Results: 2

1.

Fig. 2. From: PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells.

DNA sequence analysis of the sites of chromosome healing at the I-SceI-induced DSB in wild type or Pif1 knockout ES cell lines. The DNA sequences are shown for rare chromosome healing events that did not occur at the site of the I-SceI-induced DSB. Comparisons are shown between the site of chromosome healing in the various Ganr subclones and the original sequence of the pPPT2-tel plasmid and telomeric repeat sequences (Tel repeats). Homologous nucleotides (*), and mutations or microhomology at the site of telomere addition (bold) are shown. The Pif1 knockout subclone PIF-9A-3 contained a mixture of two different healing events that occurred near each other following degradation of the DNA at the site of the DSB. The Pif1 knockout subclone PIF-9D-41 contains an inverted repeat consisting of murine genomic DNA 1275 bp from the plasmid integration site on chromosome 11, which is located between the pPPT2-tel plasmid sequences and the newly added telomere. The size of the deletions or inversion, and the amount of microhomology at the site of telomere addition or fusion is shown for each subclone.

Gloria E. Reynolds, et al. DNA Repair (Amst). ;10(11):1164-1173.
2.
Fig. 1

Fig. 1. From: PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells.

Analysis of rearrangements resulting from a DSB near a telomere in wild type or Pif1 knockout ES cell lines. (A) The structure of the telomeric pPPT2-tel plasmid sequences and the types of rearrangements resulting from I-SceI-induced DSBs in murine ES cell lines used in this study. The pPPT2-tel plasmid in ES cell clone 10P seeded the formation of a new telomere upon integration 1.4 Mbp from the end of chromosome 11. The telomere contains an ampicillin-resistance gene and plasmid origin of replication (amp/ori), a puromycin-resistance gene (Puro), an HSV-tk gene, an I-SceI endonuclease recognition site, and telomeric repeat sequences (telomere). The locations of the XbaI restriction sites used for Southern blot analysis are also shown. I-SceI endonuclease-induced DSBs near the telomere result in chromosome healing involving the addition of telomeric repeat sequences at the site of the break (lower left), inverted repeats resulting from sister chromatid fusion (lower right), or complete loss of the plasmid sequences (not shown). PCR using primers specific for the Puror gene and telomeric repeat sequences (arrows) and DNA sequence analysis are used to confirm the presence of a new telomere at the I-SceI site. (B) Southern blot analysis was performed on genomic DNA from I-SceI-induced Ganr subclones of the wild type 10PWT-CP and Pif1 knockout PIF-2H ES cell lines. DNA was digested with XbaI and hybridization was performed using the pNPTΔ plasmid as a probe (with no telomeric repeat sequences). Molecular weight markers consisting of Lambda bacteriophage HindIII fragments, as indicated. The unrearranged telomeric plasmid sequences in the parental wild type 10PWT-CP (10P) and Pif1 knockout PIF-2H (2H) ES cell lines produce two plasmid-specific bands, a 3 kbp band containing the HSV-tk gene, and a 4.3 kbp band containing the internal portion of the plasmid and adjacent cellular DNA (see Figure S1). In contrast, the I-SceI-induced Ganr subclones demonstrated a large XbaI plasmid-specific band at the limit of resolution (LOR) as a result of chromosome healing, variable-sized bands containing inverted repeats, or no plasmid-specific bands due to complete loss of the plasmid sequences. The hybridization of the murine Pgk gene promoter and polyA addition sequences in the pNPTΔ plasmid probe to the endogenous murine Pgk gene results in a light band of 4.4 kbp (pgk), while the plasmid sequences used to disrupt the Pif1 gene result in two bands of 8 and 10 kbp (KO).

Gloria E. Reynolds, et al. DNA Repair (Amst). ;10(11):1164-1173.

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