dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process

Summary The thermosensitivity of dnaA(Ts) mutations can be suppressed by integration of plasmid F (integrative suppression). In the light of the recent finding that F requires DnaA protein for both establishment and maintenance, integrative suppression of 11 dnaA(Ts) mutations by a mini-F, pML31, integrated near oriC was examined. The plating efficiency of integratively suppressed strains was dnaA(Ts) allele-dependent and medium-dependent. The initiation capability of suppressed dnaA(Ts) strains lacking the oriC site and their F^- counterparts was determined at various temperatures between 30°C and 42°C. The degree of integrative suppression measured by the initiation capability varied in a dnaA(Ts) allele-dependent manner. F-directed DNA replication was most affected by the dnaA(Ts) mutations mapping in the middle of the gene whereas oriC-dependent replication was most thermosensitive in strains carrying mutations mapping in the carboxy-terminal half of the gene. The results indicated that the integrative suppression by F plasmid is a DnaA-dependent process and suggested that the requirements for DnaA protein in the oriC-dependent replication and F replication processes are qualitatively different.     

Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli

Summary Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible pL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

Dam methylated and hemimethylatedoriC plasmids are replicated symmetrically; a novel and general test of replication symmetry

Summary Deoxyadenosine methylation (dam) of the numerous GATC sequences present in theEscherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleasesMboI,MboII andDpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylatedoriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part ofMboII restriction sites.     

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage λgtll called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997)191–198)found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.     

Asymmetric replication of an oriC plasmid in Escherichia coli

Summary Plasmid pTSO118 containing the Escherichia coli origin of replication, oriC, initiated replication simultaneously with the chromosome when temperature-sensitive host cells were synchronized by temperature shifts. Replicating intermediates of the plasmid as well as of the chromosome were isolated from the outer membrane fraction of the cell. Plasmid DNA with eye structures was enriched when cytosine-1--arabinofuranoside was introduced into the culture during replication. Electron microscopy of the replicating molecules, after digestion with restriction endonucleases, showed that the replication fork proceeds exclusively counter-clockwise towards the unc operon. We conclude that the replication of the oriC plasmid is unidirectional or, if bidirectional, is highly asymmetric.     

In vitro roles of Escbericbia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids

Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.     

Requirement of the Escherichia coli dnaA gene function for integrative suppression of dnaA mutations by plasmid R100-1

Summary The phenotype of Escherichia coli dnaA missense and nonsense mutations was integratively suppressed by plasmid R100-1. The suppressed strains, however, could not survive when the dnaA function was totally inactivated. This was demonstrated by the inability of replacing the dnaA allele in the suppressed strain by a dnaA::Tn10 insertion using phage P1-mediated transduction. When the intact dnaA ⁺ allele was additionally supplied by a specialized transducing phage, imm ²¹ dnaA ⁺, which integrated at the att site on the E. coli chromosome, then the dnaA::Tn10 insertion, together with a oriC deletion, were able to be introduced into the suppressed strain. Thus, the mechanisms of dnaA function for oriC and for the replication origin of R100-1 may not be quite the same.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12

Summary Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (oriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh ^– dnaA: Tn10 and rnh ^– oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm^–) of the rnh ^– dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA ⁺only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pasn20) in the absence of required DnaA⁺ protein nor inhibited dnaA ⁺–dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.     

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters

Summary A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active. It was further shown that the composite plasrnid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh. Neither pBR322 nor mini-F alone produced these inhibitory effects. Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla. Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect. Introduction of a deletion and a frameshift mutation into bla abolished the inhibition. Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, -lactamase.     

猴面包树的离体快繁条件筛选

该文以猴面包树(Adansonia digitata)种子为外植体,首先筛选合适的种子预处理及消毒方法,然后经过启动培养获得无菌外植体后在增殖培养基中进行丛生芽诱导,将丛生芽切成单株进行生根壮苗培养,最终建立猴面包树离体快繁技术体系.结果表明:75％酒精浸泡3 min+0.1％升汞消毒15 min消毒效果较佳,污染率为...     

Replication of minichromosomes in a host in which chromosome replication is random

Minichromosomes are plasmids with the origin of chromosome replication, oriC, as their only origin of replication. In Escherichia coli, minichromosomes are compatible with the chromosome and replicate in a cell-cycle-specific manner at the same time as oriC located on the chromosome initiates replication. In int strains, oriC has been inactivated and replaced by a plasmid origin. Because plasmids control their own replication, chromosome replication is uncoupled from the normal cell-cycle control and is random with respect to the cell cycle in the int strains. We have used an intP1 strain to address the question of whether minicromosome replication is coupled to the replication of the chromosome or is governed by cell-cycle-specific signals. Minichromosome replication was analysed by density-shift experiments and found not to be random in the randomly replicating intP1 host. This suggests that the cell-cycle-specific control functions of oriC replication are operating also in the intP1 strain.     

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.     

Correlation of activity with phenotypes of Escherichia coli partial function mutants of rnh, the gene encoding RNase H

Summary The rnh gene of Escherichia coli encodes RNase H. rnh mutants display at least two phenotypes: (1) they require functional RecBCD enzyme for growth; thus rnh-339::cat recB270 (Ts) and rnh-339::cat recC271 (Ts) strains are temperature sensitive for growth; (2) rnh mutants permit replication that is independent of the chromosomal origin, presumably by failing to remove RNA-DNA hybrids from which extra-original replication can be primed. We report here that manifestation of these two phenotypes occurs at different levels of RNase H function; we have examined partially functional rnh mutants for their in vitro RNase H activity, their ability to rescue viability in recB or recC cells and their ability to permit growth of mutants incapable of using oriC [dnaA (Ts)].     

Chloramphenicol releases a block in initiation of chromosome replication in a dnaA strain of Escherichia coli K12

Summary DNA-DNA hybridisation experiments show that chloramphenicol induces a burst of initiation from the oriC region of a dnaA46 mutant of Escherichia coli at 36.5° C but not from the isogenic dnaA ⁺ strain. Following this stimulation of initiation, DNA replication proceeds normally towards the terminus. The temporal pattern of the extra initiation is in parallel with the induced stimulation of RNA synthesis caused by chloramphenicol in the same strain. This is consistent with the hypothesis that the stimulation of initiation in the dnaA mutant is the result of the stimulation of the synthesis of an RNA species.