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Mechanisms of post-transcriptional control are critical to deploy RNAs and proteins asymmetrically to a discrete region of cytoplasm at the posterior of the Drosophila oocyte and embryo, called the pole plasm and thus allow differentiation of the germline. Research presented in this thesis investigates the post-transcriptional control of Drosophila pole plasm component germ cell-less (gcl ). Maternal gcl activity is required for germ cell specification and gcl RNA and protein accumulate asymmetrically in the pole plasm. gcl RNA, but not Gcl protein, is also detected in somatic regions of the embryo, and ectopic expression of Gcl in the soma causes repression of somatic patterning genes suggesting that gcl RNA is subject to translational control. I find that Gcl is expressed during oogenesis, where its expression is regulated by translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru is reduced (i.e., in an arrest heterozygote) and Bru overexpression reduces the amount of Gcl. Consistent with this, reduction of the maternal dosage of Bru leads to ectopic Gcl expression in the embryo, which, in turn, causes repression of anterior huckebein RNA expression. Bruno binds directly to the gcl3'UTR in vitro, but surprisingly, this binding is largely independent of a Bruno Response Element (BRE) in the gcl 3'UTR and depends upon a novel site. Furthermore, the gcl BRE-like region is not required to repress Gcl expression during oogenesis or embryogenesis. I concluded that Bru regulates gcl translation in a BRE-independent manner. In addition, I established the role of the gcl 3'UTR in gcl RNA localization and translation using transgenes that replace the endogenous 3'UTR with the alpha-tubulin 3'UTR or place it in tandem to the bicoid 3'UTR. I find that accumulation of gcl RNA in the embryonic pole plasm requires the gcl 3'UTR. Moreover, Gel is restricted to the pole plasm by translational repression mediated by the gcl 3'UTR and a limiting pool of trans-acting translational repressors. The phenotypic consequences of loss of this translational control are relatively mild, suggesting that gcl translation does not require stringent repression in the soma.

Fluctuations in intracellular (Ca2+) calcium levels generate signaling events and regulate different cellular processes. Whilst the implication of Ca2+ in plant cell responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary meesenger in the fungal symbiont. The molecular basis of fungal calcium homeostasis in the AM symbiosis was analyzed by investigating the expression of Ca2+-related fungal genes. In a first study, G. mosseae genes putatively encoding a MAP3k-like protein kinase (Gm2) and a P-type ATPase (Gm152) were investigated. Both Ca2+-related genes were up-regulated by A. sinicum root exudates, suggesting a role in early interactions prior to symbiosis establishment. The full-length cDNA sequence of Gm152 obtained from germinating spores of G. mosseae confirmed its identity. The role of Ca2+ in fungal processes leading to establishment of an AM symbiosis was investigated in more detail in G. intraradices-M. truncatula interactions. Enhanced expression of genes encoding six membrane transport proteins and one nuclear protein kinase, selected from the G. intraradices transcriptome database, was related to colonization of wild-type M. truncatula (line J5) roots and not observed with the mycorrhiza-resistant mutant dmi3/Mtsym13. Laser microdissection mapping of transcripts indicated that the Ca2+-related G. intraradices genes were differentially up-regulated in arbuscules and/or in intercellular hyphae. The tempo-spatial variations in fungal gene expression suggest different roles in the development or functioning of the AM symbiosis. Full-length cDNA of three G. intraradices genes putatively encoding a PMR-like endoplasmic reticulum P-type ATPase, a VCX1-like vacuolar Ca2+ ion transporter and a nuclear CCaMK were obtained for functional analyses in yeast mutants to gain insight into their role in the mycorrhizal symbiosis. Possible mechanisms are discussed in which Ca2+-related proteins of G. intraradices may play a role in the mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots

The five member Inhibitor of Growth (ING) gene family has been proposed to participate in the regulation of cell growth, DNA repair, inflammation, chromatin remodeling, and tumor suppression. All ING proteins contain a PHD motif implicated in binding to methylated histones and are components of large chromatin remodeling complexes containing histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes, suggesting a role for ING proteins in regulating gene transcription. Additionally, forced overexpression studies performed in vitro have indicated that several ING proteins can interact with the p53 tumor suppressor protein and/or the NF-кB protein complex. Since these two proteins play well-established roles in numerous biological processes, several models have been proposed in the literature that ING proteins act as key regulators of cell growth and tumor suppression not only through their ability to modify gene transcription but also through their ability to alter p53 and NF-кB activity. However, these models have yet to be substantiated by in vivo experimentation. Research described in this dissertation utilizes a genetic approach to analyze the functional role of two ING proteins, Ing1b and Ing4, in regulating cell growth, inflammation, and tumorigenesis. Loss of p37Ing1b increased proliferation and DNA damage-induced apoptosis irrespective of p53 status in primary cells and mice. However, all other p53 responses were unperturbed. Additionally, p37Ing1b suppressed the formation of spontaneous follicular B-cell lymphomas in mice. Analysis of B-cells from these mice indicates that p37Ing1b inhibits the proliferation of B cells regardless of p53 status, and loss of p53 greatly accelerates the rate of B-cell lymphomagenesis in p37Ing1b-null mice, with double null mice presenting with aggressive diffuse large B-cell lymphomas (DLBL). Marker gene analysis in p37Ing1b/p53 null tumors indicates that these mice develop both non-germinal center and germinal center B cell-like DLBL, and also documents upregulation of NF-кB activity in both B-cells and tumors. Similarly, Ing4 -/- mice did not have altered p53 growth arrest or apoptosis, and did not develop spontaneous tumors. However, Ing4 -/- cells displayed reduced proliferation, and Ing4 -/- mice and macrophages were hypersensitive to treatment with LPS and exhibited decreased IкB gene expression and increased NF-кB activity. These studies demonstrate that Ing proteins can function to suppress spontaneous tumorigenesis and/or inflammatory responses without altering p53 activity, and identifies NF-кB as a biologically-relevant in vivo target of Ing1 and Ing4 signaling.

No description available.

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.

We used flow cytometry in a retrospective study of pleomorphic adenoma and carcinoma arising in pleomorphic adenoma, using paraffin-embedded tissue, to assess the relationship among proliferative activity, ploidy, and recurrence or malignant transformation. Twenty-four specimens obtained from 22 tumors were acceptable for analysis (co-efficient of variation, < or = 7.0), including multiple samples from two tumors. Fourteen tumors (13 benign and one malignant) were diploid. Six tumors were aneuploid: four benign pleomorphic adenomas and two carcinomas arising in pleomorphic adenoma. Two tetraploid tumors were malignant recurrences from the same patient. Of the recurrent tumors (nine benign and four malignant), 54% were aneuploid. The highest S-phase fractions were observed in recurrent and malignant pleomorphic adenomas. Immunostaining with p105, a nuclear proliferation antigen, revealed increased proliferative activity in a majority of pleomorphic adenomas. Increased proliferative activity and aneuploidy occurred in benign pleomorphic adenomas.

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).