The R-Type Pyocin of Pseudomonas aeruginosa C Is a Bacteriophage Tail-Like Particle That Contains Single-Stranded DNA
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ABSTRACT Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C.Keywords:
Primer (cosmetics)
Restriction fragment
Extrachromosomal DNA
The ordering of restriction endonuclease fragments of HSV-2 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by EcoRI, Hind III, Bgl II, Xba and Hpa I have been constructed. The mol. wt. of the various regions which constitute HSV-2 genome are very similar to the corresponding mol. wt. in the HSV-1 genome.
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Cleavage (geology)
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HSL and HSV
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A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.
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Restriction endonucleases are indispensable tools in molecular biology and biotechnology.Type II restriction endonucleases are part of restriction modification systems.DNA fragment extraction and restriction mapping are the basis for several biotechnological activities.WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences.WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases.It will also provide frequency of restriction for each restriction endonuclease.
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This protocol, which describes how 3C libraries are made from formaldehyde-fixed mammalian cells, starts with cells that were previously cross-linked. Before embarking on a 3C-based analysis, decide which restriction enzyme will be used. Two parameters must be considered. First, the frequency with which a restriction enzyme cuts will determine the size of the restriction fragments and thus the resolution with which chromatin interactions can be detected. In most studies, researchers use restriction enzymes that cut once every ∼4 kb. If the intention is to analyze the composition of the 3C library using the 4C method, then a frequently cutting restriction enzyme that has a recognition sequence of four bases should be chosen. This is to ensure efficient inverse PCR-mediated amplification of ligation fragments. Second, using a restriction enzyme that cuts between elements of interest will result in each being assigned to a different restriction fragment. In that case, interactions can be studied for each individual genomic element of interest. It is not recommended to use restriction enzymes at temperatures higher than 37°C because prolonged incubation at higher temperatures will result in reversal of formaldehyde-induced cross-links. Finally, to avoid biased digestion of differentially methylated DNA, use a restriction enzyme that is not sensitive to DNA methylation.
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Genomic DNA were prepared from 5 sibling species of Anopheles hyrcanus group including An. sinensis (ASS), An. anthropophagus (ALA), An. liangshanensis (ALS), An. crawfordi (ACW) and An. xiaokuanus (AXK). High molecular weight DNA from each species were cut with three restriction endonucleases (Bgl II, Hae III and Pst I) and the DNA fragments analysed by agarose gel electrophoresis and ethidium bromide staining. Three enzymes (Bgl II, Hae III and Pst I) produced unique fragments in all sibling species tested. A diagnostic restriction fragment length of DNA from 5 sibling species of An. hyrcanus group may be derived from a single restriction enzyme pattern (i.e. Bgl II). The results demonstrated that the restriction fragment length differences of repetitive DNA could used as a tool to distinguish sibling species of An. hyrcanus group.
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genomic DNA
Ethidium bromide
Agarose gel electrophoresis
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Restriction fragment
Restriction site
Restriction map
Multiple cloning site
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Cosmid
Cloning (programming)
Linker
Recognition sequence
Exonuclease III
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Abstract Long-range restriction maps are important tools to determine the arrangement and distance of gene markers, and a prerequisite before committing oneself to costly and time-consuming cloning and sequencing projects in order to identify gene organization and rearrangements. For the construction of a low-resolution physical map of a genome or chromosomal region, the intact DNA is digested with a restriction endonuclease that cleaves infrequently and the fragments are separated by pulsed-field gel electrophoresis (PFGE) (1, 2). Restriction fragment patterns of small genomes, such as those of prokaryotes or lower eukaryotes, can be visualized directly by gel staining. With larger genomes, the complex mixture of fragments is blotted onto membranes after electrophoresis, and hybridized with suitable probes to detect fragments of interest. The order of probes and of fragments is established from the combinatorial analysis of partial and complete digests with one or more restriction enzymes. In the simplest case, two probes will hybridize to the same restriction fragment, thus revealing physical linkage between them. However, independent confirmation must always be sought to exclude hybridization to distinct but co-migrating bands. This may be accomplished by, for example, hybridization of the two probes in question to the fragments produced by partial digestion with the same enzyme as was used in the complete digest: both probes should detect the same bands (and with the same relative intensities) in this more complex mixture of fragments. Combined usage of several different enzymes should then allow construction of a macrorestriction map around both loci, which may be refined by double digestion analysis.
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genomic DNA
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