Pre-mRNA splicing has been shown to be regulated in a cell- and tissue-specific as well as a developmentally specific manner. A substantial number of proteins have been identified that play a specific role in pre-mRNA processing. Included in these, are serine/arginine-rich (SR) proteins, which are thought to be involved in the regulation of splicing. SR protein activity is believed to be modulated by their state of phosphorylation. A family of autophosphorylating dual-specific nuclear kinases termed Clk (CDC2/CDC28-like kinases) have been shown to interact with, phosphorylate, and cause the nuclear redistribution of, SR proteins. Previous studies in our laboratory have shown that catalytically active Clks influence their own pre-mRNA splicing and that active Clks also influence the alternative splicing of an E1A adenovirus expression vector in vivo. Here, we describe the development of a triple primer RT-PCR assay to measure the spliced and unspliced RNA transcripts produced from the HIV-1 TAT expression vector pgTAT. The splicing of this vector has previously shown to be influenced by the SR protein ASF/SF2 in both in vitro and in vivo studies. Using the RT-PCR approach, we show that the over expression of any of the four known human full-length Clks (hClk1 to 4) does not alter the RNA ratio of spliced to unspliced TAT transiently or stably expressed in 293T cells. As well, by this approach, we show that the splicing of this same vector, stably transfected in P19 embryonal carcinoma cells, is altered upon neuronal and endodermal differentiation with retinoic acid or dimethyl sulfoxide respectively, whereby differentiation caused an increase in the unspliced transcript.

The endothelium, a critical modulator of vascular resistance and blood pressure, can release at least three factors that relax vascular smooth muscle, including nitric oxide (NO), prostacyclin (prostaglandin I2, PGI 2), and endothelium-derived hyperpolarizing factor (EDHF). EDHF-induced relaxation is correlated with a strong hyperpolarization and may activate vascular smooth muscle cell (VSMC) K-channels. The research presented studied ACh-induced EDHF release from rat tail artery (TA) and superior mesenteric artery (SMA). Calcium-activated potassium (KCa) channels in the rat mesenteric resistance artery (MRA), putative targets of EDHF, were characterized. ACh-induced EDHF release from rat TA or SMA hyperpolarized target single TA VSMCs current-clamped with the nystatin perforated patch technique. In the absence of donor tissue, ACh induced depolarization. TA and MRA VSMCs were patch clamped in the whole-cell configuration and macroscopic K-currents were elicited by depolarizing pulses. Calcium-activated (KCa) and voltage-dependent (Kdr) potassium currents were observed in both TA and MRA VSMCs. Apamin (300 nM) significantly inhibited MRA (79% of control at +50 mV) but not TA whole-cell KCa Current. Iberiotoxin (IbTX) or charybdotoxin (ChTX) (150 nM) abolished KCa current in TA and MRA VSMCs. Single rat MRA VSMCs were patch clamped using the inside-out technique. Four KCa channels were observed and named: BK. IK, SK, and miniK (big, intermediate, small, and mini KCa channels). In symmetrical potassium (150 mM), the single channel conductances were linear (BK, 197: IK, 94; SK, 50; miniK, 31 pS) and similar to those observed in rat TA. ChTX (150 nM) completely blocked the BK, IK, and miniK channels, and dramatically inhibited the SK channel (inside-out. +40 mV, [Ca2+] i = 500 nM). IbTX (150 nM) completely blocked all MRA KCa channels. 4-AP (2 mM) completely blocked the BK and IK channels, partially blocked the SK channel, and dramatically inhibited the miniK channel. Apamin (300 nM) selectively blocked the BK channel. (Abstract shortened by UMI.)

