首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
3.
The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. Until now, this hypothesis has been investigated using multicopy ColE1 plasmids. However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation. Here we used a modified lambda plasmid, pTC lambda 1. The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication. Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model.  相似文献   

4.
Summary Plasmid mutants (svir), insensitive to inhibition by the repressor of initiation of pMB1 replication, have been selected by exploiting their ability to support growth in the presence of repressor and inhibitor of plasmid replication. The alteration in the mechanism that controls plasmid replication causes a change in the plasmid copy number. svir mutants are dominant, as expected for mutants in the target of a repressor, but at the same time they are unable to synthesise a repressor active on the wild-type target. This lack of cross interaction between svir mutants and a co-resident wild-type plasmid results in their compatibility. These findings are explained by postulating that the target of the inhibitor of pMB1 replication coincides with part of the DNA segment that codes for the inhibitor itself. As a consequence single base pair changes in the target result in altered repressor molecules.  相似文献   

5.
6.
7.
The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.  相似文献   

8.
9.
Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.  相似文献   

10.
Escherichia coli mutants completely defective in ColE1 cer-mediated site-specific recombination have been mapped to two genes, xerA and xerB. In this study, xerA xerB double mutants were constructed by gene replacement with a lambda dv plasmid and were shown to be both viable and defective in ColE1 site-specific recombination.  相似文献   

11.
K Matsubara  Y Otsuji 《Plasmid》1978,1(3):284-296
Various lambdoid phages, including those carrying the immunity region of 434, 21, and φ80, were found to give rise to fragments of DNA that can be perpetuated in the plasmid state. The plasmids, imm434dv, imm21dv, and φ80dv, were similar to the already known plasmid λdv in size, genetic constitution, oligomeric state, copy number, and stability. Cells carrying two kinds of dv plasmids were constructed by transformation. It was shown that a pair of plasmids is compatible in a cell if they originate from phages differing in immunity region. On the other hand, a pair of plasmids is incompatible if they are derived from phages carrying the same immunity region. These observations were taken to imply that the incompatibility of a pair of plasmids is determined by the “immunity region” of the plasmid genome that contains an autorepressor gene and a promoter-operator. The region that carries initiator genes and a site for initiation of plasmid replication is not primarily important for determination of the incompatibility. Plasmids λdv and imm434dv, which are very closely related to each other, behaved in an intermediate fashion.  相似文献   

12.
Genetic and physical map of a P1 miniplasmid   总被引:23,自引:11,他引:12       下载免费PDF全文
The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.  相似文献   

13.
14.
Serban Iordanescu 《Plasmid》1983,10(2):130-137
A chromosomal mutation leading to an important increase in the copy number of plasmid pT181 and its derivatives has been isolated from Staphylococcus aureus strain 8325. The amplification effect in the mutant strain SA1350 was found to be specific for plasmids of the Inc3 group, to which belongs pT181. There are some other differences in the behavior of Inc3 plasmids between SA1350 and 8325, including stable maintenance in SA1350 at high copy number of temperature-sensitive replication mutants at restrictive temperatures, and altered incompatibility properties. Derivatives of SA1350 carrying only Inc3 plasmid mutants with high copy numbers (Cop mutants) could not be obtained, suggesting a lethal runaway plasmid replication in this situation. SA1350 expressed also a temperature-sensitive phenotype. The relationship of this character to the plaC1 mutation determining the amplification of Inc3 plasmids has not yet been elucidated.  相似文献   

15.
Plasmid R1 replication was studied in shifts between two steady states of copy number. The copy number was varied in two ways. First, we utilized the fact that it decreases with increasing growth rate. To minimize the metabolic effects of changes in the growth rate, the downshifts were obtained by adding α-methylglucoside to cultures growing in glucose-minimal medium, and the upshifts were obtained by adding glucose to cultures growing in the presence of glucose plus α-methylglucoside. Second, we used a temperature-dependent copy mutant of plasmid R1 (pKN301). Plasmid pPK301 shows a threefold higher copy number at 40 than at 30°C. In both types of shift, plasmid replication immediately adjusted to the postshift differential rate. The copy number asymptotically adjusted to the new steady state. Hence, the system that controls plasmid R1 replication sets the frequency of replication without measuring the actual copy number. It has been suggested that plasmid R1 replication is under negative control by an R1-mediated repressor protein. Among the replication control models that involve negative control, the Pritchard inhibitor dilution model, the Sompayrac-Maaløe autorepressor model, and the plasmid λdv system all predict gene dose-independent copy number control.  相似文献   

16.
17.
18.
Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.  相似文献   

19.
L Berg  M Lusky  A Stenlund  M R Botchan 《Cell》1986,46(5):753-762
Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.  相似文献   

20.
The lambda O and P gene products are required for the initiation of lambda DNA replication. In order to study the biochemistry of this process, we have constructed plasmids that carry the lambda O gene, P gene, and half of the O gene coding for the amino-terminal half of the O protein. Each is under the control of the inducible lambda promoter, PL. We have purified these three proteins from induced cells carrying the plasmids. Our results show that the amino-terminal portion of the O protein binds to the lambda origin of replication in a manner similar to the intact lambda O protein, demonstrating that the amino-terminal portion of O protein contains the DNA binding domain. Using chromatographic procedures, we have isolated a complex of lambda O and P proteins with lambda dv DNA. The amino-terminal portion of the O protein does not complex with P protein under the same conditions. This suggests that the specificity of the lambda O protein for P protein resides in the carboxyl-terminal half of the lambda O protein. Our results also show that, while the intact O protein is active in in vitro replication of lambda dv plasmid DNA, the amino-terminal portion of the O protein is inactive and is a competitive inhibitor of the lambda O protein in this reaction. These results confirm previous genetic observations that were interpreted as indicating a bifunctional structure for the lambda O protein with the amino-terminal domain recognizing the lambda origin of replication and the carboxyl-terminal domain interacting with the lambda P protein.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号