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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Retrons are genetic elements that encode multicopy single-stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single-stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem-loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem-loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: It has been shown that retrons, retro-elements in bacteria, produce a reverse transcriptase (RT) and multicopy single-stranded DNA(msDNA) whose 5′ end is covalently linked to RNA (msdRNA) by a 2′-5′ phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79-nucleotlde-long single-stranded DNA with monophosphate on its 5′ terminus. When the retron in strain 161 is cloned into E. coli K-12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K-12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA-DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5′ side. From the analysis of the RNA-DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83-nucleotide-tong DNA covalently linked to RNA. This RNA-DNA compound is further processed to the final product, the 79-nucleotide-long msDNA with a terminal 5′ monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA-DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA-RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA-RNA compound is autoca-talytically processed.
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli, such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli. To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Escherichia coli, RecA protein regulates the DNA damage-inducible survival-enhancing SOS response. Mutant aliele recA730, which causes constitutive SOS expression, is lethal at high temperatures in B/r, a derivative of wild-type B, but not in K-12 or in certain B/r-K-12 hybrids. We present evidence that killing is due to SOS induction of a defective retronphage, φR86, which is integrated into the B/r chromosome at 19 min, but is absent in K-12. φR86 contains retron EC-86 which encodes reverse transcriptase and a small multicopy DNA-RNA complex, msDNA-RNA. Induction of φR86 in recA730 B/r strains results in inhibition of host DNA replication before cell death. A retronphage ‘killer’ gene, ORF336, when overexpressed from a plasmid, causes similar effects without SOS induction. φR86 is not detectably u.v.-inducible in recA* strains.
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