Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Analysis of differential gene expression in nontumorigenic glioblastoma multiforme

Loss of chromosome 10 represents the most common cytogenetic abnormality in high grade gliomas (glioblastoma multiforme). To identify genes involved in the malignant progression of human gliomas, a subtractive hybridization was performed between a tumorigenic glioblastoma cell line (LG11) and a nontumorgenic hybrid cell (LG11.3) containing an introduced chromosome 10. LG11 mRNA was subtracted from LG11.3 cDNA to produce cDNA probes enriched for sequences whose expression differs quantitatively from the parental tumorigenic cells. Both known and novel sequences were identified as a result of the subtraction. Northern blot analysis was then used to confirm differential expression of several subtracted clones. One novel clone, clone 17, identified a 2.6 kb message that showed a consistent two to four fold increase in expression in the LG11.3 nontumorigenic cells. Clone 17 (340 bp) was used successfully to screen for a near full-length version, RIG (regulated in glioma), which was 2,569 bp in size. The RIG cDNA sequence showed homology to clone 17 and to an anonymous EST (IB666), but to no previously identified genes. This screening effort also identified several independent clones representing novel sequences, most of which failed to show increased expression in the nontumorigenic GBM cells. Tissue distribution studies of RIG indicated highest levels of expression in human brain with appreciably lower levels in heart and lung. In vitro transcription and translation experiments demonstrated the ability of RIG to direct the synthesis of a 13 kD protein product. However, open reading frame analysis revealed no identify with previously described motifs or any known proteins. Using a combination of somatic cell hybrid panels and in situ hybridization, the RIG gene was mapped to chromosome 11p14-11p15. Further study of RIG and related gene products may provide insight into the negative regulation of glial oncogenesis. ^

Characterization of the human tissue transglutaminase gene promoter

Transglutaminases are a family of calcium-dependent enzymes, that catalyze the covalent cross-linking of proteins by forming $\varepsilon(\gamma$-glutamyl)lysine isopeptide bonds. In order to investigate the molecular mechanisms regulating the expression of the tissue transglutaminase gene and to determine its biological functions, the goal of this research has been to clone and characterize the human tissue transglutaminase promoter. Thirteen clones of the tissue transglutaminase gene were obtained from the screening of a human placental genomic DNA library. A 1.74 Kb fragment derived from DNA located immediately upstream of the translation start site was subcloned and sequenced. Sequence analysis of this DNA fragment revealed that it contains a TATA box (TATAA), a CAAT box (GGACAAT), and a series of potential transcription factor binding sites and hormone response elements. Four regions of significant homology, a GC-rich region, a TG-rich region, an AG-rich region, and HR1, were identified by aligning 1.8 Kb of DNA flanking the human, mouse, and guinea pig tissue transglutaminase genes.^ To measure promoter activity, we subcloned the 1.74 Kb fragment of the tissue transglutaminase gene into a luciferase reporter vector to generate transglutaminase promoter/luciferase reporter constructs. Transfection experiments showed that this DNA segment includes a functional promoter with high constitutive activity. Deletion analysis revealed that the SP1 sites or corresponding sequences contribute to this activity. We investigated the role of DNA methylation in regulating the activity of the promoter and found that in vitro methylation of tissue transglutaminase promoter/luciferase reporter constructs suppressed their basal activity. Methylation of the promoter is inversely correlated with the expression of the tissue transglutaminase gene in vivo. These results suggest that DNA methylation may be one of the mechanisms regulating the expression of the gene. The tumor suppressor gene product p53 was also shown to inhibit the activity of the promoter, suggesting that induction of the tissue transglutaminase gene is not involved in the p53-dependent programmed cell death pathway. Although retinoids regulate the expression of the tissue transglutaminase gene in vivo, retinoid-inducible activity can not be identified in 3.7 Kb of DNA 5$\sp\prime$ to the tissue transglutaminase gene.^ The structure of the 5$\sp\prime$ end of the tissue transglutaminase gene was mapped. Alignment analysis of the human tissue transglutaminase gene with other human transglutaminases showed that tissue transglutaminase is the simplest member of transglutaminase superfamily. Transglutaminase genes show a conserved core of exons and introns but diverse N-terminuses and promoters. These observations suggest that key regulatory sequences and promoter elements have been appended upstream of the core transglutaminase gene to generate the diversity of regulated expression and regulated activity characteristic of the transglutaminase gene family. ^

Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^

Play in Rats: Association across Contexts and Types , and Analysis of Structure.

