maintenance of plasmid psc101 in escherichia coli requires the host primase.the abilities of three escherichia coli strains with thermosensitive dnag alleles to maintain plasmids psc101 or pbr322 or an rp4 derivative were studied at elevated growth temperatures. under these conditions, psc101 segregated from cells to a greater extent than did pbr322. no segregation of the primase-encoding rp4 derivative was observed.19853900047
identification of the mob genes of plasmid psc101 and characterization of a hybrid psc101-r1162 system for conjugal mobilization.similarities in dna base sequence indicate that psc101 and r1162 encode related systems for conjugal mobilization, although these plasmids are otherwise very different. the mob region of psc101 was cloned, and two genes that are required for transfer were identified. one gene, moba, encodes a protein similar in amino acid sequence to the dna processing domain of the r1162 moba protein. the other gene, mobx, is within the same transcriptional unit as the psc101 moba and is located just downstream ...200010940031
the structure and function of the replication initiator protein (rep) of psc101: an analysis based on a novel positive-selection system for the replication-deficient mutants.plasmid psc101 encodes a 37.5 kda rep (repa) protein, which binds to three 21-base repeats (dr-1, dr-2, and dr-3) in the replication origin region (ori) of the plasmid to initiate replication. rep also binds to two palindromic sequences (ir-1 and ir-2) which overlap the rep promoter. the binding of rep to ir-2 represses the production of rep itself. it is highly likely that the balance of these functions of rep plays a major role in controlling the copy number of psc101. in this study, we develo ...200111530016
effect of nalidixic acid and novobiocin on pbr322 genetic expression in escherichia coli minicells.the effects of two deoxyribonucleic acid (dna) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pbr322 in escherichia coli minicells were studied. quantitative estimates of the synthesis of pbr322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactam ...19817031033
overinitiation of chromosome and plasmid replication in a dna acos mutant of escherichia coli k12. evidence for dnaa-dnab interactions.the dnaacos mutations are phenotypic suppressors of dnaats46 that are co-transduced with dnaa, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees c to 32 degrees c. we have used pulse labelling and dna-dna hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids psc101, rtf-tc, and lambda dv in such strains. after a shift of a dnaacos strain from 4 ...19846389885
a 37 x 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid psc101 and its temperature-sensitive derivative phs1.nucleotide sequences were determined for a region essential for autonomous replication and partitioning of psc101, a plasmid whose replication is dependent on the escherichia coli dnaa gene product. the essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 x 10(3) mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequenc ...19846327996
structure of a promotor on plasmid pmb9 derived from plasmid psc101.the dna sequence of a 354 basepair ecori-hindiii fragment of plasmid pmb9 which has originally been derived from plasmid psc101 has been resolved. this fragment contains a promoter for transcription directed towards the ecori site. escherichia coli rna polymerase binds to a region within the ecori-hindiii fragment which contains the heptamer 5' tatggtg (132-126) and the duodecamer 5' tgatgaacatca (158-147). based on commonalities with other promotors these dna sequences probably function as, res ...19806253940
genome construction between bacterial species in vitro: replication and expression of staphylococcus plasmid genes in escherichia coli.genes carried by ecori endonuclease-generated fragments of staphylococcus plasmid dna have been covalently joined to the e. coli antibiotic-resistance plasmid psc101, and the resulting hybrid molecules have been introduced into e. coli by transformation. the newly constructed plasmids replicate as biologically functional units in e. coli, and express genetic information carried by both of the parent dna molecules. in addition, electron microscope heteroduplex analysis of the recombinant plasmids ...19744598290
regions associated with the stable maintenance of plasmid psc101 and its tetracycline resistance.two regions tentatively called unsa and unsr were identified on psc101. one, unsa, corresponds to less than 650 bp of the n-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of psc101. the other, unsr, is defined within the 1 kb xhoi-ecori region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetr (unger et al. 1984); it serves as a suppressor of the unsa function.19863018437
the replication initiator protein of plasmid psc101 is a transcriptional repressor of its own cistron.the plasmid-encoded replication initiator protein of psc101 specifically repressed initiation of transcription of its own cistron from its natural promoter. addition of the purified initiator had little or no visible effect on transcription initiated from a heterologous promoter. dnase protection experiments revealed that the rna polymerase recognition sequence was overlapped by the initiator protein recognition sequences, which are vicinal to the replication origin. using the labeled promoter s ...19852986108
the partition locus of plasmid psc101 is a specific binding site for dna gyrase.a protein in extracts of escherichia coli that specifically binds the stabilizing par sequence of psc101 was identified as dna gyrase. the purified enzyme protects par against digestion by dnase i and exonuclease iii. competition assays demonstrate that gyrase has a 40-fold higher affinity for the 100-bp par sequence than for nonspecific dna and that par is the major gyrase-binding site in psc101 derivatives used in this and other studies. within par, at-rich sequences occur with a pronounced 10 ...19882844527
involvement of integration host factor (ihf) in maintenance of plasmid psc101 in escherichia coli: mutations in the topa gene allow psc101 replication in the absence of ihf.integration host factor (ihf), encoded by the hima and himd genes, is a histonelike dna-binding protein that participates in many cellular functions in escherichia coli, including the maintenance of plasmid psc101. we have isolated and characterized a chromosomal mutation that compensates for the absence of ihf and allows the maintenance of wild-type psc101 in him mutants, but does not restore ihf production. the mutation is recessive and was found to affect the gene topa, which encodes topoisom ...19892539359
exonuclease iii promotes in vitro binding of the replication initiator protein of plasmid psc101 to the repeated sequences in the ori region.purified rep protein, a replication initiator protein of plasmid psc101, has less binding affinity for the direct repeats (dr) in the replication origin region (ori) than that for the inverted repeats (ir) in the promoter region of the structure gene of rep (rep) (sugiura, s. et al. (1990) j. biochem. 107, 369-376). we found a protein factor that promotes binding of purified rep to the dr sequence in the cell extract of escherichia coli. in the presence of the factor, dna fragments containing th ...19911859836
boundaries of the psc101 minimal replicon are conditional.the dna segment essential for plasmid replication commonly is referred to as the core or minimal replicon. we report here that host and plasmid genes and sites external to the core replicon of plasmid psc101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. missense mutations in the plasmid-encoded repa protein or mutation of the escherichia coli topoisomerase i gene enable autonomous replication of a 310-bp psc101 dna fragment that contains only th ...19957665462
the effects of an escherichia coli dnaats mutation on the replication of the plasmids cole1 psc101, r100.1 and rtf-tc.the rate of replication of the plasmids cole1, psc101, r100.1 and par132 (an rtf-tc derivative of the drug resistance factor r100.1) has been investigated directly by dna:dna hybridization. these rates have been compared, in a dnaats strain, to that of various markers of the host chromosome at permissive and non-permissive temperatures. chromosome initiation in the dnaats strain stops rapidly after a shift to the non-permissive temperature, but plasmids r100.1 and par132 do not seem to be affect ...1979386040
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