1,25-dihydroxyvitamin D$\sb3$ (1,25(OH)$\sb2\rm D\sb3$), the hormonally active form of vitamin D, mediates its effects, at least partially, via binding to its nuclear receptor, the vitamin D receptor (VDR) which then interacts with vitamin D-response elements in the promoter region of vitamin D-regulated genes. Our studies have focused on the regulation of calbindin D-28K, a vitamin D-dependent calcium-binding protein, in Madin-Darby bovine kidney (MDBK) cells. MDBK cells are renal epithelial cells which display distal tubular characteristics including the expression of calbindin D-28K and VDR. Consistent with data derived in primary cultures and in vivo models, we have characterized the dose- and time-dependence of calbindin D-28K regulation by 1,25(OH)$\sb2\rm D\sb3$, offering the first established in vitro model system for studying the molecular regulation of calbindin D-28K. Our data also emphasize the role of post-transcriptional mechanisms of regulation in calbindin D-28K induction by 1,25(OH)$\rm\sb2D\sb3$. The observation that protein kinase C (PKC) activators and inhibitors can modulate the expression of vitamin D-dependent proteins implicates this signalling pathway in 1,25(OH)$\rm\sb2D\sb3$ regulation. We have investigated the potential role of PKC in the regulation of calbindin D-28K in MDBK cells. Time course analysis with 1,25(OH)$\rm\sb2D\sb3$ and TPA, a well-characterized PKC modulator, suggests a temporal correlation between PKC activity and calbindin D-28K expression in MDBK cells. More precisely, both long term treatment with 1,25(OH)$\rm\sb2D\sb3$ and short term treatment with TPA induce PKC activity, PKC$\alpha$ immunoreactivity and calbindin D-28K expression. Long term treatment with TPA which down-regulates PKC activity and expression also causes a decrease in calbindin D-28K levels. The observation that phosphatase inhibitors blunt the down-regulating effect of TPA on calbindin D-28K expression further suggests a role for phosphorylation in this regulation. Amino acid sequence analysis of calbindin D-28K reveals the presence of five casein kinase II (CKII) and two PKC phosphorylation sites. In vitro phosphorylation assays demonstrate that PKC, and more precisely PKC$\alpha$, phosphorylates calbindin D-28K in a calcium- and phospholipid-dependent manner. In agreement with the amino acid sequence, the phosphorylated form of calbindin D-28K is detected with an anti-phosphothreonine antibody. CKII does not phosphorylate calbindin D-28K under our experiment conditions. Immunoprecipitation studies of radiolabeled MDBK cells further support the phosphorylation of calbindin D-28K. Both TPA and 1,25(OH)$\rm\sb2D\sb3$ treatments enhance the phosphorylation state of 28 kDa protein specifically immunoprecipitated in the presence of calbindin D-28K antibodies. These data have been compiled into a hypothetical model involving the phosphorylation of calbindin D-28K as a 1,25(OH)$\rm\sb2D\sb3$ regulatory step. Our results strongly implicate the PKC signalling pathway in calbindin D-28K regulation.

Mouse-adapted influenza A virus, FM-MA, interferes with the replication of wild-type strains on coinfection of MDCK cells to the extent that $>$99% of the infectious progeny virus are FM-MA. The interference phenotype was previously mapped to FM-MA segment 2 which encodes the PB1 polymerase protein with a single amino acid substitution relative to the parent, FM, at position 538. To identify the point at which FM-MA interferes with wild-type A/HK/1/68 (HK), the relative levels of FM-MA and HK transcription and genome replication from PB1, NP and M1 genes were determined in coinfected MDCK cells using RT-PCR. All stages of FM-MA macromolecular synthesis (primary and secondary transcription, genomic RNA, complementary RNA and protein) were enhanced relative to HK, a phenotype which mapped to FM-MA segment 2. The kinetics of viral RNA synthesis in single or mixed infections indicated not only that the presence of FM-MA specifically compromised HK transcription and replication in coinfected cells but also that FM-MA's ability to interfere was due in part to its capacity for increased primary transcription relative to HK. FM-MA genomes were also selectively assembled into progeny virus from cells coinfected with HK and FM-MA, a step which was distinct from the capacity for enhanced RNA synthesis. This suggests that interference of wild-type virus growth by FM-MA in mixed infections resulted from two distinct events: a preferential synthesis of FM-MA-specific macromolecules which was then augmented by a preferential assembly of FM-MA genomes. The ability of FM-MA to selectively amplify its own genes in cells coinfected with HK did not depend on recognition of an FM-MA-specific promoter by the mutant polymerase. Alternate explanations for this selectivity are proposed.

