Morphological differences in flowers have important evolutionary consequences; they influence the plants relationship with pollinators and are strongly correlated with sexual function in some breeding systems. Here, I explore the functional relationship between flower morphology and pollination dynamics (e.g. pollen receipt / export) in Solanum carolinense (Solanaceae) and evaluate whether this relationship varies with pollinator taxa. I also investigate if flower morphology determines fruit setting ability of flowers under different pollination regimes. Solanum carolinense has been characterized as having an andromonoecious sexual system where individual plants bear both hermaphroditic and male flowers. This species presents an ideal system to study the relationship between floral morphology, functionality and pollinators because flowers in natural populations vary in their style length and grow in diverse array of environments that vary in their pollinator fauna composition. I conducted a series of greenhouse experiments, pollinator observations and natural population surveys to test these relationships. My results demonstrate that long styled flowers serve as pollen recipients and short styled flowers as pollen donors. However, only bumblebees when (Bombus impatiens) are the pollinators I observe a positive relationship between style length and pollen deposition and a negative relationship with pollen removal. These findings support the female/male interference hypothesis and suggest that when plants are visited by species of species of Bombus, the differences in fitness could favor the evolution of andromonoecy. In contrast, when plants are visited either by Augochloropsis metallica or Lassioglossum spp. there is no selection for the dimorphism (or any particular style length). I also found that flower morphology, in particular style length, determines the fruit setting ability of the flowers in S. carolinense under different pollination regimes. However, in some flowers sexual functionality varies and does not accord with traditional classification of the flowers. The variation observed for style length, functionality and production of staminate flowers among individuals in natural populations of S. carolinense could be due to variation in abundance and visitation rate of pollinator taxa. Future studies should not neglect taxa-specific plant-pollinator interactions because the evolution of plant breeding systems can be determined by taxa specific interactions.

RNA Polymerase II (Pol II) transcription is a highly regulated process. Many factors associate with Pol II to ensure that transcription occurs as efficiently as possible. One of these factors is the Paf1 complex, which consists of the subunits Paf1, Ctr9, Rtf1, Cdc73, and Leo1. This complex has been shown to be important for the regulation of chromatin modifications that promote active transcription. Rkr1 was identified in a genetic screen to uncover factors that function in parallel with the Paf1 subunit Rtf1. My work has focused on characterizing a role for Rkr1 in transcription and chromatin function. I have shown that strains lacking RKR1 have transcription-related phenotypes. Genetic analysis has shown that Rkr1 functions in parallel with Rtf1-dependent histone modifications, particularly histone H2B ubiquitylation and histone H3 lysine 4 methylation. Strains lacking RKR1 have telomeric silencing defects, further connecting Rkr1 to chromatin function. Rkr1 is a nuclear protein that contains a RING domain at its extreme carboxy terminus. RING domain proteins often act as ubiquitin-protein ligases, which determine substrate specificity in the ubiquitylation pathway. Subsequent analyses have shown that Rkr1 does possess ubiquitin ligase activity in vitro, and mutational analysis shows that the RING domain of Rkr1 is required for in vivo activity. In an attempt to identify a functional process for Rkr1, a yeast two-hybrid screen was performed using an amino-terminal fragment of Rkr1 as bait. Twenty proteins were identified to interact with this region of Rkr1, many of which are functionally connected to transcription and chromatin. Microarray analysis shows that Rkr1 is required for proper expression of a subset of genes in yeast. Taken together, my work has identified a new ubiquitylation pathway within the nucleus that acts to regulate transcription and chromatin function.

Antitrypsin Deficiency is a primary cause of juvenile liver disease and arises from expression of the Z variant of the alpha-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retro-translocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed and a gene, ADD66, was identified that facilitates A1PiZ turn-over. I report here that ADD66 encodes an ~30 kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Ä mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also appears to be a proteasome substrate. Consistent with its role in ER associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC) and provide the first link between PAC activity and ERAD.

