LOCALIZATION OF DNA REPAIR FUNCTIONS TO THE NUCLEAR MATRIX

The nucleus of a eukaryotic cell contains both structural and functional elements that contribute to the controlled operation of the cell. In this context, functional components refers to those nuclear constituents that perform metabolic activities such as DNA replication and RNA transcription. Structural nuclear components, designated nuclear matrix, organize the DNA into loops or domains and appear to provide a framework for nuclear DNA organization. However, the boundary between structural and functional components is not clear cut as evinced by reports of associations between metabolic functions and the nuclear matrix. The studies reported here attempt to determine the relationship of another nuclear function, DNA repair, to the nuclear matrix.^ One objective of these studies was to study the initiation of DNA repair by directly measuring the UV-incision activities in human cells and determine the influence of various extractable nuclear components on these activities. The assay for incision activities required the development of a nuclear isolation protocol that produced nuclei with intact DNA; the conformation of the nuclear DNA and its physical characteristics in response to denaturing conditions were determined.^ The nuclei produced with this protocol were then used as substrates for endogenous UV-specific nuclease activities. The isolated nuclei were shown to contain activities that cause breaks in nuclear DNA in response to UV-irradiation. These UV-responsive activities were tightly associated with nuclear components, being unextractable with salt concentration of up to 0.6 M.^ The tight association of the incision activities with salt-extracted nuclei suggested that other repair function might also be associated with salt-stable components of the nucleus. The site of unscheduled DNA synthesis (UDS) was determined in salt-extracted nuclei (nucleoids) using autoradiography and fluorescent microscopy. UDS was found to occur in association with the nuclear matrix following low-doses (2.55 J/M('2)) of ultraviolet light, but the association became looser after higher doses of ultraviolet light (10-30 J/m('2)). ^

Nuclear functions of dynein light chain 1 in hormone action

It is widely accepted that the process of breast cancer tumorigenesis involves estrogen receptor-alpha (ER)-regulated stimulatory pathways, which feed into survival, cell cycle progression and proliferative response. Recent data from Kumar laboratory indicate that dynein light chain 1 (DLC1) plays a role in survival, motility and invasiveness, all of which are required for a successful tumorigenesis process. In the present research, we have discovered a mechanistic bidirectional regulatory link between the DLC1 and ER. We found that DLC1 facilitates ligand-induced ER transactivation involving the recruitment of the DLC1-ER complex to ER-target genes. To gain insights into the mechanism by which DLC1 regulates the ER pathway, we set out to identify novel DLC1-interacting proteins. Among other proteins, we identified KIBRA and Ciz1 as two novel DLC1-interacting proteins. We found that the KIBRA-DLC1 complex is recruited to ER-responsive promoters, and that KIBRA-DLC1 interaction is needed for the recruitment of ER to its targets as well as for ER's transactivation function. Finally, we found that KIBRA utilizes its histone H3interacting glutamic acid-rich region to regulate the transactivation activity of ER. During the course of this work, we also discovered that DLC1 interacts with Cdk2 and Ciz1, and such interactions play a direct accelerating role in the G1-S transition of breast cancer cells. While delineating the role of Ciz1 in hormone-responsive cancer cells, we found that Ciz1 is an estrogen-responsive gene, and acts as a co-regulator of ER. Accordingly, Ciz1 overexpression in breast cancer cells conferred estrogen hypersensitivity, promoted the growth-rate, anchorage-independency and tumorigenic properties. Collectively, findings made during the course of the present dissertation research introduced two new molecular players in the action of ER in breast cancer cells, with a particular focus on cell cycle progression and ER-chromatin target regulation. In addition, findings presented here provide novel mechanistic insight about the contribution of DLC1 and its interacting proteins in amplifying the hormone action and promoting the process of breast cancer tumorigenesis. ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

