Effects of hairpin sequences on retroviral recombination and construction of gfp/neo and gfp/hyg fusion genes

Effects of hairpin sequences on retroviral recombination. Retroviral particles contain two copies of genomic RNA. During reverse transcription, reverse transcriptase (RT) frequently changes its template between two RNA molecules. This results in a new virus with the genetic information from two viral RNA molecules, a phenomenon known as retroviral recombination. Recombination plays an important role in viral diversity and evolution. Secondary structures of the nucleic acid template can enhance strand transfers. Most evidence of the effects of secondary structure on strand transfers have been observed from in vitro reverse transcription assays, which do not have the exactly same physiological conditions in which retroviral recombination occurs in vivo. Therefore, it is necessary to determine whether secondary structures of the template correlate with recombination by using a single round viral replication assay in a tissue culture system. In this project, a series of vectors that contain two 290-nt homologous sequences were constructed. After a single round of viral replication, a recombination event results in a deletion of the sequence between the two homologous sequences. The rate of recombination is increased when a single hairpin was inserted 3' to the downstream homologous sequence. Based on these data, it is concluded that retroviral recombination occurred in infected cells is also affected by secondary structures.; Construction of the gfp/neo and gfp/hyg fusion genes. Linking two genes in the same reading frame may provide an active bifunctional fusion protein. Fusion proteins are currently used in generating antibody libraries, yeast two hybrids, protein tagging, etc. Among these, a fusion protein containing the green fluorescent protein (GFP) is convenient to study cellular events or track target fusion proteins in real time. Generating a fusion protein is still an empirical process that is based on trial and error. In this project, combinatorial linkers were used to fuse the gfp gene to either the neomycin resistance gene (neo) or the hygromycin resistance gene (hyg). Through selection for antibiotic resistance and green fluorescence, three gfp/neo fusion genes and two gfp/hyg fusion genes were constructed. The cells expressing these fusion genes were drug resistant, and emitted green fluorescence when analyzed under fluorescence microscopy.