Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats

Abstract

The plasmid mini-Rts1, consisting of an EcoRI/HindIII fragment of about 1.8 kilobases (kb), contains an incompatibility determinant in its EcoRI/AccI region (0.5 kb). The nucleotide sequence of this incompatibility fragment was determined. A most striking feature of the sequence is the presence of five 24-base pair direct repeats. Four out of the five repeating units, which are contained in a 0.2-kb EcoRI/HincII fragment, were cloned en bloc in pACYC184 and found to express Rts1-specific incompatibility. In addition, the copy number of the mini-Rts1 plasmid appeared to be increased threefold upon removal of the 0.2-kb incompatibility region (incI) from the plasmid. This deletion derivative of mini-Rts1, as well as mini-Rts1, was maintained stably at 37 degrees C, but was cured at a high frequency at 42 degrees C. A possible role of the repeated nucleotide sequence was discussed. By subcloning the mini-Rts1 DNA, a second inc determinant (incII) was found on the AccI fragment, which is contiguous to the 0.5-kb EcoRI/AccI fragment.

A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

“…These are nested within short (heptamer) direct repeat elements (Figure 1). Although this feature is limited to a small group of low copy number plasmids that show homology to P1 (Kamio and Terawaki, 1983;Saul et al, 1988;Gabant et al, 1993), clustered Dam sites are common in the origins of bacterial chromosomes (Zyskind et al, 1983), including the Escherichia coli origin, oriC (Meijer et al, 1979). These origins also have GATC sites nested within repeat sequences, in this case 13mers (Figure 1).…”

Nucleotide Sequence of the Replication Region of Plasmid R401 and Its Incompatibility Function

“…The plasmid R401 belongs to the same incompatibility group (IncT) as Rtsl (5). The replication region of Rtsl contains two clusters of direct repeated sequences flanking the RepA coding region (13,14). In several plasmids or bacteriophages, R6K (21), F (18, 26), A dv (8,17), and P1 (1), direct repeated sequences and the coding region for a protein (Rep protein) are also known to be essential for autonomous replication.…”

“…In another group of plasmids, R6K (21), F (18, 26), and Rtsl (13,14), direct repeated sequences located close to the replication origin are involved in the incompatibility function. These repeated sequences are supposed to titrate the protein (RepA) essential for plasmid replication, resulting in control of initiation of the plasmid replication.Recently the nucleotide sequence of mini-Rtsl, the basic replicon of Rtsl consisting of 1,855 base pairs (bp), was determined (13,14). Two clusters of direct repeats were detected, between which the RepA protein coding region consisting of 288 amino acids was assigned (14).…”

“…Recently the nucleotide sequence of mini-Rtsl, the basic replicon of Rtsl consisting of 1,855 base pairs (bp), was determined (13,14). Two clusters of direct repeats were detected, between which the RepA protein coding region consisting of 288 amino acids was assigned (14).…”

Complete nucleotide sequence of mini-Rts1 and its copy mutant

“…Mini-Rtsl consists of a 1.8-kilobase (kb) EcoRI-Hindlil fragment which is capable of autonomous replication in a polA Escherichia coli host and is maintained stably at 37°C (10,22). Recently, the nucleotide sequence of the 0.5-kb EcoRI-AccI fragment of mini-Rtsl was determined, and the presence of five 24-base-pair (bp) direct repeats showing incompatibility and apparent copy control functions was demonstrated on the fragment (12). Such a cluster of direct repeats is found in the replication origin region of plasmids R6K (21), X dv (8,16), F (17,23), and bacteriophage P1 (A. L. Abeles, K. M. Snyder, and D. K. Chattoraj, J. Mol.…”