding of AAF along the DNA. Our data support the results of previous studies showing a preferential binding on the linker region.     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Specificity of deletion events in pBR322

The reversion of mutations due to inserts of identical palindromic DNAs just 1-bp apart in the amp gene of plasmid pBR322 varied up to 3000-fold (U. DasGupta, K. Weston-Hafer, and D.E. Berg (1987) Genetics 115, 41-49). The experiments reported here show that the intrinsic frequencies of deletion from these sites are truly very different. Deletions were selected by the joint loss of sacB (sucrose sensitivity) and lacZ alpa genes cloned together at these sites, without requiring restoration of the ampr allele. We found that greater than 90% of deletions at each of these sites do restore the ampr allele. This result reinforces the view that the probability of forming a particular deletion depends strongly on the DNA sequence at its prospective endpoints.     

The site-specific deletion in plasmid pBR322

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.     

Synthesis of some O-, S- and N-glycosides of hept-2-ulopyranosonamides

(O-Peracylated α-d-gluco- and -galacto-hept-2-ulopyranosylbromide)onamides gave the corresponding (alkyl β-d-glyco-hept-2-ulopyranoside)onamides under Koenigs-Knorr conditions, and similar aryl glycosides were obtained with sodium phenolates; (aryl and hetaryl 2-thio-β-d-gluco-hept-2-ulopyranoside)onamides were formed with thiophenols in the presence of K₂CO₃ in acetone, and reactions with aniline in CH₂Cl₂ furnished (N-phenyl β-d-glyco-hept-2-ulopyranosylamine)onamides. Some deprotected derivatives of d-gluco configuration obtained by the Zemplén protocol showed no significant inhibition against rabbit muscle glycogen phosphorylase b.     

Stability of pBR322-derived plasmids

The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics. Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions. An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability). Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability). This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene. This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids. There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature. The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells.     

Construction of pBR322-ara hybrid plasmids by in vivo recombination

Summary In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analysis demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%–3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araCmutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.     

Analysis of pBR322 replication kinetics and its dependency on growth rate

Accurate estimates of plasmid copy number in a cell are a prerequisite for predicting plasmid stability and protein production. A refined version of a structured model for the pBR322 plasmid replication mechanism is described. The model is capable of accurately predicting pBR322 plasmid copy number in Escherichia coli B/r for a wide range of growth rates. The refinements include better estimates of promoter strength, the degradation rate of RNA species, binding constant of RNAI-RNAII reaction, and dependency of promoter strength on growth rate. The predictions of the model are verified by recent experimental observations but differ from some previous reports. This model can also be used to predict the binding constant of the RNAI-RNAII reaction of ColE1 type plasmids. At 37 degrees C, the binding constant is estimated to be 77 +/- 11 x 10(-13) mL/molecule-h for pBR322.     

Fate of pBR322 DNA in a wastewater matrix

Recombinant organisms used in biopharmaceutical production processes are destroyed prior to environmental release into a private or municipal wastewater treatment system. However, concern over the fate of recombinant DNA used in these processes may adversely affect product regulatory approval. This study examined the fate of DNA from the plasmid pBR322 in an activated sludge-derived matrix. DNA suitable for PCR amplification was extracted from the activated sludge matrix and a 1042-bp fragment from pBR322 rapidly decreased in concentration from 0 to 2 h after it was spiked into the activated sludge matrix at an initial DNA concentration of 25 ng ml⁻¹. While some evidence of the 1042-bp fragment was observed at 4 h, no evidence of amplified DNA was observed at 6 h. Plasmid DNA in buffer that served as a positive control exhibited no significant reduction in concentration over time. The intensity of each DNA band over the first 4 h was analyzed. A linear regression of the natural log transformation of these results yielded a mean first-order rate constant of 3.55 h⁻¹ and half-life of 0.2 h. This study demonstrated that recombinant DNA released from industrial processes into wastewater treatment systems should be rapidly degraded. Received 14 August 1998/ Accepted in revised form 19 February 1999     

UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides

An efficient method of cross-linking DNA to protein is described. The method is based on the incorporation of photoactive 8-azidoadenine 2'-deoxyribonucleotides into DNA. We have found that 8-N3dATP is a substrate for E. coli DNA polymerase I and that 8-N3dATP can be incorporated into plasmid pBR322 by nick-translation. Subsequently we were able to cross-link a set of different proteins to 8-azido-2'-deoxyadenosine-containing pBR322 by UV irradiation (366nm). No DNA-protein photocross-linking was observed under the same conditions when the non-photoactive plasmid pBR322 was used.     

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu

Summary The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.     

超螺旋DNA的碱变构研究

对超螺旋ＤＮＡ（ＤＮＡⅠ）的碱处理产物进行了琼脂糖凝胶电泳,氯化铯－溴化乙锭密度梯度超离心分析,紫外吸收光谱分析和电镜观察。实验结果表明超螺旋ＤＮＡ在碱性环境中的结构改变发生在很窄的ｐＨ范围内（ｐＨ１２．８８─１３．００）．超过ｐＨ临界点的超螺旋ＤＮＡ碱变构产物紫外吸收高于同浓度天然ＤＮＡ紫外吸收的２９％。变构产物在ＣｓＣｌ－ＥＢ密度梯度超离心中的高密度区形成稳定的区带．用透射电镜的观察表明碱变构的超螺旋ｐＢＲ３２２ＤＮＡ具有高电子密度并呈中空颗粒状,以上事实表明,ＤＮＡ在高ｐＨ下可产生一种结构有序的相对稳定的产物．这些结果意味着在碱处理过程中,超螺旋ＤＮＡ在构象上发生了改变,使其分子由扭曲线形变成球形颗粒状。根据实验事实本文对超螺旋ＤＮＡ的碱变构产物（ＤＮＡⅣ）提出一个新的结构模型──压缩模型。这个模型能更合理地解释一些实验现象。     

Maintenance of pBR322-derived plasmids without functional RNAI

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.     

Tetracycline resistance element of pBR322 mediates potassium transport

High concentrations of choline and phosphorylcholine blocked the adsorption of pneumococcal autolytic enzyme to homologous cell walls and inhibited enzymatic cell wall hydrolysis in a noncompetitive manner. Enzyme adsorption had an absolute requirement for the presence of choline residues in the wall teichoic acid. Other amino alcohols and derivatives such as ethanolamine, monomethylaminoethanolamine , and phosphorylethanolamine had no effect on enzyme adsorption or hydrolytic activity. It is proposed that enzymatic hydrolysis of cell walls requires prior adsorption of enzyme molecules to the insoluble wall substrate and that cholin residues of the wall teichoic acid have the role of adsorption ligands in this process.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.