Acetylcholinesterase (AChE) is responsible for the rapid hydrolysis of acetylcholine into acetic acid and choline, thereby ensuring precise temporal control of synaptic transmission. The role of AChE has been well documented with respect to the neuromuscular junction where it performs this classic function. However, it has recently been discovered that AChE is also expressed in a variety of non-cholinergic tissues such as haematopoietic cells and selected populations of neurons. The role of the enzyme in non-cholinergic neuronal cells is currently unknown and its regulation in this population of cells has been generally overlooked. In this study, we established a cell model to study the developmental and trophic regulation of AChE in neuronal cells. In addition, we examined the regulation of the acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT) to elucidate the relationship between the two cholinergic enzymes. In a first series of experiments, we tested several hybrid cell lines for AChE activity. Next, we examined the developmental regulation of AChE and ChAT using NSC-34 motoneurons. (Abstract shortened by UMI.)

The childhood spinal muscular atrophies (SMAs) are autosomal recessive neurodegenerative conditions characterized by degeneration of lower motor neurons classified based on the age of onset and clinical severity. Type I is the most common and severe form of SMA with clinical presentation either in utero or immediately after birth. The gene encoding NAIP (Neuronal Apoptosis Inhibitory Protein) has been proposed to be a modulator of the severity of SMA and is frequently deleted in type I SMA. In this study I have assessed Naip (murine homologue of NAIP) transcript levels during mouse embryogenesis. Naip mRNA is present in the developing brain and spinal cord of E9.5 to E14.5 mouse embryos as detected by various in situ hybridization techniques. It is also found in the embryonic liver, the branchial arches, the nasal epithelium and in the future digits. At E16.5 Naip transcripts were found in the marginal zone of the lateral ventricle, the follicles of the vibrissae, in the retina and in the intestinal villi. These results are the first report of Naip gene transcript levels in embryogenesis. One model of SMA pathogenesis involves motor neuron attrition in the second and possibly third trimester of gestation. Our observation of Naip transcripts in the spinal cord between E9.5 and E14.5 (equivalent to the second trimester) is consistent with a role for Naip in modifying SMA severity.

Sporadic clear cell renal carcinomas (RCC) frequently harbor inactivating mutations in exon 2 of the von Hippel-Lindau (VHL) tumor suppressor gene. In this work, we examine the effect of the loss of exon 2-encoded beta-domain function on VHL biochemical properties. Exon 2-encoded residues are not essential for VHL ability to assemble with elongin BC/Cullin-2 and to display E3-ubiquitin ligase activity in vitro. However, exon 2-encoded beta-domain is required for VHL-mediated NEDD8 conjugation on Cullin-2, proper formation of an extracellular fibronectin matrix, assembly with fibronectin and elongation factor-1alpha (EF-1alpha), a protein that we recently found to be associated with wild-type VHL in vivo. Exon 2-encoded residues are also needed for VHL binding to hypoxia-inducible factor alpha (HIF-alpha) and for its subsequent ubiquitination. Localization studies in HIF-1alpha-null embryonic cells suggest that exon 2-encoded beta-domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1alpha and oxygen concentration. Therefore, we suggest that exon 2-encoded sequences of VHL are essential for VHL nuclear/cytoplasmic shuttling and for substrate HIF-alpha recognition and ubiquitination.

Apoptosis or programmed cell death is a genetically controlled process that eliminates harmful or unwanted cells. This process plays an important role during development by contributing to tissue sculpting and in maintaining homeostasis of cell number in a variety of tissues. Recently, we have identified a family of apoptotic inhibitory proteins, naip, hiap1 & hiap2, and xiap, which are capable of suppressing caspases, key proteolytic mediators of apoptosis. To elucidate a potential role for xiap during development, we have analyzed the expression patterns of the murine equivalent, miap3, at various stages of mouse development. In situ hybridization studies have revealed a ubiquitous expression pattern for miap3 mRNA in a variety of developing organs, including brain, heart, lung, liver, pancreas, intestines and skin. Similar results were observed with wholemount in situ hybridization. In addition, immunohistochemical analysis demonstrated that miap3 is widely expressed during embryonic days 9.5 to 16.5. Furthermore, miap3 protein expression was observed in many of the adult mouse tissues and in day 9 to 13 mouse embryos by western blot analysis. These results demonstrate that miap3 is ubiquitously distributed suggesting that it is an essential regulator of cell survival during murine development.

