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Sulfolobus mutants, generated via PCR products, which lack putative enzymes of UV photoproduct repair.

Sakofsky CJ, Runck LA, Grogan DW - Archaea (2011)

Bottom Line: In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays.The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR.The success of the gene-disruption strategy, which used 5' extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Cincinnati, 614 Rieveschl Hall, Clifton Court, Cincinnati, OH 45221-0006, USA.

ABSTRACT
In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5' extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

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Related in: MedlinePlus

Stimulation of marker exchange (SME) by UV. Relative frequency of recombinants in response to indicated UV fluence  UV was from an unfiltered germicidal lamp and the doses correspond to the short-wavelength portion of the output (λ < 300 nm). (a) CS1 (circles) versus DG185 (squares).  (b) CS3 (circles) versus DG185 (squares).
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fig4: Stimulation of marker exchange (SME) by UV. Relative frequency of recombinants in response to indicated UV fluence UV was from an unfiltered germicidal lamp and the doses correspond to the short-wavelength portion of the output (λ < 300 nm). (a) CS1 (circles) versus DG185 (squares). (b) CS3 (circles) versus DG185 (squares).

Mentions: Using this approach, we first compared the magnitude of SME as a function of UV dose in each disruptant to that of wild-type S. acidocaldarius (Figure 4). The results indicate no obvious impact of either the Saci_1227 or Saci_1096 gene disruption. Recombinant yields from the UV-treated mutants were generally higher than the control, but the differences were smaller than the standard deviation at each UV dose. Similarly, the kinetics of SME decay suggested only slightly slower decay in the Phr− strain, and the apparent difference was less than the standard deviation at each time point (Figure 5).


Sulfolobus mutants, generated via PCR products, which lack putative enzymes of UV photoproduct repair.

Sakofsky CJ, Runck LA, Grogan DW - Archaea (2011)

Stimulation of marker exchange (SME) by UV. Relative frequency of recombinants in response to indicated UV fluence  UV was from an unfiltered germicidal lamp and the doses correspond to the short-wavelength portion of the output (λ < 300 nm). (a) CS1 (circles) versus DG185 (squares).  (b) CS3 (circles) versus DG185 (squares).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139894&req=5

fig4: Stimulation of marker exchange (SME) by UV. Relative frequency of recombinants in response to indicated UV fluence UV was from an unfiltered germicidal lamp and the doses correspond to the short-wavelength portion of the output (λ < 300 nm). (a) CS1 (circles) versus DG185 (squares). (b) CS3 (circles) versus DG185 (squares).
Mentions: Using this approach, we first compared the magnitude of SME as a function of UV dose in each disruptant to that of wild-type S. acidocaldarius (Figure 4). The results indicate no obvious impact of either the Saci_1227 or Saci_1096 gene disruption. Recombinant yields from the UV-treated mutants were generally higher than the control, but the differences were smaller than the standard deviation at each UV dose. Similarly, the kinetics of SME decay suggested only slightly slower decay in the Phr− strain, and the apparent difference was less than the standard deviation at each time point (Figure 5).

Bottom Line: In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays.The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR.The success of the gene-disruption strategy, which used 5' extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Cincinnati, 614 Rieveschl Hall, Clifton Court, Cincinnati, OH 45221-0006, USA.

ABSTRACT
In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5' extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

Show MeSH
Related in: MedlinePlus