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  • Analysis of DNA fragmentation We followed a modified

    2020-01-19

    Analysis of DNA fragmentation. We followed a modified Hirt extraction procedure for analysis of DNA fragmentation. Cells were suspended in TNE buffer (1M NaCl, 20mM Tris–HCl, pH 7.4, 2mM EDTA, and 0.6% SDS) at a density of 106 cells/ml and incubated overnight on ice. The next day, cellular debris was spun down at 20,000g, at 4°C for 30min. The supernatant was digested with RNAseA (100μg/ml) for 30min at 37°C, extracted with phenol and chloroform, and precipitated with ethanol. The pellets were suspended in 100μl TE buffer. Tenμl of this extracted DNA was analyzed on a 1.5% agarose gel in TBE buffer, stained with ethidium bromide, and photographed under UV illumination. Western blotting. DT40 Phos-tag and heterozygous mutants cells were grown and cytoplasmic and nuclear extracts were prepared. Equal amounts of proteins were boiled in SDS sample buffer, separated by 15% SDS–PAGE, and transferred onto a nitrocellulose membrane. The membranes were blocked in TBST (10mM Tris–HCl, pH 8.0, 150mM NaCl, and 0.05% Tween 20) with 3% BSA and then incubated with anti-Chk-YB-1b antibody (1:2000) overnight at 4°C. Membranes were washed in TBST and incubated with alkaline phosphatase-conjugated secondary antibody (1:7500) for 1h and protein bands were visualized using 5-bromo-4-chloro-3 indolyl phosphate/nitro blue tetrazolium. Microarray analysis. Total RNA from DT40 and DT40YB1+/− cells was isolated using guanidine isothiocyanate (GITC)–phenol method [25] and poly(A+) RNA was purified using oligo(dT) cellulose column following standard procedures [24]. Two DNA microarrays of 120 human cell cycle-related genes were purchased from Clontech (Palo Alto, CA). Equal amounts of mRNA from DT40 or DT40YB1+/− cells were labeled with [α-]dATP as suggested by the manufacturer. By this method, we obtained cDNA probes with highly comparable specific activity. Each of the microarrays was then hybridized with the cDNA probe from DT40mRNA or DT40YB1+/− mRNA, washed, and exposed to a phosphorimager as suggested by Clontech. The results obtained were analyzed using the Atlas Image 1.5 software from Clontech. Data presented are an average of three experiments.
    Results
    Discussion Our results suggest that disruption of one allele of YB1 gene resulted in abnormal phenotypes in the DT40-YB-1b+/− cells, which include slower growth of cells, increased genomic DNA content, and morphological changes. YB-1 is a multifunctional protein with a significant role in RNA metabolism and transcriptional regulation of several genes. To date, proteins demonstrated to be capable of interacting with YB-1 include p53 [26], heterogeneous nuclear ribonucleoprotein K [27], single-stranded DNA binding proteins purα and purβ [28], and proliferating cell nuclear antigen [29]. Many of these YB-1 interacting proteins play an important role in cell proliferation. We have not observed any reduction in the amount of Chk-YB-1b mRNA or protein. This is likely because of the tetraploid phenotype of DT40YB-1b+/− cells. Since there are still two functional alleles of Chk-YB-1b in a tetraploid genome of DT40YB-1b+/− cells, the levels of Chk-YB-1b transcripts and protein may not have dropped below those seen in the wild-type DT40 cells. In such a scenario, one possible reason for the altered phenotype seen in DT40YB-1b+/− cells is the dominant negative effect exerted by the product of the disrupted allele. The mutant allele can potentially encode a truncated protein of about 12kDa, which may interact with other proteins and cause a dominant negative effect. Recently, it has been shown that YB-1 interacts with p53 protein through its C-terminal 14 amino acid sequence [26]. One of the domains is located at the N-terminus of YB-1. p53 has been implicated in a G2/M checkpoint. In the absence of wild-type p53, rereplication of DNA synthesis can occur, resulting in increased ploidy [30]. Some of our results (Fig. 3) can be explained by the absence of p53. Delayed mitosis can lead to rereplication of DNA and apoptotic cell death. Normally, this rereplication is inhibited by p53. In the absence of p53 [31], any perturbation in cell-cycle events may result in rereplication and apoptosis by a p53-independent pathway.