Regulation of genetic information occurs through many intricate and varied mechanisms. We have explored two such mechanism, splicing and translation initiation, by studying two kinase families involved in these processes. The work in this thesis represents our ongoing efforts to understand the role of these proteins and the mechanisms by which they work. In the first section of this thesis, we present evidence that the Clk family of kinases are able to influence the regulatory process known as alternative splicing. In the second portion, we discuss PKR, a regulator of protein translation, and demonstrate it to be an essential mediator of antiviral defence. In the final chapter, we consider a possible application of the data from our PKR studies, and propose an oncolytic therapy based on putative defects in regulatory pathways controlling PKR and other members of the IFN pathway.

Transcription of the human parainfluenza virus type 3 (hPIV3) genome occurs by a sequential stop-start mechanism which is directed by short conserved sequence elements found at the boundary of the each of the hPIV3 genes, yielding several monocistronic mRNAs. The general aim of this project was to begin to elucidate the role of these cis acting transcription regulatory sequences. A technique called minigenome rescue, which allows the in vitro manipulation and analysis of cDNA representing minigenome analogs of the hPIV3 genome, was used. The requirement of the rule of six for efficient rescue of hPIV3 minigenomes was verified using a series of monocistronic cDNAs. Minigenomes whose total length was a multiple of six nt (6n) were rescued more efficiently than minigenomes that were not 6n nt in length, as assayed by CAT activity. Removal of the eight extra nt from the M/F junction reduced the frequency of transcriptional readthrough to the level seen for the other hPIV3 gene junctions. (Abstract shortened by UMI.)

Recombinant hepatitis B virus (HBV) core particles have been successfully used as particulate carriers exposing viral and bacterial antigens on their surface. The objective of this research was to explore the use of recombinant core particles from HBV and its close relative the woodchuck hepatitis virus for edible vaccine technology. This was accomplished in two parts. Part 1 was the transformation of a truncated HBV core protein gene into transgenic tobacco plants and characterization of the gene's expression with respect to mRNA levels, protein levels, and particle self-assembly. Part 2 was the construction of a "universal antigen carrier" based on the woodchuck hepatitis virus (WHV) core protein and generation and characterization of chimeric WHV core proteins carrying two different epitopes from the hepatitis C virus (HCV) core protein. In conclusion, it appears that a different approach may be required to express core proteins from HBV-like viruses in transgenic tobacco plants. (Abstract shortened by UMI.)

Apoptosis is a natural cellular function which is tightly controlled along its multiple pathways by positive and negative regulator proteins. Among the negative regulators are the gene family called the IAPs (inhibitor of apoptosis proteins) which were initially identified in baculoviruses. To date, five vertebrate members of this family have been identified (NAIP, HIAP1, HIAP2, XIAP and survivin). The X-linked inhibitor of apoptosis protein (XIAP), is proteolytically processed during the course of apoptosis in Jurkat lymphoma cells when triggered with anti-Fas monoclonal antibodies and cycloheximide (CHX). Native 55 kDa XIAP undergoes proteolytic processing producing a single 30 kDa fragment containing the carboxy terminus of the protein. In vitro cell-free studies, the type I calpain isoform is capable of cleaving XIAP. Calpains are calcium-dependent cysteine proteases, which are activated during apoptosis. Calpain and 26S proteasome specific inhibitors are ineffective in blocking both XIAP cleavage and cell death in Fas/CHX-stimulated Jurkat cells when used individually. However, when used together these two classes were able to block death, but not XIAP cleavage. Caspase specific inhibitors block both cell death and XIAP cleavage. In this study, I have shown that XIAP cleavage is likely induced by a member of the caspase family and that the event occurs upstream of calpain and 26S proteasome activation during the apoptotic cascade. The proteolysis of XIAP during Fas/CHX mediated apoptosis may represent an important step in the propagation of the apoptotic cascade.