CELLULAR AND DEVELOPMENTAL FUNCTIONS OF THE XENOPUS ARVCF-CATENIN:KAZRIN COMPLEX

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. In considering the intracellular localization of the xARVCF-catenin:xKazrin complex, I did not resolve xKazrinA in a larger ternary complex with cadherin, nor did I detect its co-precipitation with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin. This suggested a potential means by which xKazrinA localizes to cell-cell junctions, and indeed, biochemical assays confirmed a ternary xARVCF:xKazrinA:xβ2-spectrin complex. Functionally, I demonstrated that xKazrin stabilizes cadherins by negatively modulating the RhoA small-GTPase. I further revealed that xKazrinA binds to p190B RhoGAP (an inhibitor of RhoA), and enhances p190B’s association with xARVCF. Supporting their functional interaction in vivo, Xenopus embryos depleted of xKazrin exhibited ectodermal shedding, a phenotype that could be rescued with exogenous xARVCF. Cell shedding appeared to be caused by RhoA activation, which consequently altered actin organization and cadherin function. Indeed, I was capable of rescuing Kazrin depletion with ectopic expression of p190B RhoGAP. In addition, I obtained evidence that xARVCF and xKazrin participate in craniofacial development, with effects observed upon the neural crest. Finally, I found that xKazrinA associates further with delta-catenin and p0071-catenin, but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin sub-family. Taken together, my study supports Kazrin’s essential role in development, and reveals KazrinA’s biochemical and functional association with ARVCF-catenin, spectrin and p190B RhoGAP.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Developmental Changes in the Structure and Composition of the Postsynaptic Density

The development of the brain and its underlying circuitry is dependent on the formation of trillions of chemical synapses, which are highly specialized contacts that regulate the flow of information from one neuron to the next. It is through these synaptic connections that neurons wire together into networks capable of performing specific tasks, and activity-dependent changes in their structural and physiological state is one way that the brain is thought to adapt and store information. At the ultrastructural level, developmental and activity-dependent changes in the size and shape of dendritic spines have been well documented, and it is widely believed that structural changes in spines are a hallmark sign of synapse maturation and alteration of synaptic physiology. While changes in spine structure have been studied extensively, changes in one of its most prominent components, the postsynaptic density (PSD), have largely evaded observation. The PSD is a protein-rich organelle on the cytoplasmic side of the postsynaptic membrane, where it sits in direct opposition to the presynaptic terminal. The PSD functions both to cluster neurotransmitter receptors at the cell surface as well as organize the intracellular signaling molecules responsible for transducing extracellular signals to the postsynaptic cell. Much is known about the chemical composition of the PSD, but the structural arrangement of its molecular components is not well documented. Adding to the difficulty of understanding such a complex mass of protein machinery is the fact that its protein composition is known to change in response to synaptic activity, meaning that its structure is plastic and no two PSDs are identical. Here, immuno-gold labeling and electron tomography of PSDs isolated throughout development was used to track changes in both the structure and molecular composition of the PSD. State-of-the-art cryo-electron tomography was used to study the fine structure of the PSD during development, and provides an unprecedented glimpse into its molecular architecture in an un-fixed, unstained and hydrated state. Through this analysis, large structural and compositional changes are apparent and suggest a model by which the PSD is first assembled as a mesh-like lattice of proteins that function as support for the later recruitment of various PSD components. Spatial analysis of the recruitment of proteins into the PSD demonstrated that its assembly has an underlying order.

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

The evolution and assessment of pharmaceutical care: Structure, processes, and outcomes

The evolution of pharmaceutical care is identified through a complete review of the literature published in the American Journal of Health-System Pharmacy, the sole comprehensive publication of institutional pharmacy practice. The evolution is categorized according to characteristics of structure (organizational structure, the role of the pharmacist), process (drug delivery systems, formulary management, acquiring drug products, methods to impact drug therapy decisions), and outcomes (cost of drug delivery, cost of drug acquisition and use, improved safety, improved health outcomes) recorded from the 1950s through the 1990s. While significant progress has been made in implementing basic drug distribution systems, levels of pharmacy involvement with direct patient care is still limited.^ A new practice framework suggests enhanced direct patient care involvement through increase in the efficiency and effectiveness of traditional pharmacy services. Recommendations advance internal and external organizational structure relationships that position pharmacists to fully use their unique skills and knowledge to impact drug therapy decisions and outcomes. Specific strategies facilitate expansion of the breadth and scope of each process component in order to expand the depth of integration of pharmacy and pharmaceutical care within the broad healthcare environment. Economic evaluation methods formally evaluate the impact of both operational and clinical interventions.^ Outcome measurements include specific recommendations and methods to increase efficiency of drug acquisition, emphasizing pharmacists' roles that impact physician prescribing decisions. Effectiveness measures include those that improve safety of drug distribution systems, decrease the potential of adverse drug therapy events, and demonstrate that pharmaceutical care can significantly contribute to improvement in overall health status.^ The implementation of the new framework is modeled on a case study at the M.D. Anderson Cancer Center. The implementation of several new drug distribution methods facilitated the redeployment of personnel from distributive functions to direct patient care activities with significant personnel and drug cost reduction. A cost-benefit analysis illustrates that framework process enhancements produced a benefit-to-cost ratio of 7.9. In addition, measures of effectiveness demonstrated significant levels of safety and enhanced drug therapy outcomes. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^