Chemistry: molecular biology and microbiology – Vector – per se
Patent
1984-05-15
1988-07-26
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Vector, per se
435 70, 435 91, 4351723, 435253, 435 68, 935 29, 935 38, 935 56, C12P 2100, C12P 1934, C12N 1500, C12N 120
Patent
active
047600225
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the stabilization of plasmids useful in the field of recombinant DNA technology for production of genes and their products.
TECHNICAL BACKGROUND
The continued maintenance of most natural plasmids, even in the absence of selection pressure (factor which secure the growth of only those ogranisms which harbour the plasmids; an example is an antibiotic in the nutrient medium in the case of a plasmid carrying a gene mediating resistance to the antibiotic), suggests that plasmid maintenance functions have been evolved to secure the continued presence of these extrachromosomal elements with high efficiency. The plasmid maintenance functions primarily consist of the replication genes including their control circuits which regulate the plasmid concentration in the cell. In growing cells the replication control system monitors the number of plasmid copies and corrects deviations from the average by increasing or decreasing the probability for plasmid replication. However, no replication control system can prevent the occurrence of cells with very few or just one copy of the plasmid, and from such cells the possibility of formation of a plasmid-free daughter cell is obvious. This problem is of course greatest for low copy number plasmids. Moreover, a passive distribution of plasmid molecules at cell division will inevitably result in a certain frequency of plasmid-free cells. Since the loss of a plasmid molecule from a cell is irreversible, the consequences of such an unstable situation will be that the entire population eventually becomes plasmid-free.
Besides a random distribution of plasmids at cell division, other factors may influence the rate of loss of plasmids from a culture of cells grown in the absence of selection for the maintenance of the plasmid. For example, some plasmids require a specific recombination system in order to resolve two newly replicated molecules (Austin et al., Cell 25, 1981, pp. 729-36). In the absence of resolution, multimers will form (interlocked), and in this way even a high copy number plasmid may appear as a low copy number plasmid at the level of distribution to daughter cells, since the probability of generating plasmid-free cells will increase with increasing numbers of plasmid molecules interlocked in multimeric structures. Another phenomenon which is often observed in recombinant DNA technology is the conversion of a stable cloning vector into an unstable hybrid plasmid as a result of insertion of a DNA fragment, whose presence either causes a decrease in plasmid copy number or gives rise to a detrimental product that negatively interferes with cell growth.
In all such cases, plasmid segregation and loss occurs with a frequency (high or low) which cannot easily be controlled from the outside.
The stability of natural plasmids, and especially of low copy number plasmids, suggest that, in addition to the replication control system, a second set of maintenance functions exists which actively takes part in an ordered distribution of the plasmid molecules at cell division. Such functions have been formed partitioning functions, and studies, e.g., Meacock et al., Cell 20, 1980, pp. 529-42, Nordstrom et al., Plasmid 4, 1980, pp. 332-49, Seelke et al., Plasmid 7, 1982, pp. 163-79, have now shown that these are at least partly encoded by the plasmids themselves (in par regions). Thus, certain plasmid deletion mutants have lost their stable maintenance or inheritance despite their normal wild-type replication behaviour, indicating that a plasmid specified function securing stability has been removed. (c.f. Nordstrom et al., op.cit.).
As many of the plasmids employed as vectors in recombinant DNA technology have been deleted of a large amount of DNA compared to the wild-type parent plasmid, they are susceptible to being unstably inherited. This poses a serious problem since instability of a plasmid will ultimately result in a complete loss of the plasmid from the cells, and in any case will reduce the relative yield of plasmid-encoded gene products.
REFERENCES:
patent: 4487835 (1984-12-01), Uhlin et al.
Nordstrom et al. (1980) Plasmid, vol. 4, pp. 332-339.
Nordstrom, K. et al. (1980) Plasmid 4:215.
Meacock, P. et al. (1980) Cell 20:529.
Remant, et al. (1981) Gene 15:81.
Miki, T. et al., Jan. 1980, J. Bacteriol. 141:87.
Gerdes, K. et al., Jan. q985, J. Bacteriol, 161:292.
P. Gustafsson in New Hosts and Vectors for Recombinant DNA, Technology, IVA-PM No. 3, Mar. 1982, pp. 75-83.
R. W. Seelke et al., Plasmid 7, 163-179 (1982).
Gerdes Kenn A.
Molin Soen
A/S Alfred Benzon
Seidman Stephanie
Wiseman Thomas G.
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