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mutational and physiological analyses of plasmid pt181 functions expressing incompatibility.plasmid pt181 is a small multicopy plasmid from staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, inc3a and inc3b. inc3a incompatibility is expressed by the primary replication control determinant, copa, which specifies two small transcripts, rna i and rna ii, that jointly inhibit the synthesis of the rate-limiting initiator protein, repc. inc3b incompatibility is expressed by the leading strand replication origin and is due to com ...19901693440
the staphylococcus aureus mutation pcra3 leads to the accumulation of pt181 replication initiation complexes.in staphylococcus aureus cells carrying the pcra3 chromosomal mutation, plasmid pt181 and its derivatives were maintained at a reduced copy number. a significant proportion of their dna migrated during agarose gel electrophoresis as nicked dna. the results obtained in the characterization of this plasmid dna species show that it represents replication initiation complexes. such complexes could not be detected in a wild-type host. the replication initiation complexes present in pcra3 cells could ...19911942047
the cloning of chromosomal dna associated with methicillin and other resistances in staphylococcus aureus.competitive hybridization was used to detect the deletion of chromosomal dna accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant staphylococcus aureus (mrsa). the method was also used to screen a partial plasmid library of chromosomal hindiii fragments from the mrsa strain. eight recombinant plasmid clones were identified as containing dna included in the deletion. these clones were used as p ...19873668502
replication of plasmid pt181 dna in vitro: requirement for a plasmid-encoded product.pt181 is a naturally occurring 4.5-kilobase staphylococcus aureus plasmid encoding resistance to tetracycline. the plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. a cell-free extract has been developed that carries out complete replication of this plasmid. extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pt181. in an extract from which endogenous ...19816946436
replication-specific inactivation of the pt181 plasmid initiator protein.replication of the staphylococcus aureus plasmid pt181, which occurs by the rolling circle mechanism, is accompanied by the covalent attachment of a approximately 12-residue oligodeoxy-nucleotide to one subunit of the dimeric plasmid-coded initiator protein, repc. this oligonucleotide represents the plasmid sequence immediately 3' to the initiating nick site. the resulting heterodimeric protein lacks the topoisomerase and replication activities of unmodified repc, suggesting that the regulation ...19938235621
double-stranded origin nicking and replication initiation are coupled in the replication of a rolling circle plasmid, pt181.the staphylococcus aureus rolling circle plasmid pt181 initiator repc is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, repc*. repc/repc* heterodimer is an inhibitor of replication. however, in order to act effectively, the initiator/inhibitor protein must be stable. we show here that repc is stable for at least 90 min, which enables it to function effectively as an inhibitor of replication. this finding also allowed us to carry out the two stages in pt181 replic ...19979228752
sequence analysis of a cryptic plasmid from flavobacterium sp. kp1, a psychrophilic bacterium.a cryptic plasmid found at high copy number was isolated from flavobacterium sp. kp1, a psychrophilic gram-negative bacterium, cloned, and sequenced. the sequence will appear in the ddbj/embl/genbank databases under the accession number ab007196. the pfl1 plasmid is 2311 nucleotides in length with 32.7% gc content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. the plasmid contains two open reading frames of significant length, orfi and orfii. o ...19999919674
antisense rna-mediated transcriptional attenuation: an in vitro study of plasmid pt181.antisense rnas regulate plasmid replication by several different mechanisms. one of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pt181, and later for the streptococcal plasmids pip501 and pambeta1. previously, we performed detailed in vitro and in vivo analyses of the pip501 system. here, we present an in vitro analysis of the antisense system of plasmid pt181. the secondary structures of antisense and sense rna species of different lengths we ...200010760147
the pcra3 mutant binds dna and interacts with the repc initiator protein of plasmid pt181 but is defective in its dna helicase and unwinding activities.plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. the repc initiator protein binds to the plasmid pt181 dso in a sequence-specific manner and recruits the pcra helicase through a protein-protein interaction. subsequently, pcra unwinds dna at the nick site followed by replication by dna polymerase iii. the pcra3 mutant of staphylococcus aureus has previously been shown to be defective in plasm ...200516122559
genetic and biochemical characterization of the streptococcus pneumoniae pcra helicase and its role in plasmid rolling circle replication.pcra is a chromosomally encoded dna helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. efficient interaction between pcra and the plasmid-encoded replication initiator (rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a rep protein to interact with heterologous pcra helicases has been invoked as a determinant of plasmid promiscuity. we characterized transcription of the streptococcus pn ...200616936036
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