Summer Undergraduate Research Symposium Thursday, 9 August 2001 McKinly Laboratory University of Delaware

BVES/Pop1A is a member of a novel gene family with no known function and no sequence similarity to any other gene family. This gene has been shown to be expressed at high levels in the developing heart as well as skeletal muscle. However, a conflict exists in the literature as to the true expression pattern of this protein. Thus, in order to resolve this conflict in the expression pattern of this protein, monoclonal antibodies were produced. These antibodies have been shown to be specific to endogenous chicken BVES/Pop1A and are useful in both western blotting as well as immunoflourescence techniques. These monoclonal antibodies will be useful in immunolocalization and immunoblotting experiments of different tissue types to determine the location and levels of BVES/Pop1A expression throughout development, as well as further analysis of the biochemistry of this protein.
 
 

Our lab hypothesizes that the gene encoding the Sperm Adhesion Molecule 1, SPAM1, is expressed in the human epididymis. This prediction is based on a recent finding in our lab that the murine homologue of this gene, Spam1, is expressed in all three regions of the mouse epididymis. The goal of my work is to gather evidence of SPAM1 expression in the human epididymis and characterize the pattern of expression. Evidence collected will include detection of SPAM1 mRNA by in situ hybridization and detection of the SPAM1 protein by immunohistochemistry. Findings from murine Spam1 suggest human SPAM1 will be localized to the principal cells of the human epididymal epithelium and expression will be region-specific within the epididymis. Comparison of pathological tissue from an obstructed epididymis to physiological tissue may also reveal that SPAM1 expression can be disrupted by blockage of the epididymis.
 
 

The mechanisms of biosynthesis of the sarcoplasmic reticulum (SR), a specialized Ca²⁺ storage organelle in skeletal muscle cells, during early myogenesis have remained elusive. However, investigation of the expression in mouse Ltk^- fibroblasts of SERCA1a (Sarco/Endoplasmic Reticulum Ca²⁺-ATPases), a member of the SR Ca²⁺-ATPase family which loads the Ca²⁺ stores within the SR has provided insight into this mystery. Immunofluorescent labeling of avian SERCA1a transient and stable transfected Ltk^- cells revealed intense fluorescent areas that were a result of SERCA1a expression in compact masses of intracellular membrane. Using the confocal microscope, living Ltk^- cells transfected with a fusion protein of GFP (Green Fluorescent Protein) and SERCA1a similarly showed high levels of SERCA1a expression in membraneous locations. Thus, SERCA expression may trigger the induction of endoplasmic reticulum (ER) biosynthesis. Western blot analysis showed that avian SERCA1a transfected Ltk^- cells were expressing high levels of the correct sized SERCA1a protein. To further understand the role that SERCA’s may play in the biosynthesis of the SR during myogenesis experiments such as electron microscopy of SERCA- and control-transfected cells, measurements of functional ER Ca²⁺-ATPase activity and quantification of endogenous ER proteins in transfected cells are underway.
 
 

Chimeric Oligonucleotides are double-hairpin RNA/DNA. They have been shown to facilitate site-specific DNA mutagenesis when mismatched to a targeted gene. The process is hypothesized to occur via a recombinase mediated pairing event, forming an intermediate called a double displacement loop (double D-loop). This process is followed by structure recognition and repair of the target DNA using the chimera as a template. We have optimized a method for synthesizing double-D loops in vitro, and have used the resulting joints to identify proteins, which bind to and process double-D loops. Our results suggest that enzymes from two separate DNA repair pathways, the Nucleotide Excision Repair (NER) and Mismatch Repair (MMR) interact in a novel way to facilitate DNA Repair. Identification and purification of proteins, which cleave this structure causing double strand breaks will not only provide insight into the mechanism of targeted gene repair, but will also provide a novel technique for cutting DNA in the absence of a restriction site.