The study of retrotransposons in yeast
Retroelements are a large class of genetic elements that multiply by the reverse transcription of an RNA intermediate. The resulting cDNA is incorporated into the genome of host cells. In eukaryotes, the wide-spread success of long terminal repeat (LTR)-containing retroelements has led to replication mechanisms that are conserved among diverse families of retrotransposons and retroviruses. The medical importance of retroviruses such as HIV has intensified the need to understand the molecular details of the mechanisms responsible for the propagation of LTR-retroelements. Since LTR-retrotransposons exist in yeast, the powerful techniques of yeast genetics can be applied to answer basic questions about the function of LTR-retroelements. In turn, this fundamental information may identify new antiviral targets or stradegies that can be used to combat the spread of retroviruses such as HIV.
The similarities of retrotransposons to retroviruses includes the presence of two long terminal repeats (LTRs) and open reading frames (ORFs) with coding sequences homologous to retroviral protease (PR), reverse transcriptase (RT), and integrase (IN). The first step in the transposition pathway as shown in Figure 1 is synthesis of a full-length mRNA with sequence that begins in the 5' LTR and terminates in the 3' LTR. This is directly analogous to the initial step in retrovirus particle formation. Retroviral and retrotransposon mRNAs are translated into proteins that assemble along with the mRNA into large particle structures. Both retroviral and retrotransposon particles undergo a maturation process that includes the proteolytic processing of precursor proteins and the reverse transcription of the mRNA. Retrotransposon particles complete transposition by simply inserting their DNA into the genome of the original host cell. Because each step in retrotransposition is directly related to a retrovirus process, results from the investigation of yeast retrotransposition are relevant to aspects of retrovirus behavior.
The retrotransposon we study is the Tf1 element of the fission yeast Schizosaccharomyces pombe. The transposon is 5 kb and contains a single ORF with coding sequences for Gag, PR, RT, and IN (5). We have previously demonstrated using an in vivo assay that uses a neo-marked version of Tf1, that at least one of our cloned copies is active and can transpose at a significant frequency (3, 4). Figure 2 shows that cells that contain transposition events grow readily on the drug G418. This system allows us to use a battery of powerful molecular and genetic techniques to identify and characterize factors that contribute to the transposition process. The following are summaries of our current research projects.
Current Research Projects: