Control of initiation of pMB1 replication: Purified rop protein and RNA I affect primer formation in vitro

Lacatena, Rosa M.; Banner, David W.; Castagnoli, Luisa; Cesareni, Gianni

doi:10.1016/0092-8674(84)90435-5

Cited by 68 publications

(30 citation statements)

References 9 publications

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“…This is probably because rop is absent, as in the pUC plasmids, leading to a higher copy number (28), which may be detrimental to some strains. Also, in some strains transformed with the P c fusion plasmids, fluorescence may be lost over time.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, although the P c promoter was based on a compilation of several putative Campylobacter promoter sequences (49), this promoter can function in several enteric taxa. No binding sites for regulatory proteins (e.g., Cap and Fur) were inserted, and no promoters for alternate factors (i.e., 28 and 54 ) are present; therefore, this promoter should be constitutive and insensitive to many environmental and nutritional regulatory signals. However, the effect of other regulatory signals, such as DNA supercoiling, on transcription from the Campylobacter promoter has not been addressed.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp , and cfp Marker Plasmids

Miller

Bates

Horn

et al. 2000

Appl Environ Microbiol

129

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We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), or cfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacter promoter sequence (P c ). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the P c fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the P c fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp , and cfp Marker Plasmids

Miller

Bates

Horn

et al. 2000

Appl Environ Microbiol

129

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“…Pvu II has a restriction site in pBR322 and cuts TnS four times. A Hpa I plus Pvu II digest of pLO88 yields seven fragments, the smallest of which was isolated and ligated into Sma I-cut M13mp8 (37) (11,(13)(14)(15). The insertion leaves 43 of the 63 amino acids intact and is in-frame with this open reading frame.…”

Section: Resultsmentioning

confidence: 99%

“…A third aspect of ColEl plasmid copy number control involves a trans-acting (10) 63 (11) has been postulated to, in the presence of RNA I (12), affect the secondary structure of the nascent RNA primer, resulting in efficient transcription termination (13). The Rom protein has been shown to increase the inhibitory action of RNA I on in vitro primer formation and to enhance binding of RNA I to RNA II (14,15).…”

Section: Introductionmentioning

confidence: 99%

A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

Lupski¹,

Projan²,

Ozaki³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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In the process of randomly mutagenizing a recombinant pBR322 clone with transposon TnS, a high copy number plasmid mutant, pLO88, has been isolated. The copy number phenotype of pLO88 is observed only at elevated temperatures, >37C, and is due to the precise position of a TnS insertion. Nucleotide sequence of the TnS-pBR322 junction reveals that TnS-88 has inserted into an open reading frame that codes for a 63 amino acid protein previously shown to negatively regulate pBR322 plasmid copy number. By deleting portions of the TnS it is shown that the copy number phenotype is due not only to the insertion of TnS in pBR322 but also to the requirement that some TnS sequences remain intact. It appears that an outwardly directed TnS promoter inlitiates the synthesis of a transcript (RNA X) that interferes with the normal repressor RNA (RNA I)-primer RNA (RNA II) interaction at elevated temperatures.Initiation of DNA replication for ColEl plasmids depends on the formation of an RNA primer by the action of RNA polymerase and RNase H. Synthesis of the primer precursor transcript (RNA II) is initiated 555 base pairs (bp) upstream of the origin of DNA replication and this then forms a persistent hybrid with the template DNA near the origin of DNA replication. RNase H cleaves the hybridized transcript at the origin and this processed RNA II is used as a primer for DNA synthesis by DNA polymerase 1 (1-4). Regulation of the promoter for RNA II appears to be sufficient to control plasmid copy number (5). Primer formation is inhibited by a plasmid-specified 108-nucleotide repressor RNA, RNA I (6-8). RNA I synthesis starts 445 bp upstream from the origin of replication and proceeds in the opposite direction from RNA II synthesis. RNA I terminates near the site where RNA II synthesis starts and is therefore a complement of RNA II. When RNA I binds to RNA II, primer formation is inhibited (7, 9). Point mutations that affect formation and structure of RNA I will also affect RNA II and these have

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“…We propose that RNA II is involved in the initiation of plasmid replication, while RNA I acts as an inhibitor. The existence of a secondary copy control system in pMU720, such as those found in the IncFII (23,24,27,32,35,36) and ColEl (9,10,14,21,39,49,52) plasmids cannot be ruled out at this stage. However, it should be pointed out that our failure to detect any promoter activity within the first 334 bp of pMU720 DNA indicates that a CopB-like protein is not encoded upstream of the RNA II promoter.…”

Section: Methodsmentioning

confidence: 99%

Role of countertranscript RNA in the copy number control system of an IncB miniplasmid

et al. 1989

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Transcriptional mapping studies of the IncB minireplicon pMU720 demonstrated the existence of a long RNA molecule, RNA II, whose 5' portion is complementary to the product of the incompatibility gene RNA I. By using gene fusion and transcriptional fusion plasmids, it was shown that RNA I regulated the expression of the RNA II gene product and that it did so primarily at the level of translation. The target of RNA I was mapped to lie within a 216-base region of RNA II containing the sequence complementary to RNA I. Introduction of the target for RNA I in trans increased the copy number of an IncB minireplicon, indicating that RNA I and RNA II form the basis of the copy number control system of IncB plasmids.

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Control of initiation of pMB1 replication: Purified rop protein and RNA I affect primer formation in vitro

Cited by 68 publications

References 9 publications

Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp , and cfp Marker Plasmids

Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp , and cfp Marker Plasmids

A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

Role of countertranscript RNA in the copy number control system of an IncB miniplasmid

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