The ability of proteins to engage in nuclear-cytoplasmic shuttling is required for their proper function. The nuclear export of the von Hippel Lindau (VHL) tumour suppressor protein is necessary for the proteasomal degradation of the hypoxia inducible factor alpha (HIFα). Studies have identified that the nuclear export of VHL and other proteins encoding a Transcription-Dependent Nuclear Export Motif (TD-NEM) is independent of the classical CRM1 nuclear export pathway but requires ongoing transcription. Furthermore, the eukaryotic elongation factor 1 alpha (eEF1A) was identified as a mandatory component of the TD-NEM-mediated nuclear export machinery. In this study, we have uncovered the ability of eEF1A to mediate the nuclear export of proteins by accessing the nuclear compartment in its inactive, GDP-bound form. Although previously thought of as a strictly cytoplasmic protein, work conducted in this thesis has shown that eEF1A is a nuclear-cytoplasmic shuttling protein and this ability is required for the effective export of proteins encoding a TD-NEM.

No description available.

Cytoplasmic male sterility (CMS) is a maternally inherited trait of higher plants that can be suppressed by nuclear restorers of fertility ( Rf) genes that normally down-regulate the expression of a CMS-associated mitochondrial gene. In Brassica napus, nap CMS is associated with the expression of the orf222/nad5c/orf2101 mitochondrial gene region and is suppressed by the restorer gene Rfn. I present here an extensive analysis of the expression of the orf222/nad5c/orf2101 region in nap CMS and fertility-restored plants. Using RT-PCR methodology, I mapped transcript initiation sites, processing sites, and 3' termini. I identified two processing events, one within and one immediately downstream of orf222, that are specific to fertility restored plants and I suggest possible mechanisms by which the Rfn protein may recognize cognate RNA substrates. Unexpectedly, I also found that levels of atp8 transcripts are much lower in CMS than in restored plants. / nad5c, one of the components of the nap CMS-associated region, is the small central exon of the nad5 gene. In higher plants, nad5c transcripts must be joined to exons b and d through two group II intron trans-splicing events. I found that in the dicot Brassica and the monocot wheat, proper splicing requires exon c and d joining occur prior to the splicing of c with b. Joining of c to a/b transcripts prior to c/d splicing results exclusively in mis-spliced products in which the 5' end of c is joined to cryptic sites within exon b. It is suggested that intron sequences downstream of c base-pair with exon a, leading to mis-folding of the b/c intron and mis-splicing. In Oenothera, where the c/d intron is further fragmented into a tri-partite intron, mis-splicing does not occur. I suggest that avoidance of mis-splicing may be a factor that drives fragmentation of trans -splicing group II introns and may have contributed to the eventual evolution of spliceosomal RNAs from a group II intron precursor.

No description available.

This thesis will discuss the 'Polima' cytoplasmic male sterility (CMS) system (pol) in Brassica napus (Canola) and detailed genetic mapping of the region surrounding restorer gene ( Rfp) for that system This fine mapping of the Rfp region will facilitate efforts to clone the gene that will eventually lead to its characterization. Knowledge of the structure of Rfp will provide insight in the molecular mechanisms governing mitochondrial gene statement as well as pollen production and may lead to the development of alternative methods of pollination control. In addition, it is possible that nuclear restorer genes for other CMS systems in other crops may be similar to that of the 'Polima' system. / Map based cloning requires the identification of DNA markers tightly linked to Rfp. Two PCR based markers which are located on either side of Rfp were developed. These markers allowed facile screening of a large population. / RFLP markers used in this study are based on the synteny between B. napus and the well known crucifer A. thaliana. (Abstract shortened by UMI.)

To identify region(s) of the mitochondrial genome that might be involved in specifying the "Polima" (pol) cytoplasmic male sterility (CMS) of Brassica napus, transcripts corresponding to 14 mitochondrial genes and DNA clones representing $>$90% of the mitochondrial genome of Brassica campestris were analyzed in nap (male fertile), pol (male sterile) and nuclear fertility-restored pol cytoplasm plants. CMS-correlated transcriptional differences among these plants were detected only with the ATPase subunit 6 (atp6) gene. Sequence analysis of the atp6 gene regions of pol and nap mitochondrial DNAs show that rearrangements in the pol mitochondrial genome upstream of atp6 have generated a chimeric 224-codon open reading frame, designated orfJ224, that is cotranscribed with atp6. In male sterile plants, most transcripts of this region are dicistronic, comprising both orf224 and atp6 sequences. In fertility restored-plants, genes at either of two distinct nuclear restorer loci specifically alter this transcript pattern, resulting in predominantly monocistronic atp6 transcripts. The effect of the restorer locus on orf224/atp6 transcripts does not seem to be tissue or developmental stage specific. orf224 comprises a portion of the mitochondrial gene, orfB, fused to sequence of unknown origin. The pol mitochondrial genome contains an apparently functional copy of orfB. The expression of the atp6 region is developmentally regulated in pol plants such that levels of monocistronic atp6 transcripts are increased in seedlings as compared to the floral tissue. Preliminary data indicate that the chimeric gene, orf224, is expressed at the protein level in pol plants.

The unusually long Cytoplasmic tail (CT) of Human Immunodeficiency Virus Type-1 (HIV-1) glycoprotein-41 (gp41) is highly conserved and engineered large truncations often render the virus non-infectious in a cell-type dependent manner. While large CT truncations occur infrequently in natural isolates, little is known about the mechanisms involved in infectious virions harbouring a large CT truncation. This thesis characterises RFgp34, a replication competent laboratory HIV-1 isolate with an acquired 100 amino acid CT truncation, and how it diverged from wildtype RF. The CT truncation and two possible compensatory mutations in Matrix (E40K and F44I) were introduced into the HIV-1 isolate NL4-3. These mutants were tested for infectivity, syncytia formation and glycoprotein incorporation into virions, alternative co-receptor usage and sensitivity to the fusion inhibitor T-20. Compared with RFwt, RFgp34-infected cultures displayed delayed viral replication kinetics in all cell types. Similar sized (MT-4 cells, PBMC) or larger and more numerous syncytia (Hut78 cells) were detected in RFgp34-infected cultures. Similar (Hut78 cells) or decreased (MT-4 cells, PBMC) amounts of glycoprotein was incorporated into RFgp34 virions, compared with RFwt virions. The increased syncytia in RFgp34-infected Hut78 cultures and the reduced glycoprotein incorporation into RFgp34 virions from MT-4 cells and PBMCs may explain the delayed RFgp34 replication kinetics. The Matrix E40K and F44I mutations were not able to directly compensate for the CT truncation to restore infectivity in Hut78 and MT-4 cells, as secondary mutations or the reversion of the CT truncation to a full-length CT were observed. In PBMCs the Matrix mutations alone were able to partially restore infectivity, suggesting specific mutations may compensate for the CT truncation in different cell types. None of the viruses utilised alternative HIV-1 co-receptors, nor were more resistant to T-20 than wildtype HIV-1 suggesting that the CT does not directly play a role in these viral functions. This thesis suggests that the sequence of mutations acquired by RFgp34 to compensate for the CT truncation and restore infectivity in multiple cell types may have occurred in a specific order and the evolution of RFgp34 to out-compete RFwt occurred over many passages.

Thesis (Ph. D.)--Lehigh University, 1996. / Includes vita. Includes bibliographical references (leaves 78-90).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 178-194). Also available in electronic version.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Cell Biology. Title from pdf file main screen (viewed on July 25, 2010). Includes bibliographical references.