DIFFERENTIAL AMPLIFICATION EFFICIENCY OF PMB1 AND P15A (COLE1-TYPE) REPLICONS IN ESCHERICHIA-COLI STRINGENT AND RELAXED STRAINS STARVED FORPARTICULAR AMINO-ACIDS

It was demonstrated previously that CoIE1-type plasmids, the most comm only used vectors in molecular cloning, can be amplified in amino acid -starved relA mutants of Escherichia coli. Subsequent studies demonstr ated that replication of at least some plasmids during amino acid star vation depends not only on the host relA allele but also on temperatur e and on the nature of deprived amino acid. Therefore, we investigated efficiency of amplification of two types of CoIE1 plasmids (pMB1- and p15A-derived replicons) in E. coli relA(+) and relA(-) hosts starved for different amino acids at 30 degrees C, 37 degrees C and 43 degrees C. We found differential amplification efficiency of plasmids pBR328 (pMB1-derived replicon) and pACYC184 (p15A-derived replicon) in the re lA mutant during starvation for particular amino acids. Although ampli fication of pBR328 was negligible in the relA(+) host, significant inc rease in pACYC184 content was observed in this strain starved for some (but not all) amino acids. The amplification efficiency of pBR328 and pACYC184 was found to be dependent on temperature. These results indi cate that for maximal amplification of particular plasmid appropriate amino acid starvation and optimal temperature should be chosen. Our fi ndings are in agreement with recently proposed model of the regulation of CoIE1-type plasmid replication in amino acid-starved E. coli cells .