Play has been proposed as a promising indicator of positive animal welfare. We aimed to study play in rats across contexts (conspecific/heterospecific) and types (social: pinning, being pinned; solitary: scampering), and we investigated its structure using behavioral sequence analysis. Group-housed (three per cage) adolescent male Lister Hooded rats (n = 21) were subjected to a Play-In-Pairs test: after a 3 hour isolation period, a pair of cage-mates was returned to the home cage and both social and solitary play were scored for 20 min. This procedure was repeated for each pair combination across three consecutive days, and individual play scores were calculated. Heterospecific play was measured using a Tickling test: rats were individually tickled by the experimenter through bouts of gentle, rapid finger movements on their underside, and the number of positive 50 kHz frequency modulated vocalizations and experimenter-directed approach behaviors were recorded. Both of the above tests were compared with social play in the home cage. While conspecific play in both the Play-In-Pairs test and home cage were correlated, both seemed to be unrelated to heterospecific play in the Tickling test. During the Play-In-Pairs test, although both solitary and social play types occurred, they were unrelated, and solitary locomotor play of one rat did not predict the subsequent play behavior of its cage mate. Analysis of play structure revealed that social play occurred more often in bouts of repeated behaviors while solitary play sequences did not follow a specific pattern. If play is to be used as an indicator of positive welfare in rats, context, type and structure differences should be taken into account.

Assessing the Potential of Multiple Imputation in Sequence Analysis

Sequence analysis and optimal matching are useful heuristic tools for the descriptive analysis of heterogeneous individual pathways such as educational careers, job sequences or patterns of family formation. However, to date it remains unclear how to handle the inevitable problems caused by missing values with regard to such analysis. Multiple Imputation (MI) offers a possible solution for this problem but it has not been tested in the context of sequence analysis. Against this background, we contribute to the literature by assessing the potential of MI in the context of sequence analyses using an empirical example. Methodologically, we draw upon the work of Brendan Halpin and extend it to additional types of missing value patterns. Our empirical case is a sequence analysis of panel data with substantial attrition that examines the typical patterns and the persistence of sex segregation in school-to-work transitions in Switzerland. The preliminary results indicate that MI is a valuable methodology for handling missing values due to panel mortality in the context of sequence analysis. MI is especially useful in facilitating a sound interpretation of the resulting sequence types.

Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses.

Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Leopard Complex and CSNB phenotype in horses. Re-sequencing of the gene and associated splice sites within the 105 624 bp genomic region of TRPM1 led to the discovery of 18 SNPs. Most of the SNPs did not have a predictive value for the presence of LP. However, one SNP (ECA1:108,249,293 C>T) found within intron 11 had a strong (P < 0.0005), but not complete, association with LP and CSNB and thus is a good marker but unlikely to be causative. To further localize the association, 70 SNPs spanning over two Mb including the TRPM1 gene were genotyped in 192 horses from three different breeds segregating for LP. A single 173 kb haplotype associated with LP and CSNB (ECA1: 108,197,355- 108,370,150) was identified. Illumina sequencing of 300 kb surrounding this haplotype revealed 57 SNP variants. Based on their localization within expressed sequences or regions of high sequence conservation across mammals, six of these SNPs were considered to be the most likely candidate mutations. While the precise function of TRPM1 remains to be elucidated, this work solidifies its functional role in both pigmentation and night vision. Further, this work has identified several potential regulatory elements of the TRPM1 gene that should be investigated further in this and other species.

Phylogenetic analysis of receptor FgfrL1 shows divergence of the C-terminal end in rodents

FGFRL1 is a member of the fibroblast growth factor receptor (FGFR) family. Similar to the classical receptors FGFR1-FGFR4, it contains three extracellular Ig-like domains and a single transmembrane domain. However, it lacks the intracellular tyrosine kinase domain that would be required for signal transduction, but instead contains a short intracellular tail with a peculiar histidine-rich motif. This motif has been conserved during evolution from mollusks to echinoderms and vertebrates. Only the sequences of FgfrL1 from a few rodents diverge at the C-terminal region from the canonical sequence, as they appear to have suffered a frameshift mutation within the histidine-rich motif. This mutation is observed in mouse, rat and hamster, but not in the closely related rodents mole rat (Nannospalax) and jerboa (Jaculus), suggesting that it has occurred after branching of the Muridae and Cricetidae from the Dipodidae and Spalacidae. The consequence of the frameshift is a deletion of a few histidine residues and an extension of the C-terminus by about 40 unrelated amino acids. A similar frameshift mutation has also been observed in a human patient with a craniosynostosis syndrome as well as in several patients with colorectal cancer and bladder tumors, suggesting that the histidine-rich motif is prone to mutation. The reason why this motif was conserved during evolution in most species, but not in mice, is not clear.