The human influenza virus A/FM/1/47 (FM) was mouse-adapted by serial lung passage to produce the more virulent variant A/FM/1/47-MA (FM-MA). Previous genetic analysis identified four genome segments, 4, 5, 7 and 8 that are statistically associated with virulence. The aim of this investigation was first, to find the mutations on these four genome segments and then to determine a role for these changes in disease production. Upon sequencing segments 4 and 7, single amino acid replacements were found at amino acid 47 of the HA2 subunit of the hemagglutinin (HA) and at amino acid 139 of the matrix protein (M1). Segments 5 and 8 had not mutated on mouse-adaption. Viral growth kinetics were studied both in the mouse lung and in cell culture with MDCK cells. For viral pathology, standard hematoxylin, phloxine, saffron staining of formalin fixed sections, fluorescent antibody labelling of frozen sections and flow cytometric analysis for infected cells and immune cell recruitment was used to detect differences between the viruses. The matrix protein has a role in both replication and pathology and its coding change can be observed as a shift on SDS-PAGE. It was shown that viruses containing the matrix protein of FM-MA could replicate as quickly and to as high a titer as FM-MA itself. In pathology, it was apparent that segment 7 contributes to the early onset of interstitial pneumonia and that these reassortants can infect a greater number of cells than FM or the segment 4 reassortants. The parental FM-MA strain infects more cells in the lung and is more virulent than FM X FM-MA reassortants containing segments 4 and 7. (Abstract shortened by UMI.)

Forty-four actin genes from five angiosperm species, whose evolutionary relationships are well characterized, were PCR-cloned and sequenced. Phylogenetic analysis of 34 of these actin genes, along with those previously published, indicate that plant actin genes are monophyletic and underwent a rapid radiation early in land plant evolution. Six sets of putative orthologues have been identified and their sequences were used to calculate rates of evolution. The synonomous rate of substitution $(5.44\times10\sp{-9}$/site/year) is similar to that of other nuclear protein-encoding genes but the non-synonomous rate $(0.13\times10\sp{-9}$/site/year) is 4-10 times higher than that of vertebrate actin genes. Relative-rate tests do not support a faster rate in plants than in vertebrates. Evidence is also provided that some members of the actin multigene family in maize are undergoing gene conversion. Finally, we show that some plant actin genes have undergone intron loss probably as a consequence of a gene conversion event between the genomic copy and the reverse transcript.

Myotonic dystrophy (DM) is the most common form of inherited neuromuscular disease in adults and is characterized by progressive muscle wasting and myotonia. The mutation responsible for DM has been identified as the amplification of apolymorphic (CTG)$\rm\sb{n}$ repeat in the 3$\sp\prime$ untranslated region of a gene encoding a serine/threonine kinase (DMK). We have produced a polyclonal rabbit antibody preparation against a fusion protein encoding C-terminal amino acids 471-629 of the human DMK gene. This antibody specifically detects products of both full length and truncated human DMK genes expressed in bacteria and in insect cells. On immunoblots, we observed protein species of $\sim$74 and 82 kDa in human and rodent cardiac muscle and skeletal muscle, as well as rodent ependyma and choroid plexus. By immunofluorescence, DMK was found to localize postsynaptically at the neuromuscular junction of skeletal muscle, at intercalated discs of cardiac tissue and at the apical membrane of the ependyma and within the choroid plexus. We have also detected 2-3 species ($\sim$45-50 kDa) in brain tissue. Neuroanatomical evidence suggests synaptic localization for DMK in rodent cerebellum, hippocampus, midbrain and medulla. These results indicate that DMK may have a role in intercellular communication. Finally, we have demonstrated that DMK is present in adult and congenital DM tissues and that its distribution is no different than that observed in normal controls.

The Esk dual specificity kinase has elevated mRNA levels in cell types that proliferate compared to terminally differentiated cells. Two isoforms of the kinase were cloned from embryonal cells suggesting that Esk may be important during murine development. esk1 and esk2 differ only by the splicing in of an additional exon present in the esk1 mRNA that is absent from esk2. This thesis assesses the importance of Esk in the normal development of the mouse. Myc epitope tagged Esk1 and Esk2 constructs enabled the properties of the two isoforms to be analyzed separately. M-Esk1 and M-Esk2 are catalytically active by in vitro autophosphorylation kinase assay and both isoforms are localized in the cytoplasm by immunostaining in COS-1 cells. Fractionation of endogenous Esk from P19 EC cells confirmed that Esk is predominantly a soluble cytoplasmic kinase. Site directed mutagenesis of Esk2 identified a kinase inhibitory phosphorylation site in the activation segment of the kinase. The mutant kinase, MT648A, T649A is activated 19 fold above M-Esk2 and 41 fold above the kinase impaired M-Esk2(LL'8'). Successful stable expression of MT648A, T649A but not WT Esk demonstrated that the mutant is regulated differently than the WT kinase. Mutation at three sites in the catalytic domain has been shown to affect the autophosphorylation kinase activity of Esk2 to different degrees. The T648A, T649A mutation leads to activated kinase activity by the removal of a negative regulatory phosphorylation site. A L(758) to R mutation in subdomain XI retains kinase activity while a L(561) to R mutation in subdomain TV is kinase inactive. (Abstract shortened by UMI.)

