Heterotic Trait Locus (HTL) Mapping Identifies Intra-Locus Interactions That Underlie Reproductive Hybrid Vigor in Sorghum bicolor

Identifying intra-locus interactions underlying heterotic variation among whole-genome hybrids is a key to understanding mechanisms of heterosis and exploiting it for crop and livestock improvement. In this study, we present the development and first use of the heterotic trait locus (HTL) mapping approach to associate specific intra-locus interactions with an overdominant heterotic mode of inheritance in a diallel population using Sorghum bicolor as the model. This method combines the advantages of ample genetic diversity and the possibility of studying non-additive inheritance. Furthermore, this design enables dissecting the latter to identify specific intra-locus interactions. We identified three HTLs (3.5% of loci tested) with synergistic intra-locus effects on overdominant grain yield heterosis in 2 years of field trials. These loci account for 19.0% of the heterotic variation, including a significant interaction found between two of them. Moreover, analysis of one of these loci (hDPW4.1) in a consecutive F2 population confirmed a significant 21% increase in grain yield of heterozygous vs. homozygous plants in this locus. Notably, two of the three HTLs for grain yield are in synteny with previously reported overdominant quantitative trait loci for grain yield in maize. A mechanism for the reproductive heterosis found in this study is suggested, in which grain yield increase is achieved by releasing the compensatory tradeoffs between biomass and reproductive output, and between seed number and weight. These results highlight the power of analyzing a diverse set of inbreds and their hybrids for unraveling hitherto unknown allelic interactions mediating heterosis

Divergent phenotypes induced by expression of bcl-XS: Cytokinetic effects and death without caspases.

bcl-XS, a member of the bcl-2 family, has been shown to induce and/or sensitize some cells to undergo programmed cell death and to negate the anti-apoptotic activity of bcl-XL and bcl-2 by mechanisms which are still uncertain. Using a tetracycline-regulated expression system, we have shown that expression of bcl-XS is able to elicit divergent phenotypic responses in different cell lines. In K12 rat colon carcinoma cells, we observe two phenotypic responses. A small fraction of cells undergo spontaneous programmed cell death while the majority of cells undergo a form of cytostasis. This stasis is the result of a redistribution of cells out of the S-phase, and into the G1-phase of the cell cycle. Expression of bcl-XS also decreased the viability of K12 cells, as demonstrated by a log decline in clonogenic survival. This decrease in viability is not prevented by caspase inhibition. Expression of bcl-XS was determined to be sufficient to induce acute cell death in 3T3 cells and the manner in which these cells die is both morphologically and biochemically different from Fas/CD95 induced apoptosis. Bcl-XS expression causes loss of the inner mitochondrial membrane potential (DeltaPsim) but does not induce caspase activation. In addition to the loss of DeltaPsim, electron microscopy showed gross structural perturbations in the mitochondria upon expression of bcl-XS. These data suggested that cytochrome c was being released from the mitochondria and should, therefore, trigger the activation of pro-caspase-9 and apoptosis. However, the lack of caspase activation. appeared to be due to a unique phenomenon also caused by expression of bcl-XS, depletion of cellular cytochrome c. In vitro pro-caspase activation assays proved that addition of exogenous cytochrome c to cytosolic extracts prepared from bcl-XS expresssing 3T3 cells is sufficient to trigger 35S-pro-caspase-9 processing. Furthermore, transient transfection experiments showed that cell death induced by bcl-XS is not inhibited by dominant negative caspase 9 but, rather, that cell death induced by bax or bak expression is inhibited. Consequently, bcl-XS must kill 3T3 cells through a pathway that does not require liberation and/or action of bax/bak nor through a caspase-dependent pathway.Ph.D.Biological SciencesCellular biologyHealth and Environmental SciencesOncologyPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132350/2/9963783.pd

Expression of Bcl-XS alters cytokinetics and decreases clonogenic survival in K12 rat colon carcinoma cells

bcl-XS, a member of the bcl-2 family, has been shown to induce and/or sensitize some cells to undergo programmed cell death, and to negate the anti-apoptotic activity of bcl-XL and bcl-2 by mechanisms which are still uncertain. To help understand these mechanisms we have established stable derivatives of the K12 rat colon carcinoma cell line that express bcl-XS in a tetracyclineregulated manner, using an autoregulatory retroviral cassette. When bcl-XS expression is induced, we observe two phenotypic responses. A small fraction of cells appear to undergo spontaneous apoptosis while the majority of cells undergo a form of cytostasis. In the latter case, the cells stop dividing (or divide a limited number of times at a retarded rate) and swell to many times their original size. These cells can take on a ghostlike appearance and subsequently detach from the culture plates and die or they may remain intact in a hindered state of proliferation. Doubling times were calculated to be 31.4 h in the presence of tetracycline and 50.4 h without tetracycline, bcl-XS expression also causes dramatic alterations in the cell cycle distribution of K12 cells manifesting as a substantial decrease (&50%) in the fraction of S phase cells with a concomitant increase in the G1 population. Continuous expression of bcl-XS, at levels approximately equal to that of bcl-XL, decreased the viability of K12 cells as demonstrated by a log decline in clonogenic survival. This decrease occurred without considerable apoptosis or a compensatory increase in the level of bcl-XL. None of these phenotypes were present in control cells expressing b-galactosidase in a similar retroviral cassette. These observations demonstrate that bcl-XS can have substantial cytokinetic eects under circumstances that produce relatively little apoptosis

A Caspase-Resistant Form of Bcl-X(L), but Not Wild Type Bcl-X(L), Promotes Clonogenic Survival After Ionizing Radiation

Bcl-2 and Bcl-X(L) belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-X(L) overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-X(L) protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-X(L) to inhibit these markers of apoptosis. When Bcl-X(L) was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-X(L) was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-X(L) by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-X(L) Bcl-X(L)Δloop. Cells overexpressing Bcl-X(L)-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-X(L)-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-X(L) overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-X(L) is not a prognostic marker of tumor response to cancer therapy