Rate Of Plasmid Loss Research Articles

The origin recognition complex in silencing, cell cycle progression, and DNA replication.

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.

2-micron vectors containing the Saccharomyces cerevisiae metallothionein gene as a selectable marker: excellent stability in complex media, and high-level expression of a recombinant protein from a CUP1-promoter-controlled expression cassette in cis.

We have constructed 2-micron-based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisiae as a semi-dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking the CUP1 gene. In copper-sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed-batch fermentation for an extended period of time. (2) Use of the CUP1-stabilized plasmids improved the production of hirudin by both copper-sensitive and copper-resistant hosts. The highest hirudin titers were obtained with a delta CUP1 host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two-fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) The CUP1 marker proved particularly useful in combination with a CUP1-promoter-controlled expression cassette on the same plasmid. In such a set-up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper-sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of the CUP1 marker in the construction of stable yeast expression vectors.

Cometabolic oxidation of polychlorinated biphenyls in soil with a surfactant-based field application vector

Polychlorinated biphenyl (PCB)-degradative genes, under the control of a constitutive promoter, were cloned into a broad-host-range plasmid and a transposon. These constructs were inserted into a surfactant-utilizing strain, Pseudomonas putida IPL5, to create a field application vector (FAV) in which a surfactant-degrading organism cometabolizes PCB. By utilizing a surfactant not readily available to indigenous populations and a constitutive promoter, selective growth and PCB-degradative gene expression are decoupled from biphenyl. Since PCB degradation via the biphenyl degradation pathway is nonadaptive in the absence of biphenyl, there is no selective pressure for PCB gene maintenance. The recombinant strains exhibited degradative activity against 25 of 33 PCB congeners in Aroclor 1248 in the absence of biphenyl. Whole-cell enzyme assays indicated that PCB-degradative activity of a recombinant strain carrying the PCB genes on a plasmid was approximately twice that of the same strain carrying the PCB genes on a transposon. Plasmid loss rates in the absence of antibiotic selection averaged 7.4% per cell division and were highly variable between experiments. Surfactant-amended slurries of PCB-contaminated electric power plant substation soil were inoculated with approximately 10(5) recombinant cells per ml. The populations of the added strains increased to greater than 10(9) cells per ml in 2 days, and cell growth coincided with PCB degradation. By 15 days, 50 to 60% of the indicator congener 2,3,2',5'-tetrachlorobiphenyl was degraded. The effectiveness of PCB degradation by the plasmid-containing strain depended on plasmid stability. The transposon-encoded PCB genes were much more stable, and in surfactant-amended soil slurries, PCB degradation was more consistent between experiments.

Variation and modeling of the probability of plasmid loss as a function of growth rate of plasmid‐bearing cells of Escherichia coli during continuous cultures

A large number of models concerning cultures of genetically engineered bacteria have been described. Among them, some are specifically adapted to continuous cultures and lead to the determination of two variables: (i) the difference in the specific growth rates between plasmid-carrying cell and plasmid-free cells (deltamicro) and (ii) the frequency of plasmid loss by plasmid-containing cells (p(r)micro(+)). Until now, studies have been performed on the global expression p(r)micro(+) and deltamicro, whose value during continuous assays have been supposed approximately constant (mean value) and not on separate values of both terms p(r) and micro(+), respectively, probability of plasmid loss and specific growth rate of the plasmid-carrying cells. So far these studies do not allow examination of the relationship between these two last parameters. Experimental results were obtained with Escherichia coli C600 galk (GAPDH), a genetically engineered strain that synthetizes an elevated quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). From data obtained during continuous cultures, it is shown that during an assay, deltamicro, and p(r)micro(+) do not remain constant. An appropriate mathematical analysis of the expression of micro(-) (specific growth rate of the plasmid-free cells) and micro(+) has been built up. This allows the evaluation of the values of micro(+) and micro(-) during the continuous cultures carried out at different dilution rates. Values of p(r) have been calculated from these data. Indeed our results show that p(r) increases with micro(+). A modeling approach which allows correct simulation of this variation is also proposed. This model is derived from the Hill equation regarding cooperative binding of enzymic type reaction.

Effect of parB on plasmid stability and gene expression in Xanthomonas campestris.

The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campestris and X.c.pv manihotis. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB+) showed that the parB locus decreased significantly the plasmid loss rate mainly by X.c.pv campestris. The lower efficiency of stabilization in X.c.pv manihotis was probably due to the incompatibility system between the native plasmids and the newly introduced pAP23. Although parB had conferred higher stability, it determined a lower rate of alpha-amylase activity even by the strain Cm where its stabilization rate was higher.

On the level of plasmid‐bearing cells in transformed cultures of Saccharomyces cerevisiae

LC1, a YIP5-derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10-20% or 30-50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hybridization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.

Determination of kinetic parameters related to the piasmid instability: For the recombinant fermentation under repressed condition

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12DeltaH1Deltatrp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (theta) describing the rate at which the plasmid-harboring cells lose plasmids and the plasmid-free cells are generated per unit time and the difference in growth rates (Delta) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (Omega) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters theta and Delta were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.

Effect of growth rate on stability of a recombinant plasmid during continuous culture of Saccharomyces cerevisiae in non-selective medium

The stability of a 2μm based plasmid, pLG669-z, was measured in a Saccharomyces cerevisiae strain, grown continuously in a non-selective medium. The apparent stability of the plasmid was found to decrease with increasing growth rate. A model was developed to predict the contributions of segregational instability and growth rate differences to the apparent stability. An increase in segregational instability was the dominant factor in the increased rate of plasmid loss.

Elevated recombination rates in transcriptionally active DNA.

