Search

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells., Mouse ES cells are derived from the inner cell mass (ICM) of blastocysts (1,2), and have similar gene expression patterns to ICM cells (3). The defining features of ES cells [...]

Hypermutation in immunoglobulin genes produces a high frequency of substitutions of all four bases, which are likely generated by low-fidelity DNA polymerases. Indeed, humans deficient for DNA polymerase (pol) [eta] have decreased substitutions of A.T base pairs in variable and switch regions. To study the role of pol [eta] in a genetically tractable system, we created mice lacking pol [eta]. B cells from [Polh.sup.-/-] mice produced normal amounts of IgG, indicating that pol [eta] does not affect class switch recombination. Similar to their human counterparts, variable and switch regions from [Polh.sup.-/-] mice had fewer substitutions of A-T base pairs and correspondingly more mutations of C.G base pairs, which firmly establishes a central role for pol [eta] in hypermutation. Notably, the location and types of substitutions differ markedly from those in Msh[6.sup.-/-] clones, which also have fewer A x T mutations. The data suggest that pol [eta] preferentially synthesizes a repair patch on the nontranscribed strand, whereas MSH6 functions to generate the patch. class switch recombination | somatic hypermutation | low-fidelity DNA polymerase | mismatch repair

The entry of a fluorescent dye, dihydrorhodamine 6G (dR6G), increases in cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) after retrovirus- and adenovirus-mediated gene transfer. Dye influx is dependent on ATP and is prevented by Cl anion removal or the addition of 10 mM SCN anions. dR6G staining allows the flourescent analysis of CFTR role and the identification of successfully corrected cells after gene therapy.

Carboxyamidotriazole (CAI), an inhibitor of calcium influx, helps study the influence of calcium on endothelial cell proliferation, which is essential for angiogenesis. The presence of CAI affects neovascularization, as revealed by the inhibition of basic fibroblast growth factor-induced proliferation and invasion of human umbilical vein endothelial cells. This implies the necessity of calcium-mediated signal pathways for neovascularization.

The uneven postmitotic apportionment of oncogene-encoded proteins were studied in computer-assisted image analysis. Results show uneven mitotic apportionment of proteins with different subcellular localization, distinct biochemical properties and dissimilar functions. There is also unequal partitioning of DNA (26, 27), total RNA (19,28) and total protein (29) contentsin cultured cells. These results suggest that postmitotic cells expressing higher levels may be closer to the G1 to S transition and that cells with low levels would have a longer time of residency in G1.