The experimental evolution of laboratory populations of microbes provides an opportunity to observe the evolutionary dynamics of adaptation in real time. Until very recently, however, such studies have been limited by our inability to systematically find mutations in evolved organisms. We overcome this limitation by using a variety of DNA microarray-based techniques to characterize genetic changes—including point mutations, structural changes, and insertion variation—that resulted from the experimental adaptation of 24 haploid and diploid cultures of Saccharomyces cerevisiae to growth in either glucose, sulfate, or phosphate-limited chemostats for ∼200 generations. We identified frequent genomic amplifications and rearrangements as well as novel retrotransposition events associated with adaptation. Global nucleotide variation detection in ten clonal isolates identified 32 point mutations. On the basis of mutation frequencies, we infer that these mutations and the subsequent dynamics of adaptation are determined by the batch phase of growth prior to initiation of the continuous phase in the chemostat. We relate these genotypic changes to phenotypic outcomes, namely global patterns of gene expression, and to increases in fitness by 5–50%. We found that the spectrum of available mutations in glucose- or phosphate-limited environments combined with the batch phase population dynamics early in our experiments allowed several distinct genotypic and phenotypic evolutionary pathways in response to these nutrient limitations. By contrast, sulfate-limited populations were much more constrained in both genotypic and phenotypic outcomes. Thus, the reproducibility of evolution varies with specific selective pressures, reflecting the constraints inherent in the system-level organization of metabolic processes in the cell. We were able to relate some of the observed adaptive mutations (e.g., transporter gene amplifications) to known features of the relevant metabolic pathways, but many of the mutations pointed to genes not previously associated with the relevant physiology. Thus, in addition to answering basic mechanistic questions about evolutionary mechanisms, our work suggests that experimental evolution can also shed light on the function and regulation of individual metabolic pathways.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer that is characterized by the paradoxical features of intense apoptosis resistance yet a marked propensity to undergo necrosis. Bcl2L12 (for Bcl2-Like12) is a nuclear and cytoplasmic oncoprotein that is universally overexpressed in primary GBM and functions to block postmitochondrial apoptosis signaling by neutralizing effector caspase-3 and caspase-7 maturation. This postmitochondrial block in apoptosis engenders the alternate cell fate of cellular necrosis, thus providing a molecular explanation for GBM's classical features. Whereas Bcl2L12-mediated neutralization of caspase-7 maturation involves physical interaction, the mechanism governing Bcl2L12-mediated inhibition of caspase-3 activity is not known. The nuclear localization of Bcl2L12 prompted expression profile studies of primary astrocytes engineered to overexpress Bcl2L12. The Bcl2L12 transcriptome revealed a striking induction of the small heat shock protein ␣-basic-crystallin (␣B-crystallin/HspB5), a link reinforced by robust ␣B-crystallin expression in Bcl2L12-expressing orthotopic glioma and strong coexpression of ␣B-crystallin and Bcl2L12 proteins in human primary GBMs. On the functional level, enforced ␣B-crystallin or Bcl2L12 expression enhances orthotopic tumor growth. Conversely, RNAi-mediated knockdown of ␣B-crystallin in Bcl2L12-expressing astrocytes and glioma cell lines with high endogenous ␣B-crystallin showed enhanced apoptosis, yet decreased necrotic cell death with associated increased caspase-3 but not caspase-7 activation. Mirroring this specific effect on effector caspase-3 activation, ␣B-crystallin selectively binds procaspase-3 and its cleavage intermediates in vitro and in vivo. Thus, ␣B-crystallin is a Bcl2L12-induced oncoprotein that enables Bcl2L12 to block the activation of both effector caspases via distinct mechanisms, thereby contributing to GBM pathogenesis and its hallmark biological properties.heat shock protein ͉ apoptosis/necrosis balance ͉ glial cells

Glioblastoma multiforme (GBM) is a lethal brain tumor characterized by intense apoptosis resistance and extensive necrosis. Bcl2L12 (for Bcl2-like 12) is a cytoplasmic and nuclear protein that is overexpressed in primary GBM and functions to inhibit post-mitochondrial apoptosis signaling. Here, we show that nuclear Bcl2L12 physically and functionally interacts with the p53 tumor suppressor, as evidenced by the capacity of Bcl2L12 to (1) enable bypass of replicative senescence without concomitant loss of p53 or p19Arf, (2) inhibit p53-dependent DNA damage-induced apoptosis, (3) impede the capacity of p53 to bind some of its target gene promoters, and (4) attenuate endogenous p53-directed transcriptomic changes following genotoxic stress. Correspondingly, The Cancer Genome Atlas profile and tissue protein analyses of human GBM specimens show significantly lower Bcl2L12 expression in the setting of genetic p53 pathway inactivation. Thus, Bcl2L12 is a multifunctional protein that contributes to intense therapeutic resistance of GBM through its ability to operate on two key nodes of cytoplasmic and nuclear signaling cascades.

Embryonic stem (ES) cells hold great promise for the future of medicine. To elucidate the molecular mechanisms that control ES cell self-renewal and differentiation, a comprehensive knowledge of the molecules involved in these processes is required. Here we describe an effective approach for genomewide identification of functionally active genes in ES cells. This approach combines genetic screens based on cDNA libraries with microarray detection methods to permit high-throughput functional analyses. We implement this strategy to identify genes whose overexpression can maintain phenotypic properties of undifferentiated mouse ES cells under differentiation-inducing conditions, specifically in the absence of leukemia inhibitory factor. The identified genes encode a variety of regulatory proteins whose function in ES cells was previously unknown. Moreover, our approach is capable of detecting genes whose overexpression promote differentiation or cell death. Overall, our studies establish a methodology for highly sensitive identification of genes that confer particular phenotypes on ES cells.cDNA library ͉ differentiation ͉ microarray ͉ phenotype ͉ self-renewal