We have examined the effect of RNA polymerase II-dependent transcription on recombination between directly repeated sequences of the GAL10 gene in S. cerevisiae. Direct repeat recombination leading either to plasmid loss or conversion was examined in isogenic strains containing null mutations in the positive activator, GAL4, or the repressor, GAL80. A 15-fold increase in the rate of plasmid loss is observed in cells constitutively expressing the construct compared with cells that are not. Conversion events that retain the integrated plasmid are not stimulated by expression of the repeats. Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmid sequences.

Data analysis of plasmid maintenance in a CSTR

A simple procedure is developed to process experimental data from plasmid maintenance studies of recombinant cells in a chemostat with nonselective medium. This procedure, based on the model proposed by Imanaka and Aiba, provides quantitative information on the rate of plasmid loss and the difference in the specific growth rate between the plasmid-carrying and plasmid-free cells. The performance of the proposed method is evaluated through simulation studies. In addition, the method is applied to a set of previously reported experimental data. The two-parameter model, together with the estimated parameter values, provides an excellent fit to the experimental data.

The effect of oxygen limitation on stability of a recombinant plasmid inSaccharomyces cerevisiae

The effect of oxygen limitation on the stability of a recombinant yeast plasmid was measured during continuous culture in a non-selective medium. The fraction of plasmid containing cells was found to decrease more rapidly after a step change to low dissolved oxygen (DOT) levels. An increased rate of plasmid loss appeared to be the major cause of the decreased stability.

A new method for quantifying the probability of segregative plasmid loss inEscherichia coli B/r

A new method for quantifying rates of segregative plasmid loss is described. The probability of plasmid loss is found from the decrease in the fraction of plasmid containing cells, following a synchronous division. Stability inEscherichiacoli B/r increased with pH and dilution rate, but decreased with temperature.

Persistence and expression of the plasmid pBR322 inEscherichia coli K12 cultured in complex medium

The stability and gene expression of plasmid pBR322 in a chemostat with complex non-selective medium at different dilution rates were studied. It was observed that pBR322 was eventually lost from the population after a long lag period. The rate of plasmid loss decreases with decreasing dilution rate. This result is different from those obtained with cells grown in defined medium, where plasmid loss was observed to decrease with increasing dilution rate. In addition, it was observed that the β-lactamase activity per ml per optical density of cell culture, independent of the dilution rates, increases with time and reaches a maximum around 4.5 units after 100 hrs of continuous culture.

Stability function in the Bacillus subtilis plasmid pTA1060

Plasmid pBB2 (11.3 kb) was constructed by genetically labeling the cryptic Bacillus subtilis plasmid pTA1060 with the pC194-derived Cm R and the pUB110-derived Km R markers. In nonselective media pBB2 was segregationally almost completely stable (loss rates ⩽ 0.02% per cell generation). In contrast, pBB3, obtained by deleting from pBB2 a region consisting of two ClaI fragments (1.45 and 0.20 kb, respectively), was unstable (loss rates ⩾ 0.5% per cell generation). This indicates that a genetic element required for stability is located on one or both of these fragments. In pBB3 cop, a mutant with a two- to threefold increased copy number, the rate of plasmid loss was reduced compared to that of pBB3. The insertion of a 4.2-kb Escherichia coli DNA fragment reduced the stability of pBB2 only slightly, suggesting that this vector may be useful for the cloning of relatively large fragments.

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA.

The designation of the yeast 2 mu circle as a "selfish" DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%-3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 mu plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.

Effect of growth rate on the maintenance of a recombinant plasmid inBacillus subtilis

The rate of fall in the proportion of plasmid-containing cells in a population ofBacillus subtilis 1A297[pVC102] grown in continuous culture was independent of growth rate. Plasmid loss could not be ascribed to faulty partitioning during cell division. At a low dilution rate, the specific rate of plasmid loss exceeded the specific growth rate of the plasmid-containing cells.

Stability of Continuous Culture with Recombinant Organisms

It was found that both poor selection pressure and a variable rate of plasmid loss were present in the system studied and that both have significant effects on continuous reactor operation. At least some of these effects were analyzed by a simple model. At this point, experimental analysis for extracellular levels of tryptophan sufficient to support X- growth (1-4 mg/l) has given contradictory results. This has at least partially indicated the effect may be an intracellular one, and thus the culture history would be critical in such experiments. Since the system studied is not atypical of recombinant cultures, it leads one to speculate on the generality of the phenomena and its extent in other cultures. If important, the use of double auxotrophs or auxotrophs that are mutant in a metabolite for which the cell has a greater growth requirement should be used. Additionally, the presence of higher copy numbers in yeast at lower growth rates also leads one to speculate on how these apparently contradictory phenomena are related.

Structural and functional analysis of the par region of the pSC101 plasmid

Three distinct segments (the partition-related, or PR segments) within the 370 bp par region of pSC101 have been shown by deletion analysis to be involved in partitioning of the plasmid to daughter cells. The two lateral segments are direct repeats, each of which potentially can pair with an inverted repeat located between them to form a hairpin-loop structure. Deletion of either lateral segment, together with the middle segment, results in plasmid instability (the Par − phenotype). Deletion of one PR segment yields a stable plasmid that nevertheless shows reduced ability to compete with a coexisting wild-type derivative of the same replicon (the Cmp − phenotype). Deletion of all three segments results in a rate of plasmid loss far in excess of that predicted from the observed copy number of the plasmid. Analysis of the segregation properties of these mutants and of temperature-sensitive and high copy number derivatives of the pSC101 replicon suggests a model in which the par function allows the nonreplicating plasmids of the intracellular pool to be counted as individual molecules, and to be distributed evenly to daughter cells. In the absence of par, the multicopy pool of plasmids behaves as a single segregation unit.

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.