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The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Mitsudome Y, Takahama M, Hirose J, Yoshida N - AMB Express (2014)

Bottom Line: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons.Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively.This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen, Kibanadai-Nishi, Miyazaki 889-2192, Japan.

ABSTRACT
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

No MeSH data available.


Related in: MedlinePlus

Gene silencing effects of antisense DNA oligonucleotides (LacZ15, 90) on β -galactosidase activity inEscherichia coliJM109 (pUC18).(A) Values indicate the means ± standard deviation of three independent experiments. (B) Comparison of β -galactosidase band intensity on SDS-PAGE gels from E. coli bearing or lacking antisense DNA (LacZ15, 90). The peak area indicates the intensity of Coomassie-stained protein bands that correspond to β-galactosidase.
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Figure 3: Gene silencing effects of antisense DNA oligonucleotides (LacZ15, 90) on β -galactosidase activity inEscherichia coliJM109 (pUC18).(A) Values indicate the means ± standard deviation of three independent experiments. (B) Comparison of β -galactosidase band intensity on SDS-PAGE gels from E. coli bearing or lacking antisense DNA (LacZ15, 90). The peak area indicates the intensity of Coomassie-stained protein bands that correspond to β-galactosidase.

Mentions: β-galactosidase activity in E. coli penetrons that had been exposed to antisense DNA oligonucleotides (LacZ15 or LacZ90) was reduced to 38.4 and 26.0% of that in control E. coli penetrons respectively (Figure 3A). These antisense DNAs would have inhibited translation of targeted mRNA by binding complementary sequences in the 5’-region of the targeted mRNA and resulted in depression of β-galactosidase activity. The β-galactosidase activity in E. coli penetrons that were exposed to B-LAC15 or B-LAC90 was reduced to 84% of that in control E. coli penetrons. This result was attributed to the possibility that the mRNA encoding β-galactosidase carried complementary sequences similar to the B-LAC15 and B-LAC90 sequences.


The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Mitsudome Y, Takahama M, Hirose J, Yoshida N - AMB Express (2014)

Gene silencing effects of antisense DNA oligonucleotides (LacZ15, 90) on β -galactosidase activity inEscherichia coliJM109 (pUC18).(A) Values indicate the means ± standard deviation of three independent experiments. (B) Comparison of β -galactosidase band intensity on SDS-PAGE gels from E. coli bearing or lacking antisense DNA (LacZ15, 90). The peak area indicates the intensity of Coomassie-stained protein bands that correspond to β-galactosidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230895&req=5

Figure 3: Gene silencing effects of antisense DNA oligonucleotides (LacZ15, 90) on β -galactosidase activity inEscherichia coliJM109 (pUC18).(A) Values indicate the means ± standard deviation of three independent experiments. (B) Comparison of β -galactosidase band intensity on SDS-PAGE gels from E. coli bearing or lacking antisense DNA (LacZ15, 90). The peak area indicates the intensity of Coomassie-stained protein bands that correspond to β-galactosidase.
Mentions: β-galactosidase activity in E. coli penetrons that had been exposed to antisense DNA oligonucleotides (LacZ15 or LacZ90) was reduced to 38.4 and 26.0% of that in control E. coli penetrons respectively (Figure 3A). These antisense DNAs would have inhibited translation of targeted mRNA by binding complementary sequences in the 5’-region of the targeted mRNA and resulted in depression of β-galactosidase activity. The β-galactosidase activity in E. coli penetrons that were exposed to B-LAC15 or B-LAC90 was reduced to 84% of that in control E. coli penetrons. This result was attributed to the possibility that the mRNA encoding β-galactosidase carried complementary sequences similar to the B-LAC15 and B-LAC90 sequences.

Bottom Line: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons.Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively.This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen, Kibanadai-Nishi, Miyazaki 889-2192, Japan.

ABSTRACT
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

No MeSH data available.


Related in: MedlinePlus