Hsp70 molecular chaperones play critical roles in the pathogenesis of many human diseases, including cancer and viral replication. Hsp70s bind polypeptides and couple ATP hydrolysis to alter substrate conformation and function. However, ATP hydrolysis by Hsp70 is weak, but can be stimulated by J domain-protein chaperones. To identify new targets of chaperone action, I performed a multi-copy suppressor screen for genes that improved the slow growth defect of yeast lacking YDJ1 but expressing a defective YDJ1 chimera. Among the genes identified were MID2, which regulates cell wall integrity, and PKC1, which encodes protein kinase C, which is also linked to cell wall biogenesis. Consistent with these data, I found that ydj1Δ yeast and yeast with temperature sensitive mutations in Hsp90 exhibit phenotypes consistent with cell wall defects but these phenotypes were improved by Mid2p or Pkc1p over-expression. Mid2p over-expression thickened the ydj1&Delta cell wall, which is likely the basis for suppression of the ydj1&Delta growth defect. These data provide the first link between cytoplasmic chaperones and cell wall integrity, and suggest that chaperones orchestrate the biogenesis of this structure. Another J domain-protein is the Large Tumor Antigen (TAg) in the polyomavirus Simian Virus 40 (SV40). TAg is required for viral replication and cellular transformation, and binds Hsp70. Because of their roles in cancer and SV40 function, small molecule modulators that inhibit Hsp70 or J-protein activity might represent novel anti-cancer and/or anti-viral agents. To identify such agents, I screened a bank of small molecules and identified a compound, MAL3-101, that had no effect on endogenous Hsp70 ATPase activity, but inhibited TAg stimulation of Hsp70 ATPase activity and reduced breast cancer cell proliferation. Forty-two derivatives of MAL3-101 were then synthesized and twelve compounds inhibited breast cancer cell proliferation at lower concentrations than MAL3-101. Reduction of cell proliferation correlated with reduced TAg stimulation of Hsp70 in vitro. Intriguingly, one compound, MAL2-11B, also inhibited the ATPase activity of TAg. This compound inhibited viral replication almost five-fold and SV40 DNA replication in vitro. These data show that J-protein inhibitors may be viable treatments for breast cancer and polyomavirus infection.

The ability of an organism to adapt to its surrounding environment is at the essence of survival. In metazoa, this ability starts at the level of the individual cell, which utilizes a specialized set of cytoskeletal proteins to determine their overall shape and the organization of their intracellular protein complexes and organelles. During embryonic development, the dynamic nature of the actin cytoskeleton is critical for virtually all morphogenic events requiring changes in cell shape, migration, adhesion, and division. The behavior of the actin cytoskeleton is modulated by a myriad of accessory proteins. Shroom3 (Shrm3) is an actin binding protein that regulates neural tube morphogenesis by eliciting changes in cell shape through a myosin II-dependent pathway. The Shroom-related gene SHROOM4 (formerly called KIAA1202) has also been implicated in neural development, as mutations in this gene are associated with human X-linked mental retardation. To better understand the function of Shrm4 in embryonic development, the mouse Shrm4 gene was cloned and its protein product was characterized both in vivo and in vitro. Shrm4 is expressed in a wide range of tissue types during mouse development, including the vascular endothelium of the lung and the polarized epithelium of the neural tube and kidney. In endothelial cells and embryo fibroblasts, endogenous Shrm4 co-distributes with myosin II to a distinct cytoplasmic population of F-actin and ectopic expression of Shrm4 in multiple cell types enhances or induces the formation of this actin-based structure. This localization is mediated, at least in part, by the direct interaction of Shrm4 and F-actin. The actin-binding motif of mShrm4 defines a novel actin-binding element that has not yet been described in other proteins. The results described here suggest that mShrm4 is a regulator of the actin cytoskeleton and may play an important role during vertebrate development, particularly in the developing vasculature.

