Cell Biology & Molecular Genetics

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

Regulation of gene function can be achieved through a variety of mechanisms. In this dissertation, I present the genetic and molecular characterization of two genes involved in two distinct mechanisms of control. Each gene was initially identified by its functional role in sperm development in the model organism Caenorhabditis elegans. The first gene, uba-1, is an essential enzyme involved in protein turnover through ubiquitin-mediated proteolysis. A temperature-sensitive allele, (uba-1)it129, was isolated in a classical genetic screen for mutations that cause sperm-specific sterility. The second gene, spe-44, encodes a putative transcription factor. Its identification by microarray screening for sperm-enriched genes led to the cytological analysis of the deletion allele spe-44(ok1400), by reverse genetics approach. it129 encodes a conditional allele of uba-1, the sole E1 ubiquitin-activating enzyme in C. elegans. E1 functions at the apex of the ubiquitin-mediated conjugation pathway, and its activity is necessary for all subsequent steps in the reaction. Ubiquitin is covalently conjugated to various target proteins. Poly-ubiquitination typically results in target protein degradation, which provides an essential mechanism for the dynamic control of protein levels. Homozygous mutants of uba-1(it129) manifest pleiotropic phenotypes, and include novel roles for ubiquitination in sperm fertility, control of body size, and sex-specific development. We propose a model whereby proteins normally targeted for proteasomal degradation instead persist in uba-1(it129^ts) and impair critical cellular processes. The second gene, spe-44, was identified as a putative sperm gene regulator in C. elegans based on its up-regulated expression during spermatogenesis and its significant sequence homology to the DNA-binding SAND domain. Genetic analysis of a deletion allele of spe-44(1400) has revealed its functional role during sperm development. Cytological analysis of spe-44(ok1400) showed developmental arrest of spermatocytes prior to spermatid differentiation. spe-44 mRNA is expressed in a narrow spatial and temporal window, just prior to spermatocyte differentiation, consistent with its functional role during spermatogenesis. Future study will be directed to find putative targets of spe-44 and the mechanisms that regulate gene expression using microarray analysis and yeast-one hybrid screens. These studies will help to understand transcriptional regulatory aspects of spermatogenesis in C. elegans.

Cell Biology & Molecular Genetics

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