Gram-positive bacteria replicon

Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales

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424 934, 424 9346, 424 93462, 4353201, 4352523, 43525231, 536 231, 536 237, 536 2371, 536 241, A01N 6300, C12N 121, C12N 1531, C12N 1563

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057665869

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BRIEF SUMMARY
CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a 371 of PCT/FR92/00711, filed Jul. 20, 1992.


BACKGROUND OF THE INVENTION

1. Field of the Invention
The invention relates to a novel type of Gram-positive replicon and its use for the construction of recombinant vectors.
2. Discussion of the Background
The naturally occurring bacterial plasmids are usually endowed with a high degree of stability. If they are distributed randomly the theoretical probability (L) of producing a plasmid-free cell is given by the equation: L=(1/2).sup.2n (where n is the number of plasmid molecules per cell and it is supposed that 2n copies of the plasmid are present immediately prior to division). Thus, high copy number plasmids can be maintained in a stable manner following random distribution, whereas efficient mechanisms are necessary to prevent the loss of low copy number plasmids in the absence of selection pressure (Nordstrom and Austin, 1989 Annu. Rev. Genet. 23:37-69).
In Gram-negative bacteria, several systems involving maintenance functions for the plasmids have been described for low copy number plasmids such as F, P1 and R1. They comprise the hok/sok and ccd systems responsible for the post-segregational destruction of the cells which lose plasmids (Jaffe et al., 1985 J. Bacteriol. 163:841-849; Gerdes et al., 1986 Mol. Microbiol. 4:1807-1818) and true partition functions which ensure that each daughter cell receives a plasmid molecule (Austin and Nordstrom, 1990 Cell 60:351-354). Furthermore, auxiliary functions which allow random distribution of the replicons have been described. Examples include the site-specific recombination system of Co1 E1 (Summers and Sherrat, 1984 Cell 36:1097-1103) and the r locus of pSC101 (Tucker et al., 1984 Cell 38:191-201).
In Gram-positive bacteria, much less information is available concerning the functions required for the stable maintenance of the plasmids. In the plasmids with high or low copy numbers which replicate by a "rolling circle" mechanism through a single-stranded DNA intermediate (te Riele et al., 1986a EMBO J. 5: 631-637, 1986b Proc Natl. Acad Sci. USA 83:2541-2545; Gruss and Ehrlich, 1989 Microbiol. Rev. 53:231-241), the stability is coupled to replication and not to a discrete function. In the case of various known plasmids pUB110, pTA1060, pMV158 (Bron et al., 1991b Plasmid 19:231-241), and pLS11 (Chang et al., 1987 J. Bacteriol. 169 :3952-3962), the regions of stability are the "negative" origins controlling the conversion of the single-stranded DNA into double-stranded plasmid DNA The replication of these plasmids is guaranteed by a protein encoded in the plasmid. These plasmids are frequently unstable from a structural and segregational point of view when they are used as cloning vectors, and this is probably due to their particular mode of replication.
A second class of Gram-positive replicons comprises the large plasmids with low copy numbers, pTB19, pIP404 and pAMbeta1 (Imanaka et al., 1986 Mol. Gen. Genet. 205:90-96; Garneir and Cole, 1988 Plasmid 19:151-160; Swinfield et al., 1990 Gene 87:79-90). Their replication requires a protein encoded in the plasmid which does not share any homology with that of the plasmids whose replication involves a "rolling circle" mechanism. These plasmids do not accumulate single-stranded DNA during replication (Gamier and Cole, 1988 Plasmid 19:151-160; Janniere et al., 1990 Gene 87:53-61) and are structurally stable (Janniere et al., 1990 Gene 87:53-61).
No information is available concerning the potential partition functions of these plasmids with low copy numbers However, these functions are essential for their maintenance in sporulating Gram-positive bacteria such as the Bacillus species. During the differentiation stage of these bacteria, the spore compartment represents only about one sixth of the cell, and the probability of producing a plasmid-free spore is: L=(5/6).sup.2n. A random distribution of the plasmids during sporulation may, consequently, lead to a serious loss of replicons (with n<10) in the s

REFERENCES:
Lereclus et al, FEMS Microbiology Letters, 1989, vol. 60: pp. 211-218.
Lereclus et al, FEMS Microbiology Letters, 1988, vol. 49: pp. 417-422.

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