VERIFICATION OF DNA PREDICTED PROTEIN SEQUENCES BY ENZYME HYDROLYSIS AND MASS SPECTROMETRIC ANALYSIS

The focus of this thesis lies in the development of a sensitive method for the analysis of protein primary structure which can be easily used to confirm the DNA sequence of a protein's gene and determine the modifications which are made after translation. This technique involves the use of dipeptidyl aminopeptidase (DAP) and dipeptidyl carboxypeptidase (DCP) to hydrolyze the protein and the mass spectrometric analysis of the dipeptide products.^ Dipeptidyl carboxypeptidase was purified from human lung tissue and characterized with respect to its proteolytic activity. The results showed that the enzyme has a relatively unrestricted specificity, making it useful for the analysis of the C-terminal of proteins. Most of the dipeptide products were identified using gas chromatography/mass spectrometry (GC/MS). In order to analyze the peptides not hydrolyzed by DCP and DAP, as well as the dipeptides not identified by GC/MS, a FAB ion source was installed on a quadrupole mass spectrometer and its performance evaluated with a variety of compounds.^ Using these techniques, the sequences of the N-terminal and C-terminal regions and seven fragments of bacteriophage P22 tail protein have been verified. All of the dipeptides identified in these analysis were in the same DNA reading frame, thus ruling out the possibility of a single base being inserted or deleted from the DNA sequence. The verification of small sequences throughout the protein sequence also indicates that no large portions of the protein have been removed after translation. ^

An analysis of the function of region 1.2 of the primary sigma factor, sigma 70, from Escherichia coli

The sigma (σ) subunit of eubacterial RNA polymerase is required for recognition of and transcription initiation from promoter DNA sequences. One family of sigma factors includes those related to the primary sigma factor from E. coli, σ70. Members of the σ70 family have four highly conserved domains, of which regions 2 through 4 are present in all members. Region 1 can be subdivided into regions 1.1 and 1.2. Region 1.1 affects DNA binding by σ 70 alone, as well as transcription initiation by holoenzyme. Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter specific binding by holoenzyme. This study primarily investigates the functional role of region 1.2 during transcription initiation. In vivo and in vitro characterization of thirty-two single amino acid substitutions targeted to region 1.2 of E. coli σ70 as well as a deletion of region 1.2, revealed that mutations in region 1.2 can affect promoter binding, open complex formation, initiated complex formation, and the transition from abortive transcription to elongation. The relative degree of solvent exposure of several positions in region 1.2 has been determined, with positions 116 and 122 likely to be located near the surface of σ70. ^ During the course of this study, the existence of two “wild type” variants of E. coli σ70 was discovered. The identity of amino acid 149 has been reported variably as either arginine or aspartic acid in published articles and in online databases. In vivo and in vitro characterization of the two reported variations of E. coli σ70 (N149 and D149) has determined that the two variants are functionally equivalent. However, in vivo and in vitro characterization of single amino acid substitutions and a region 1.2 deletion in the context of each variant background revealed that the behavior of some mutations are greatly affected by the identity of amino acid 149. ^

Analysis of BMP signalling through the receptor type IA in embryogenesis using conditional gene inactivation in mice

Although bone morphogenetic proteins (BMPs) were initially identified for their potent bone-inducing activity, their precise roles in processes of endochondral and intramembranous bone formation are far from being clear. Tissue-specific loss-of-function experiments using the BMP receptor type IA (BMPR-IA) are particularly attractive since this receptor is thought to be essential for signaling by the closely related BMPs -2, 4, and 7. To ablate signaling through this receptor during chondrogenesis, we have generated transgenic mice expressing Cre recombinase under the control of the collagen type II (Col2a1) gene regulatory sequences. Mice lacking BMPR-IA function in chondrocytes display a number of skeletal abnormalities, including defects in bones of the chondrocranium, abnormal dorsal vertebral processes, scapulae with severe hypoplasia of dorsal elements, and shortening of the long bones. Alterations in the growth plate of long bones in mutants suggest that BMPR-IA is not required for early steps of the chondrocyte specification, but is rather important in regulation of terminal differentiation. Molecular analysis revealed noticeable downregulation of the Ihh/Ptch signalling pathway, decreased chondrocyte proliferation rate and deregulation of hypertrophy. ^ In order to elucidate the role of BMP signalling in development of the limb and intramembranous ossification, we have used mice expressing Cre recombinase under control of the Prx1 (MHox) regulatory elements (M. Logan, pers comm.). Cre activity was found in those mice in the developing limb bud mesenchyme, as well as in a subset of cranial neural crest cells. Prx1-Cre-induced conditional mutants display prominent defects in distal limb outgrowth, as well as ossification defects in a number of neural crest-derived calvarial bones. Intriguingly, mutant limbs displayed alterations in patterning along all three axes. Molecular analysis revealed ectopic anterior Shh/Ptch signalling pathway activation and expression of some Hox genes. Observed loss of Msx1 and Msx2 expression in the progress zone correlates with downregulation of Cyclin D1 and decreased distal outgrowth. Abnormal ventral localization of Lmx1b-expressing cells along with observed later morphological abnormalities suggest a novel role for BMP signalling in establishment or maintaining of the dorso-ventral polarity in the limb mesoderm. ^

