Showing papers by "Phillip A. Sharp published in 1971"

Studies on the molecular nature of r factors

Abstract: There are at least five potential experimental approaches to the problem of control of transferable drug resistance in pathogenic bacteria: (1) a search for new antimicrobials; (2) elimination of R factors by selective inhibition of R-factor replication; (3) interference with genetic expression of R-factors, either at the level of gene transcription or at the stage of translation of messenger RNA into protein; (4) prevention of formation of new R factors; and ( 5 ) inhibition of transfer of drug resistance. Although the first approach can be largely empirical, rational use of the last four approaches depends upon a fundamental understanding of extrachromosomal drug resistance at a molecular level. With this in mind, we have been attempting to elucidate the molecular nature of antibiotic resistance factors in the Enterobacteriaceae. Considerable controversy has existed about the molecular nature of R factors.'D2 Transduction experiments carried out in Salmonella and E. coli almost a decade ago led Watanabe and FukasawaS to propose that R factors consist of a transfer unit (RTF segment) that controls transmission of the plasmid, linearly linked, as shown in FIGURE la, to drug resistance determinants which carry genetic information specifying resistance to antimicrobial agents. More recent studies by Anderson and his collaborators,\" and later by Mitsuhashi and coworkers,5 have shown that the transfer units of at least certain classes of R factors can be transmitted alone as well as in combination with resistance determinants. Furthermore, these investigators have found that ordinarily nontransmissible R determinants can interact with transfer units (although not necessarily covalently as depicted in this Figure), which are then able to mediate their passage by conjugation (FIGURE la) . Once within a recipient cell, the linked transfer and resistance units can replicate autonomously. The earliest studies of the molecular nature of R factorsas7 indicated that heterogeneous satellite bands of DNA are associated with the presence of R factors in Proteus mirabilis and certain other bacterial species. However, the methods of nucleic acid extraction used in these early experiments yielded DNA having a maximum molecular weight of 10-15 million daltons, whereas the molecular weight of R-factor DNA in virro was estimated at more than three times this size.6 Because the R factor was not intact in these studies, it could not be determined whether the several R-factor satellite bands identified by cesium chloride gradient centrifugation reflected intramolecular heterogeneity within a single fragmented R-factor species, as in the case of bacteriophage X,8 or whether there were in fact functionally associated, but physically distinct, R-factor subunits. More recently, procedures have been developed that have made it possible to obtain high molecular weight DNA from bacteria by detergent lysis and phenol

Isolation of catenated forms of R factor DNA from minicells.

Abstract: STUDIES1–6 of the molecular nature of antibiotic resistance (R) factors in Escherichia coli have shown that several of them consist of covalently closed molecules of circular DNA. Use was made of this property in the separation of the R factor from chromosomal DNA of E. coli, which has a similar nucleotide-base composition. In selecting for covalently circular R factor DNA molecules, however, the procedures used in these earlier experiments-caesium chloride-ethidium bromide centrifuga-tion7 and bulk nitrocellulose adsorption2—necessarily selected against isolation of other (non-circular) forms of R factor DNA that might have been present.