The Kinesin-5 subfamily of the kinesin superfamily of molecular motors has been shown to play an integral role in the transfer of genetic material from mother-cell to daughter-cell. These homotetrameric kinesins function by crosslinking two microtubules in the mitotic spindle and imparting a force necessary to both assemble and maintain the spindle. The purpose of this dissertation has been to gain a better understanding of how Eg5/KSP, a member of the Kinesin-5 subfamily, coordinates the biochemical activities of its motor domains, to fulfill its cellular role. This dissertation focuses on a truncation of the human Eg5 gene that produces a dimeric motor. Analysis of this motor has indicated that the two motor domains, which interact with the same microtubule, function cooperatively. In some respects, dimeric Eg5 resembles conventional kinesin. Both motors are capable of translocating along the microtubule by taking successive steps before dissociating. To achieve this phenomenon, both motors couple the turnover of a single molecule of ATP to each advancement while maintaining the two motor domains out of phase through alternating catalytic cycles. Also, both motors have their stepping gated by ATP binding. The mechanistic commonalities between dimeric Eg5 and conventional kinesin, however, do not reach beyond a similar mechanism of stepping. This work has uncovered a novel biphasic, microtubule associated mechanochemical cycle. Dimeric Eg5 is the first kinesin known to begin the microtubule associated phase of its ATPase cycle with both motor domains associated with the microtubule. Furthermore, the transition to this two-motor-domain-bound state is the slow step governing steady-state ATP turnover. This slow transition only occurs once in the cycle and prior to processive movement. During processive movement, the catalytic step governs the rate of motor stepping. Dimeric Eg5 is also the first kinesin motor to have a rate-limiting catalytic step.

Inferences of organismal molecular evolution have been dominated by comparisons of their constituent genes. Yet the evolutionary histories of genes within Bacterial genomes are not necessarily congruent. Here, Horizontal Gene Transfer (HGT) of sequences across species boundaries can confound these analyses. There does appear to be phylogenetic cohesion, where members of higher taxonomic groups share genotypic similarity despite gene transfer. Herein I examine the rules for governing HGT to determine the impact this process has played in the evolution of Bacteria and Archaea. Bacterial chromosomes are more than simple lists of genes. Genomes must maintain information beyond component genes to direct efficient replication and segregation of their chromosomes. I propose that this structure constrains the process of HGT so that transfer among certain pairs of donors and recipients is favored. I present methods to detect this structure and new theories of bacterial cell biology and evolution based on what this structure reveals. I present evidence that bacterial chromosomes are structured by repetitive sequences termed Architecture IMparting Sequences (AIMS). AIMS are found primarily on leading strands and increase in abundance towards the replication terminus. Bacteria with robustly-identified replication origins and termini all have AIMS, and related AIMS are conserved amongst families of bacteria. We propose that AIMS are under selection to provide DNA binding proteins with polarity information, facilitating identification of the location of the replication terminus. Although AIMS evolved to direct the biology of cell division and replication, the conservation of AIMS among related taxa leads to a secondary effect. Because AIMS are counterselected when in nonpermissive orientations, AIMS constrain both intragenomic and intergenomic rearrangements. Thus HGT frequency will depend on AIMS compatibility between different species. We predict that HGT is most common between bacterial genomes which are more closely related and will impede transfer between species which have dissimilar genome architecture. The additional level of selection reflected by AIMS has resulted in cohesive bacterial groups that reflect common gene pools as a result of biased rates of gene transfer.