This thesis is concerned with the mechanism that underlies the apparent atherogenic capacity of low density lipoproteins (LDL). This work provides a means for determining the effects of LDL phospholipids on the conformation of the protein moiety of LDL, apolipoprotein B (apoB). Reconstituted LDL (rLpB) particles were prepared from apoB and palmitoyloleoyl phosphatidylcholine (POPC). Intact apoB was isolated from native LDL retained $\sim$70% of the triglyceride (TG) from LDL. Aqueous soluble apoB-TG complexes exhibited a significantly reduced amphipathic $\alpha$-helical content (17%) and net negative charge ($-$2.9 mV) as compared to LDL-bound apoB (49% and $-$6 mV). Reconstitution of apoB-TG with phospholipid was accomplished through spontaneous complexation with POPC vesicles. apoB-TG was able to rapidly (10 min.) solubilize POPC at temperatures ranging from 4 to 24$\sp\circ$C. Electrophoresis on 4-15% gradient acrylamide gels showed the rLpB particles to be homogeneous and to exhibit a single discrete band. Inclusion of 300 molecules of POPC significantly increased the $\alpha$-helical content of apoB to 34% and the net negative charge to $-$4.9 mV. Phospholipidation of apoB also significantly decreased the effectiveness of GdnHCl in unfolding the apoprotein. Similar observations were seen with native LDL. The immunoreactivity of rLpB was assessed using various monoclonal antibodies. apoB-TG was immunoreactive with antibodies 1D1, 2D8, 3F5, 4G3 and 5E11. The inclusion of 300 molecules of POPC significantly increased the immunoreactivity of the conformation-specific antibodies 2D8 (p 0.001) and 4G3 (p 0.01) but had no major effect on the epitope accessibility or affinity of the other monoclonal antibodies studied. (Abstract shortened by UMI.)

The nuclear lamina consists of a filamentous network of proteins situated beneath the inner nuclear membrane and apposed to peripheral chromatin. As a consequence of its location, the nuclear lamina has been proposed to be involved in a number of different cellular functions. I have used a human antiserum to further characterize a novel set of nuclear lamina proteins, termed the MAN antigens. These antigens comprise three major polypeptides with relative mobilities of 78, 58 and 40 kDa. During interphase, the MAN antigens co-localized with the lamins at the nuclear periphery, but were absent from intranuclear foci of lamin B. In cells which possessed micronuclei, both the MAN antigens and lamins A/C were observed to segregate within these structures, separate from lamin B. Through mitosis, lamins A/C were seen to disassemble in late prophase and reassemble in telophase. Conversely, lamin B and the MAN antigens began to disassemble only during late prometaphase and then reformed around segregating chromosomes in anaphase, prior to lamins A/C. The human antiserum was used to screen a P19 embryonal carcinoma cDNA expression library for clones which encoded polypeptides immunologically related to the MAN antigens. (Abstract shortened by UMI.)

