Journal: Applied and Environmental Microbiology

Article Title: Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

doi: 10.1128/AEM.01381-18

Figure Lengend Snippet: The transposon integration sites were confirmed by amplification of the region of the genome flanking the insertion sites. Panel A shows the genomic PCR amplification scheme for the AFKI1 strain. The primers flanking the insertion site were designed from the published genome sequence for ATCC 23270. In the wild-type genome, the PCR amplicon is of a known size. In the transposon-integrated strains, the same PCR primers were used to amplify the transposon region as well, resulting in a 3.5-kb increase in amplicon size. Panel B shows the results of the method applied to the three transposon locations identified in the mutant strains and visualized on an agarose gel, confirming the successful identification of integration sites.

Article Snippet: Using the NCBI Nucleotide blastx program, sequences were compared against the published ATCC 23270 genome to identify integration loci. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Primer Sequence (5′–3′) Tn5Fwd TAT TAT CTG CGG CCG CCA TCG ACT GCA CGG TGC AC Tn5Rev AGA TAT CTG CGG CCG CTG TCA CTT T Tn5Rev2 AGA TAT CTC GCG GCC GCA AAA AGG CCA TCC GTC AGG ATG HypTnpFwd TAC ACA AGT AGC GTC GCA TGC CAT CGA CTG CAC HypTnpRev TTA GGC GGG CTA CTA TCT AGA TGT CAC TTT GCT TGA TAT ATG AGA ATT ATT TAA C pBAMFwd GAC GCT ACT TGT GTA CTG TCT CTT ATA CAC ATC TGA CGT CTT GTG T pBAMRev TAG TAG CCC GCC TAA TGA GCG pBAM2F AAG CGG GGT AAG CGC AAG AAT pBAM2R ATC GCC CAT GTT ATG CAG AAA tn1RFwd CCA CTA CCG GCA AGT TCT CCG tn2RFwd CAG TTC ACC GAC ACC AAA GGT G tn1RRev TAT GAA GAT GCA TGA GCC GGT C tn1LFwd TCG TCG ACC GAG CTT TTG C tn1LRev GAA AGA GGA TGC GCC GAA AGT G tn2LRev GGG AAA GCT CTT CGC CGA AC tnFSeq TGC ACA GCC ATA CCA CAG CTT C tnRSeq GGC TAC AGC TCG TTT CAC GCT G AFKI1Fwd TCG CCG TTC GTT TTC TCG AFKI1Rev GCC ACC GCA TCC AGT AAT C AFKI2Fwd ATG GTT CAC ACC GAA ATC AAT GC AFKI2Rev CAT CCA TGC TAC AGC CTA AGT TGC C AFKI3Fwd CCT GAT GTA GTC GTT GGC GTC C AFKI3Rev GTT CGT CAA CAG CAA AGT GGA AC Open in a separate window Primers used in this study (iii) Confirmation of integration loci.

Techniques: Amplification, Sequencing, Mutagenesis, Agarose Gel Electrophoresis

Journal: Applied and Environmental Microbiology

Article Title: Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

doi: 10.1128/AEM.01381-18

Figure Lengend Snippet: Location of the chromosomal integration sites. Panel A shows the integration loci for KDC-integrated strains. The insertion locus identifies the 9-bp sequence duplicated by the transposase to insert the transposon. Panel B shows the approximate locations of the transposon insertions for the mutant strains in relation to the whole A. ferrooxidans 23270 genome.

Article Snippet: Using the NCBI Nucleotide blastx program, sequences were compared against the published ATCC 23270 genome to identify integration loci. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Primer Sequence (5′–3′) Tn5Fwd TAT TAT CTG CGG CCG CCA TCG ACT GCA CGG TGC AC Tn5Rev AGA TAT CTG CGG CCG CTG TCA CTT T Tn5Rev2 AGA TAT CTC GCG GCC GCA AAA AGG CCA TCC GTC AGG ATG HypTnpFwd TAC ACA AGT AGC GTC GCA TGC CAT CGA CTG CAC HypTnpRev TTA GGC GGG CTA CTA TCT AGA TGT CAC TTT GCT TGA TAT ATG AGA ATT ATT TAA C pBAMFwd GAC GCT ACT TGT GTA CTG TCT CTT ATA CAC ATC TGA CGT CTT GTG T pBAMRev TAG TAG CCC GCC TAA TGA GCG pBAM2F AAG CGG GGT AAG CGC AAG AAT pBAM2R ATC GCC CAT GTT ATG CAG AAA tn1RFwd CCA CTA CCG GCA AGT TCT CCG tn2RFwd CAG TTC ACC GAC ACC AAA GGT G tn1RRev TAT GAA GAT GCA TGA GCC GGT C tn1LFwd TCG TCG ACC GAG CTT TTG C tn1LRev GAA AGA GGA TGC GCC GAA AGT G tn2LRev GGG AAA GCT CTT CGC CGA AC tnFSeq TGC ACA GCC ATA CCA CAG CTT C tnRSeq GGC TAC AGC TCG TTT CAC GCT G AFKI1Fwd TCG CCG TTC GTT TTC TCG AFKI1Rev GCC ACC GCA TCC AGT AAT C AFKI2Fwd ATG GTT CAC ACC GAA ATC AAT GC AFKI2Rev CAT CCA TGC TAC AGC CTA AGT TGC C AFKI3Fwd CCT GAT GTA GTC GTT GGC GTC C AFKI3Rev GTT CGT CAA CAG CAA AGT GGA AC Open in a separate window Primers used in this study (iii) Confirmation of integration loci.

Techniques: Sequencing, Mutagenesis

Journal: Nature communications

Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

doi: 10.1038/s41467-019-13667-4

Figure Lengend Snippet: Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.

Article Snippet: U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073).

Techniques: Transfection, Western Blot, Staining, Standard Deviation, Expressing, SDS Page, Plasmid Preparation, Labeling

Journal: Nature communications

Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

doi: 10.1038/s41467-019-13667-4

Figure Lengend Snippet: Fig. 5 ATAD5 promotes generation of MUS81-mediated single-stranded DNA-associated breaks in response to replication stress. a, b U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for analysis by pulsed field gel electrophoresis. a Representative data from three independent experiments. Asterisk (*) indicates a DNA break. b DNA breaks were quantified and displayed (N = 4). c U2OS or HeLa cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for a neutral COMET assay. The tail moment was calculated from ~200 cells and plotted. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for another 6 h before being collected for a neutral COMET assay. Two independent experiments were performed, and one representative result is displayed. e U2OS-TetOn-ATAD5 cells treated with doxycycline for 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. f U2OS cells transfected with siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected to immunoblotting. g U2OS cells transfected with a combination of ATAD5 and MUS81 siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. The tail moment was calculated from ~300 cells and plotted. h HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min, washed, and treated with 2 mM HU for the indicated times. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. b–e, g Error bars represent standard deviation of the mean. Statistical analysis: two-tailed Student’s t test; *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.

Article Snippet: U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073).

Techniques: Transfection, Nucleic Acid Electrophoresis, Neutral Comet Assay, Expressing, Western Blot, Labeling, SDS Page, Standard Deviation, Two Tailed Test