SV40 large T antigen (TAg) is a dominant acting oncoprotein that elicits transformation of many cell types and induces tumors in rodents. TAg induces transformation, in part, by disabling the functions of tumor suppressors such as pRb and p53. This dissertation is aimed to determine if inactivation of Rb and p53 are the major TAg activities required for transformation or if additional activities contribute. To determine whether Rb-family protein inactivation by the J domain of TAg is required for induction of intestinal hyperplasia, we have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type T antigen, the D44N mutant is unable to induce enterocyte proliferation. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. To determine if Rb inactivation is sufficient for the induction of hyperplasia or if progression to dysplasia requires some activity in the C-terminus of TAg (independent of p53), I have screened several transgenic lines expressing an amino-terminal mutant of TAg (N136) in villus enterocytes. I found that these mice develop intestinal hyperplasia, although not as early as wild-type TAg does, suggesting that the inactivation of Rb family members is sufficient to induce this phenotype. Furthermore, the appearance of signs of dysplasia was significantly delayed. I performed global analysis of gene regulation in MEFs and in mouse intestinal epithelium expressing TAg or various mutants. In mouse intestine most of the gene regulation is dependent on binding and inactivation of Rb-proteins by the LXCXE motif and J domain. Regulated genes are involved in cell cycle and proliferation. In MEFs genes belonging to cell cycle, apoptosis and growth factors are differentially regulated by TAg and its mutants. Additionally, we found upregulation of immune response genes by TAg requires the LXCXE motif and some activity mapping to the C-terminus of TAg for their regulation. Significant numbers of genes were found to be regulated independently of the LXCXE motif, J domain and p53 binding domain. This suggests activity independent of these functions.

Apolipoprotein B (ApoB) is the major structural protein component of chylomicrons and very low and low density lipoproteins, the major cholesterol carrying particles in the blood. High levels of ApoB have been directly linked to the risk of developing coronary artery disease. Pre-secretory degradation is one important mechanism regulating the assembly and secretion of ApoB. A major pre-secretory degradation pathway regulating ApoB production is Endoplasmic Reticulum Associated Degradation (ERAD), a pathway in which molecular chaperones play key roles. Molecular chaperones assist with protein folding, assembly, translocation, and targeting misfolded proteins to the proteasome for degradation. Previous work in mammalian cells and using cell-free systems with yeast cytosols established that the cytosolic chaperones, Hsp70 and Hsp90, promote ApoB ERAD. To identify additional chaperones involved in ApoB ERAD, I employed a cell-free system in which Hsp70, Hsp90 and proteasome-dependent degradation of the ApoB48 isoform is recapitulated with yeast cytosol. In addition, I developed an in vivo yeast expression system for the ApoB29 isoform. The shorter ApoB29 isoform is localized to the ER and associates with chaperones in yeast. Using both the in vitro and in vivo systems I found that a yeast Hsp110, Sse1p, associates with and stabilizes ApoB, which contrasts with data indicating that Hsp70 and Hsp90 facilitate ApoB degradation. Because Sse1p is reported to associate with Ssa1p, an Hsp70 known to regulate ApoB degradation in vitro, and Ssb1p and Ssb2p, Hsp70s in the ribosome associated complex (RAC), I tested the contributions of Ssb1p and Ssb2p on ApoB ERAD in vitro and in vivo but observed no difference in ApoB degradation rates compared to wildtype strains. Together, these data indicate that Sse1p, but not RAC, contributes to ApoB stabilization. To determine whether my results are relevant in mammalian cells, Hsp110 was over-expressed in hepatocytes and enhanced ApoB secretion was observed. This study indicates that chaperones within distinct complexes can play unique roles during ER-associated degradation (ERAD), establishes a role for Sse1/Hsp110 in ERAD, and identifies Hsp110 as a target to lower cholesterol.

A century of fire suppression and overbrowsing by deer have likely altered patterns of forest regeneration, with detrimental consequences for the future diversity, composition, and function of old-growth stands. We quantified the diversity and composition of tree species in the canopy and understory layers of 19 old-growth stands in Pennsylvania to evaluate the consequences of existing regeneration patterns for the future composition of these communities. Despite relatively high canopy diversity across all stands, the understory composition of all stands converged to a homogeneous subset of the canopy species. In addition, understory layers had unusually low stem densities, lower species diversity than their respective canopies, and showed a significant lack of mid-tolerant species. Oak species, which often require fire to regenerate, were common in the canopy of 12 stands, but absent from the understory layer of all stands. Our findings suggest that deer browsing and fire suppression are partly responsible for this dramatic alteration of understory species composition. The lower diversity and homogenized species composition of the understory indicates that the future composition of these stands will not resemble the current composition. Alteration of tree species composition is likely to have detrimental effects on the survival of species dependent upon old-growth habitat.