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Maintenance of pBR322-derived plasmids without functional RNAI.
Plasmid 1991; 26(3):186-200P

Abstract

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.

Authors+Show Affiliations

Molecular and Cell Biology Programs, University of Texas at Dallas, Richardson 75083-0688.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

1721720

Citation

Chiang, C S., and H Bremer. "Maintenance of pBR322-derived Plasmids Without Functional RNAI." Plasmid, vol. 26, no. 3, 1991, pp. 186-200.
Chiang CS, Bremer H. Maintenance of pBR322-derived plasmids without functional RNAI. Plasmid. 1991;26(3):186-200.
Chiang, C. S., & Bremer, H. (1991). Maintenance of pBR322-derived plasmids without functional RNAI. Plasmid, 26(3), pp. 186-200.
Chiang CS, Bremer H. Maintenance of pBR322-derived Plasmids Without Functional RNAI. Plasmid. 1991;26(3):186-200. PubMed PMID: 1721720.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Maintenance of pBR322-derived plasmids without functional RNAI. AU - Chiang,C S, AU - Bremer,H, PY - 1991/11/1/pubmed PY - 1991/11/1/medline PY - 1991/11/1/entrez SP - 186 EP - 200 JF - Plasmid JO - Plasmid VL - 26 IS - 3 N2 - pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers. SN - 0147-619X UR - https://www.unboundmedicine.com/medline/citation/1721720/Maintenance_of_pBR322_derived_plasmids_without_functional_RNAI_ DB - PRIME DP - Unbound Medicine ER -