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Query: UNIPROT:P06889 (Mol)
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A derivative of plasmid F which is packageable in lambda phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus plasmid that carries the beta-lactamase gene, and a portion of guaA (B) transducing lambda phage DNA carrying lambda cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7 x 10(7) daltons, about 84% size of lambda phage genome, and can be packaged in lambda coat when helper phage replicates in the plasmid-carrier cell. The packaged plasmid and the helper lambda phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F- cell and establishes a plasmid state with normal efficiency. In F+ or Hfr cells, the resident F factor hinders this process.
Mol Gen Genet 1979 Jan 16
PMID:Construction and some properties of packageable plasmid F. 28 45

The staphylococcal penicillinase plasmid pI524 and a series of derivatives have been extensively mapped by restriction endonuclease digestion and by heteroduplex analysis. We report here the identification of a 2.2 kb region that undergoes a reversible, rec-independent inversion. This sequence is bounded by a pair of inverted repeats 650 base pairs in length, and has asymmetrically located recognition sites for at least three restriction endonucleases. A series of deleted derivatives and one naturally occurring, closely related plasmid, were studied. Two of these retain the inversion; the remainder are incapable of inverting and were all found to be locked in the same orientation of the inversion. The invertible sequence is adjacent to the region of the plasmid encoding beta-lactamase (bla); this entire region appears to be transposable and the inversion may be involved in the regulation of beta-lactamase expression or in translocation.
Mol Gen Genet 1979 Aug
PMID:Physical mapping of Staphylococcus aureus penicillinase plasmid pI524: characterization of an invertible region. 31 96

Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutnat phenotypes. Whereas all plasmid examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicells by ColE1 and ColE1 :: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be beta-lactamase. ColE1 :: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.
Mol Gen Genet 1977 Mar 07
PMID:The transposon Tn1 as a probe for studying ColE1 structure and function. 32 63

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.
Mol Gen Genet 1977 Apr 29
PMID:Restriction endonuclease mapping and mutagenesis of the F sex factor replication region. 32 74

An ampicillin transposon Tn901 was used as a "mutagen" to isolate insertion mutants of the bacteriocinogenic plasmid Clo DF13. By combining the obtained heteroduplex and restriction maps of the Clo DF13::Tn901 plasmids (van Emboden et al., 1977b) with their polypeptide pattern in minicells, we were able to map five genes on the Clo DF13 genome. These five genes designated A (cloacin gene), B, C, D, and G cover 55% of the coding capacity of Clo DF13 DNA. Since integration of Tn901 within these five genes did not result in a loss of the Clo DF13::Tn901 plasmids involved, it is suggested that these genes do not play an essential role in the maintenance of these plasmid insertion mutants. In addition, the described methods allowed us to indicate the initiation site of cloacin synthesis and to propose the counter-clockwise direction of transcription of the cloacin gene. The Tn901 DNA directed the synthesis of at least three polypeptides one of which is shown to be a TEM-1 beta-lactamase.
Mol Gen Genet 1978 Mar 20
PMID:Genetic map of the bacteriocinogenic plasmid CLO DF13 derived by insertion of the transposon Tn901. 34 43

A ColEl hybrid plasmid, pNUl, carrying the amp operon coding for chromosomal beta-lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning. By reciprocal recombination between pNUl and chromosome of two unstable beta-lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the beta-lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.
Mol Gen Genet 1979 Jun 07
PMID:Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12. 38 30

In two unrelated plasmids of incompatibility groups FII and N the gene for the SHV-1 beta-lactamase exists as part of a transposable element of molecular weight 9.5 megadaltons. This transposon has moved onto plasmids of at least three incompatibility groups; PI, Ialpha and J. This confirms the suggestion that the recent spread of the SHV-1 beta-lactamase has been associated with the transposition of the genetic determinant of this enzyme between unrelated plasmids.
Mol Gen Genet 1979 Oct 01
PMID:The nature of the genetic determinant for the SHV-1 beta-lactamase. 39 25

The E. coli recA gene was cloned from the phage lambda precA into the vector pBR313. A plasmid, pJL3, was also isolated by cloning a portion of the recA gene into the vector pBR322. pJL3 coded for a fragment of the recA protein 34 Kd (kilodaltons) in size (compared to 40 Kd for the intact protein). This fragment was antigenically related to the recA protein and its synthesis was subject to the same controls as that of the recA protein. The fragment did not express any detectable recA function. When wild-type cells with pJL3 were treated with nalidixic acid, the 34 Kd fragment and the beta-lactamase, made from a gene located downstream from the recA segment, were expressed at very high levels. Moreover, in these cells the rate of synthesis of intact recA protein from the chromosome was inhibited about 2-fold, relative to other chromosomal proteins, when compared to wild-type cells with the pBR322 vector. High level expression of the recA protein fragment and/or the beta-lactamase appeared to be lethal. The size of the 34 Kd fragment, taken together with the location of chain-termination codons in pJL3, localizes the regulatory region of the recA gene within 100 base pairs.
Mol Gen Genet 1979
PMID:Construction and characterization of a plasmid coding for a fragment of the Escherichia coli recA protein. 39 10

An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their beta-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33 degrees C, but carrier strains grow well at 28 degrees C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.
Mol Gen Genet 1976 Apr 23
PMID:Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids. 77 4

Mutant penicillinase plasmids, in which penicillinase synthesis is not inducible by penicillin or a penicillin analogue, were examined by biochemical and genetic analyses. In five of the six mutants tested, penicillinase synthesis could be induced by growth in the presence of 5-methyltryptophan. It is known that the tryptophan analogue 5-methyltryptophan is readily incorporated into protein by S. aureus and that staphylococcal penicillinase lacks tryptophan. 5-methyltryptophan seems to induce penicillinase synthesis in wild-type plasmids by becoming incorporated into the repressor and thereby inactivating the operator binding function of the penicillinase repressor. Therefore, induction of penicillinase synthesis in the mutant plasmids by 5-methyltryptophan strongly suggests that the noninducible phenotype of these five plasmids is due to a mutation that inactivates the effector binding site of the penicillinase repressor (i.e., the five mutant plasmids carry an iS genotype for the penicillinase repressor). This conclusion was supported by heterodiploid analysis. The mutant plasmid that did not respond to 5-methyltryptophan either produces an exceedingly low basal level of penicillinase or does not produce active enzyme. This plasmid seems to carry a mutation in the penicillinase structural gene or in the promoter for the structural gene. Thus, a genetic characterization of many mutations in the penicillinase operon can be accomplished easily and rapidly by biochemical analysis.
Mol Gen Genet 1976 Aug 10
PMID:Characterization of mutations in the penicillinase operon Staphylococcus aureus. 95 3


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