The increased tumor incidence in telomerase null mice suggests that telomere dysfunction induces genetic instability. To test this directly, we examined mutation rate in the absence of telomerase in S. cerevisiae. The mutation rate in the CAN1 gene increased 10- to 100-fold in est1Delta strains as telomeres became dysfunctional. This increased mutation rate resulted from an increased frequency of terminal deletions. Chromosome fusions were recovered from est1Delta strains, suggesting that the terminal deletions may occur by a breakage-fusion-bridge type mechanism. At one locus, chromosomes with terminal deletions gained a new telomere through a Rad52p-dependent, Rad51p-independent process consistent with break-induced replication. At a second locus, more complicated rearrangements involving multiple chromosomes were seen. These data suggest that telomerase can inhibit chromosomal instability.

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.We have recently generated barcoded, microRNA-based shRNA libraries targeting the entire human genome that can be expressed efficiently from retroviral or lenti-viral vectors in a variety of cell types for stable gene knockdown (1,2). Furthermore, we have also developed a method of screening complex pools of shRNAs using barcodes coupled with microarray deconvolution to take advantage of the highly parallel format, low cost, and flexibility in assay design of this approach (2,3). Although barcodes are not essential for enrichment screens (positive selection) (3-5), they are critical for dropout screens (negative selection) such as those designed to identify cell-lethal or drug-sensitive shRNAs (6). Hairpins that are depleted over time can be † To whom correspondence should be addressed. selledge@genetics.med.harvard.edu. * These authors contributed equally to this work. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript identified through the competitive hybridization of barcodes derived from the shRNA population before and after selection to a microarray (Fig. 1A).We previously described the use of 60-nucleotide barcodes for pool deconvolution (2,3). To provide an alternative to these barcodes that enables a more rapid construction and screening of shRNA libraries, we have developed a methodology called half-hairpin (HH) barcoding for deconvoluting pooled shRNAs (7). We took advantage of the large 19-nucleotide hairpin loop of our mir30-based platform and designed a polymerase chain reaction (PCR) strategy that amplifies only the 3′ half of the shRNA stem (Fig. 1B). As compared with full-hairpin sequences for microarray hybridization (8,9), HH barcodes entirely eliminate probe selfannealing during microarray hybridization (Fig. 1C and fig. S1, A and B), providing the critical dynamic range necessary for pool-based dropout screens. HH barcode signals are highly reproducible in replicate PCRs (R = 0.973, fig. S1A), highly specific (0.5% cross-reaction) ( fig. S1C), and display a reasonable, although slightly compressed, dynamic range in mixing experiments w...