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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30 degrees C but not at 42 degrees C, clones are isolated resistant to kanamycin at 42 degrees C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30 degrees C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42 degrees C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained. About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10(-10). The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn1 in many Gram-negative bacterial species.
Mol Gen Genet 1981
PMID:Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4. 627 48

A system where the transposition of MupApl (a derivative of phage Mu carrying a determinant coding for ampicillin resistance) is followed from the small plasmid pML2 into the conjugative plasmid R388 has been used to investigate the influence on Mu transposition of B, an early Mu gene which is involved in normal phage DNA synthesis. In the absence of active B protein a low level (about 1% of normal) of transposition was detected. Roughly a third of these transpositional events was found to lead to the formation of cointegrate DNA structures which were shown to consist of R388, two complete copies of Mu and part only of pML2. The pML2 deletions vary in size but all those investigated appear to originate at an end of Mu. An explanation of these observations is proposed which envisages the B protein as part of the normal transposition complex.
Mol Gen Genet 1982
PMID:Abnormal cointegrate structures mediated by gene B mutants of phage Mu: their implications with regard to gene function. 628 19

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.
Mol Cell Biol 1982 Aug
PMID:Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells. 629 Aug 75

The structures of two R-plasmids pWP14a and pWP12a (Tra-, Ap, Gm; 21 kb) and of several cointegrates they form with bacteriophages P1Cm and P1-15 were analyzed. In each case, replicon fusion was mediated by the element IS140 (about 0.8 kb), one copy of which resides on both plasmids adjacent to the gentamicin resistance determinant (AAC(3)-III). pWP14a cointegrated preferentially into or near the invertible C-loop structure of the P1 genome. Cointegrational mobilization of pWP14a was observed also with several conjugative R-factors. The process of replicon fusion is independent of the host's rec+ functions. Sequences homologous to IS140 are constituents of many R-factors, including RA1, R40a, R124, R144, Rts1, N3, and pJR255. IS140 also shows homology to two other sequences, IS15 delta and Tn2680, but not to other, well studied transposable elements. The ampicillin resistance determinant of pWP14a is within a Tn3-like transposon, Tn3651.
Mol Gen Genet 1983
PMID:Cointegrational transduction and mobilization of gentamicin resistance plasmid pWP14a is mediated by IS140. 630 69

DNA from bacteriophage PRD1 was extracted and partially digested with restriction endonuclease HaeII. The digest was cloned into the PstI site of plasmid pBR322 by homopolymer tailing with guanidylate tails on the plasmid and cytidylate tails on the phage DNA. Insert bearing plasmids were isolated by transforming E. coli strains for tetracycline resistance and screening for ampicillin sensitivity. These strains were then screened for the ability to accomplish marker rescue of nonsense mutants of bacteriophage PRD1. Additional clones were isolated by screening transformants with radioactively labeled probe PRD1 DNA fragments using colony hybridization. A genetic map was generated by the marker rescue capabilities of overlapping cloned inserts. This map allowed the ordering of fourteen of the known PRD1 complementation groups.
Mol Gen Genet 1983
PMID:Molecular cloning of bacteriophage PRD1 genomic fragments. 630 88

Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid. Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.
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PMID:A vehicle for DNA transfer and for recovery of transferred genes: lambda Charon phage-pBR322 hybrid. 631 39

We previously reported the existence of a series of chemically induced trans recessive copy-number mutations (cop) for mini-F plasmids and the existence of a similar series of cop mutations induced by insertion of the ampicillin resistance transposon Tn3. In this paper we describe the experiments showing that these two series of mutations are in different genes. Briefly, the experiments show that the one mutant series can complement the other, that the mutations map in distinct but adjacent regions, that the copy numbers of double mutants are the products of the copy numbers determined by the single mutations, and that Tn3 does not elevate copy number by a polar effect on the adjacent cop gene defined by chemical mutagenesis. We term the latter gene copA and the gene mutated by Tn3, copB. We also demonstrate here that copB mutations are recessive to the wild type allele. Further, we have characterized copB by deletion and recombinational analysis as the series of five 19- to 22-base-pair directly repeated sequences that had previously been designated incC-that is, one of the incompatibility genes. The evidence for this conclusion is that plasmids lacking two, three or five direct repeats have their copy number elevated proportionately. Possible mechanisms for copB control of replication are discussed.
Mol Gen Genet 1983
PMID:Identification and characterization of a second copy number control gene in mini-F plasmids. 631 37

We report that plasmid R46 provides a function which promotes recA-independent deletion, replicon fusion, and resolution of the fusion. R46 belongs to the incompatibility group N and specifies resistance to ampicillin, tetracycline, streptomycin and sulfonamide. Four kinds of deletion derivatives were observed by selection for susceptability to tetracycline from ampicillin-resistant clones. A common region, will be called alpha region thereafter, was postulated to be involved in these deletions. The replicon fusion occurred by a conjugative mobilization of each derivative with plasmid R388. The fusion was suggested to contain both replicons linked at each junction by the sequence in the alpha region in direct orientation. The resolution of the replicon fusion was found between two alpha regions and a consequently generated, parental deletion derivative and an R388 derivative which gained one alpha region. It is possible that the alpha region contains one potential Insertion Sequence (IS) element. These events were also speculated to occur as a consequence of insertion of the potential IS onto the intramolecular or intermolecular target sequence, or reciprocal recombination between two potential IS elements.
Mol Gen Genet 1984
PMID:Plasmid R46 provides a function that promotes recA-independent deletion, fusion and resolution of replicon. 631 64

Hybrid plasmids were constructed by combining in vitro the Escherichia coli plasmid pGA22, which carries the genes determining resistance to kanamycin, tetracycline, chloramphenicol and ampicillin, with the cryptic plasmids, pCG1 and pCG2, of Corynebacterium glutamicum. The hybrid plasmids were introduced into C. glutamicum and E. coli and replicated in both hosts. They expressed all the E. coli resistance phenotypes except ampicillin resistance in C. glutamicum. The levels of antibiotic inactivating enzymes encoded on these plasmids were about four to ten times lower in C. glutamicum than in E. coli. Despite the lack of expression of ampicillin resistance, beta-lactamase activity was detected in C. glutamicum carrying hybrid plasmids.
Mol Gen Genet 1984
PMID:Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum. 638 27

The extent of induction and approximate amount of DNA replication of a Mu prophage carrying a gene for ampicillin resistance can be monitored by assaying the level of beta-lactamase. The expression of the lacZ gene adjacent to either end of an induced Mu prophage remains virtually unaffected, until late in the Mu lytic cycle, while Mu DNA is replicating and transposing.
Mol Biol Rep 1980 Dec 31
PMID:Effects of prophage Mu induction on expression of adjacent host genes. 645 99


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