Functional analysis of BMP4 signaling in mouse neural development

During early mouse neural development, bone morphogenetic protein (BMP) signaling patterns the dorsal neural tube and defines distinct neural progenitor cell domains along the dorsoventral axis. Unlike the ventral signaling molecule Sonic hedgehog, which has long-range activity by establishing a concentration gradient in the ventral neural tube, these dorsally expressed BMPs appear to have a limited domain of action. This raises questions as to how BMP activity is restricted locally and how restricted BMP signaling directs dorsal neural patterning and differentiation. I hypothesize that BMPs are restricted in the dorsal neural tube for correct dorsoventral patterning. ^ Previous studies have shown that the positively charged basic amino acids located at the N-terminus of several BMPs are essential for heparin binding and diffusion. This provides a novel tool to address these questions. Here I adapted a UAS/GAL4 bigenic mouse system to control the ectopic expression of BMP4 and a mutant form of BMP4 that lacks a subset of the N-terminal basic amino acids. The target genes, UAS-Bmp4 and UAS-mBmp4 , were introduced into the Hprt locus by gene targeting in mouse embryonic stem cells. The expression of the GAL4 transactivator was driven by a roof plate specific Wnt1 promoter. ^ The bigenic mouse embryos exhibit phenotype variations, ranging from mid/hindbrain defects, hemorrhage, and eye abnormalities to vasculture formation. Embryonic death starts around E11.5 because of severe hemorrhage. The different expression levels of the activated transgene may account for the phenotype variation. Further marker analysis reveals that mutant BMP4 induces ectopic expression of the dorsal markers MSX1/2 and PAX7 in the ventral neural tube. In addition, the expression of the ventral neural marker NKX2.2 is affected by the expanded BMP4 activity, indicating that ectopic BMP signaling can antagonize ventral signaling. Comparison of the phenotypes of the Wnt1/ Bmp4 and Wnt1/mBmp4 bigenic embryos that express transgenes at the same level, respectively, shows that mutant BMP4 causes the expansion of dorsal neural fates ventrally while wild type BMP4 does not, suggesting that mutant BMP4 acts farther than wild type BMP4. Together, these data suggest that the N-terminus basic amino acid core controls BMP4 long-range activity in neural development, and that BMP signaling patterns the dorsal neural tube through a secondary signaling pathway that involves homeodomain transcription factors MSX1/2 and PAX7. ^

Deciphering the C-Type Lectin Receptor Signaling Pathway in Macrophages in Response to Candida albicans

Candida albicans causes opportunistic fungal infections in humans and is a significant cause of mortality and morbidity in immune-compromised individuals. Dectin-2, a C-type lectin receptor, is required for recognition of C. albicans by innate immune cells and is required for initiation of the anti-fungal immune response. We set out to identify components of the intracellular signaling cascade downstream of Dectin-2 activation in macrophages and to understand their importance in mediating the immune response to C. albicans in vivo. Using macrophages derived from Phospholipase-C-gamma 1 and 2 (PLCγ1and PLCγ2) knockout mice, we demonstrate that PLCγ2, but not PLCγ1, is required for activation of NF-κB and MAPK signaling pathways after C. albicans stimulation, resulting in impaired production of pro-inflammatory cytokines and reactive oxygen species. PLCγ2-deficient mice are highly susceptible to infections with C. albicans, indicating the importance of this pathway to the anti-fungal immune response. TAK1 and TRAF6 are critical nodes in NF-κB and MAPK activation downstream of immune surveillance and may be critical to the signaling cascade initiated by C-type lectin receptors in response to C. albicans. Macrophages derived from both TAK1 and TRAF6-deficient mice were unable to activate NF-κB and MAPK and consequently failed to produce inflammatory cytokines characteristic of the response to C. albicans. In this work we have identified PLCγ2, TAK1 and TRAF6 as components of a signaling cascade downstream of C. albicans recognition by C-type lectin receptors and as critical mediators of the anti-fungal immune response. A mechanistic understanding of the host immune response to C. albicans is important for the development of anti-fungal therapeutics and in understanding risk-factors determining susceptibility to C. albicans infection.

Control of the master virulence regulatory gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.