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Almost all methods for transformation of the social amebaDictyostelium discoideumrely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure. Axenic growth requires several mutations. Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria. The method relies on a new selection...
In a range of salt-tolerant bacteria isolated from soil, rhizosphere, and phyloplane, cross-hybridization tests revealed a group of seven plasmids which were chosen for further study. Two plasmids (pSH1418 and pSH1451) were picked as representative examples of the two related subgroups. Restriction mapping and Southern blotting identified a region common to the two plasmids which later was identified...
pHS-2 is a 3-kb plasmid originally isolated fromShigella flexneriinfections associated with reactive arthritis in humans. This plasmid is stably maintained in many clinical isolates ofShigella flexneri.The nucleotide sequence of this plasmid displays two closely linked regions that may play a role in the maintenance of this plasmid. One region consists of a 250-bp locus showing a significant homology...
Suggestions are made for the estimation of plasmid loss rates in bacterial populations that multiply by binary fission. The plasmid loss rate is defined as the probability of a division of a plasmid-carrying individual giving birth to one plasmid-free and one plasmid-carrying daughter cell. The unit of time is consequently the interdivision time for plasmid-carrying individuals. The cautions that...
The highly conjugative pAD1 plasmid encodes a mating response to the peptide sex pheromone cAD1 secreted by recipient strains ofEnterococcus faecalis.The location of the origin of transfer (oriT) was determined through the ability to mobilize theEscherichia coli–E. faecalisshuttle plasmid pAM401 carrying specific segments of pAD1. TheoriTsequence was found to be completely within therepAdeterminant...
pSN22 is an 11-kbp, high-copy-numberStreptomycesplasmid which replicates via a single-stranded intermediate by the rolling circle replication (RCR) mechanism. We identified an unidirectional single-strand origin (SSO) of pSN22,sso1,where the initiation of second-strand synthesis takes place, located between thespdAandtraRgenes in a noncoding region which is functional in its natural orientation. The...
The complete nucleotide sequence of a naturally occurring 5.36-kb streptomycin and sulphonamide resistance plasmid, designated pIG1, isolated from type DPasteurella multocidawas determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were shown by sequence homologies and functional characterizations to be involved in the replication and mobilization of pIG1, respectively...
Clinical isolates ofEscherichia coliwere found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants ofE. coliDH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small,...
Tn5252is a promiscuous streptococcal element capable of madiating horizontal spread of multiple antibiotic resistance. To begin understanding the functional role of a transfer-related region in Tn5252,its nucleotide sequence was determined. Sequence of this 3.3-kb DNA segment revealed the presence of six open reading frames. The predicted amino acid sequence of one of the open reading frames, ORF9,...
Electroporation ofSpiroplasma citristrain GII3 with plasmid pMUT containing theStaphylococcus aureustransposon Tn4001resulted in random insertion of Tn4001into the spiroplasmal genome. Transformation frequencies reached 10 −8 per colony-forming unit (CFU) when 100 μg of plasmid DNA and 3 × 10 9 S. citriCFU were used. Three other strains ofS. citrifailed to be transformed under the...
A putative insertion sequence (IS1473) has been cloned and sequenced. The 1087-bp element was found between themoaAand thendhAgenes in the upstream region of the nicotine dehydrogenase (ndh) operon in the 160-kb pAO1 plasmid ofArthrobacter nicotinovorans.It is flanked by an imperfect repeat of 33 bp and carries two overlapping open reading frames which, by programmed −1 translational frameshifting,...
We describe the construction of restriction and genetic maps of plasmid pMOL28, which has a size of approximately 180 kb. To do so, partialBamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20–30 kb inserted DNA. Subsequently, a cosmid walking strategy, combined withBamHI orEcoRI restriction analysis and hybridization, was used to construct the restriction maps for...
A plasmid, pBP614 (5.647 kb), has been isolated from the New ZealandBacillus popilliaestrain 17 and characterized by physical mapping, cloning, and sequencing. An open reading frame was found which could encode a protein with homology to the replication (Rep) proteins of rolling-circle plasmids. The predictedB. popilliaeRep protein shows closest homology to Rep proteins fromBacillusplasmids of the...
We describe a vector and a streamlined procedure for isolating high-producing stable mammalian cell transfectants. The vector encodes theEscherichia coli lacZgene as a reporter. We show that levels of β-galactosidase activity, assayedin situin clonal isolates, can be used to identify clones producing high levels of the protein of interest (in this case, soluble human CD4 protein).
The 2.3-kb erythromycin resistance (Em R ) plasmid pPV141 ofStaphylococcus chromogenes3688 was isolated and characterized. Nucleotide sequence analysis identified ORF1 and ORF2 separated by a 445-bp spacing, encoding a 158-residue replication protein (Rep141) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern...
pACM1 is an 85-kb conjugative plasmid from a clinical isolate ofKlebsiella oxytocathat encodes resistance to β-lactams (mediated by SHV-5 extended spectrum β-lactamase), trimethoprim, sulfonamides, tetracycline, aminoglycosides, and mercuric chloride. The expression of the aminoglycoside resistance is difficult to detect, which could have clinical implications. A region of pACM1 containing five resistance...
Bacteriophages that produced turbid or clear zones of lysis in strains ofActinomyceswere isolated from 22 of 124 samples of fresh human dental plaque. All human and nonhuman strains ofActinomyces viscosusorActinomyces naeslundiitested in this study were sensitive to infection by one or more of these phages. In contrast, none of theActinomyces odontolyticus, Actinomyces israelii,orActinomyces bovisstrains...
The cyanobacteriumSynechocystissp. strain PCC 6803 contains several cryptic plasmids of 2.4, 5.2, and about 50 or 100 kbp. The complete nucleotide sequence of the 5.2-kbp plasmid, pCC5.2, has been analyzed and is reported here. This plasmid contains 5214 bp and 53.1% A+T. Six open reading frames, ORFs A–F (encoding peptides of larger than 90 amino acid residues), were located on both strands of pCC5...
After integration into the bacterial chromosome an exogenous gene may be stably expressed without continued selection for the recombinant locus. However, chromosomal integration events occur infrequently, requiring the concomitant integration of a drug resistance marker in order to identify colonies of recombinant cells. The generation of a drug-resistant recombinant strain can both reduce thein vivoapplicability...
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