The gene encoding the glycolytic enzyme phosphoglycerate kinase is transcriptionally regulated at two levels. Expression of enzyme is related to the glycolytic activity of the cell, and is highest in transcriptionally active cells. Expression is also regulated by X chromosome inactivation, as the somatically expressed Pgk-1 gene is X-linked. The role of 5$\sp\prime$-flanking cis-acting DNA elements and trans-acting factors in the regulation of Pgk-1 expression was examined. The murine Pgk-1 gene contains an upstream activator sequence (UAS) in its 5$\sp\prime$-flanking region. This region was found to be responsible for elevating transcription levels at least ten-fold above basal Pgk-1 promoter levels in P19 embryonal carcinoma (EC) cells. Part of this activity was attributed to the R2 protein binding site, first identified by DNase 1 footprinting techniques. Mutation of the middle region of R2 resulted in a 5-fold reduction in expression of a Pgk-1 driven construct in stable transfection experiments into P19 cells. It was also determined that the R2 site was not important for transcription in P19 cells induced to differentiate with retinoic acid (RA). In undifferentiated P19 cells, another UAS protein binding site, R1, was identified by band shift analysis. R1 could not be detected by footprint analysis, suggesting that the affinity of binding at R1 was lower than at R2. The mutation of the R2 site did not abolish protein binding, which led to the hypothesis that multiple factors were binding the DNA at R2. R1 was also hypothesized to interact with multiple factors. However, fractionation of the P19 nuclear extract and use in band shift studies against the R1 DNA resulted in a single fraction with binding activity, suggesting a single R1 DNA binding protein and a non-DNA binding component. This non-DNA binding component at R1 was found to be tissue- or species-specific. Southwestern analysis in conjunction with fractionation experiments suggested that one of the R2 DNA binding proteins was approximately 70 kD and that the R1 DNA binding protein was 120 kD. Treatment of the P19 cells with RA led to a reduction in gene expression. Two days after exposure to the drug, the contribution to expression from the UAS was reduced by 50%, and four days after exposure the UAS no longer contributed to gene expression. Protein binding to the UAS was also altered after RA-treatment. A new site of protein interaction was detected in the distal region of the UAS, at R3, and binding at R1 was altered. There was, therefore, a correlation between protein interaction within the UAS and gene expression during differentiation. The results presented in this thesis demonstrate that the regulation of PGK-1 occurs, at least in part, at the level of gene expression, and that the UAS has an important role to play in regulating expression levels of the gene during differentiation. The results also suggest that the transcriptional stimulatory activity of the UAS depends on higher-order interactions between multiple low affinity DNA binding proteins which change upon differentiation.

Interleukin 2 (IL-2), one of the most important lymphokines secreted by activated helper T-cells, plays a pivotal role in the generation and regulation of the immune response. The role of protein kinase C (PKC) in the signal transduction of IL-2 and the mechanisms of PKC activation were investigated in the cytotoxic T-cell line, CTLL-2. IL-2 is critical for CTLL-2 cell survival and cytolytic activity. CTLL-2 cells possess measurable levels of PKC activity and this activity was responsive to IL-2 withdrawal and addition. The IL-2 stimulated PKC activation was not due to the PKC translocation from cytosol to the membrane, but rather from the activation of inactive PKC already resident on membranes. Secondly, IL-2's ability to maintain cell viability was dependent on PKC activity since specific PKC inhibitors were able to block IL-2's ability to suppress apoptosis. An early and transient PKC activation was needed for IL-2 induced suppression of apoptosis. Finally, the mechanism(s) responsible for the IL-2 induced activation of PKC in CTLL-2 cells was investigated. While tyrosine kinases were activated after IL-2 stimulation, they were probably not linked to the activation of PKC. On the other hand, a pertussis toxin sensitive-G-protein was likely involved in PKC activation since pertussis toxin blocked IL-2 stimulated PKC activation. DAG, but not IP$\sb3$ and intracellular calcium, increased after IL-2 stimulation, indicated that DAG was unlikely generated from the breakdown of PI, but more likely via the PC-PLC or PC-PLD pathways. The increase in DAG by IL-2 was likely responsible for the PKC activation since exogenously applied DAG stimulated PKC activation in both intact cells and in isolated membranes. IL-2 also stimulated the levels of AA in CTLL-2 cells. This increase likely resulted from increased PLA$\sb2$ activity since PLA$\sb2$ inhibitors effectively blocked the IL-2 stimulated activation of PKC. As was the case with DAG, the addition of exogenous AA to intact cells and to isolated membranes caused a rapid increase in membrane PKC activity, suggesting that the endogenous production of AA by IL-2R stimulation was likely linked to PKC activation in CTLL-2 cells. The possible involvement of a cytoplasmic factor in IL-2 stimulated PKC activation was investigated. This compound, designated factor X, is likely a protein(s) with a molecular weight of less than 10 kDa. Factor X was rapidly generated following IL-2 stimulation of CTLL-2 cells, and the cytosolic fraction isolated from IL-2 treated, but not control cells, potently stimulated PKC activity in isolated membranes. Its role in the pathway leading to PKC activation after IL-2R stimulation is unclear, although it appears not to act upstream or to be linked to the G-protein or the generation of AA and DAG. In summary, IL-2 stimulated PKC in CTLL-2 cells, not through the translocation of cytosolic enzyme to membrane, but via activation of inactive membrane associated PKC. This PKC activation was involved in IL-2's ability to suppress apoptosis in these cells. The mechanism(s) of PKC activation by IL-2 is substantially more complicated than we initially thought, and it likely involved multiple second messengers, including G-proteins, DAG, AA and factor X. (Abstract shortened by UMI.)