We used several diutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functidnafly aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demdnstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse trnstriptase. These results indicate that the Ty protease, like its retroviral cohnterpart, plays an important role in particle assembly, replication, and transposition of these elements.

Transposition of Ty elements in the yeast Saccharomyces cerevisiae occurs through an RNA intermediate. Although Ty RNA accounts for 5 to 10% of the total polyadenylated RNA in a haploid cell, the transposition frequency is only i0' to 10-8 per gene. To determine whether Ty elements native to the yeast genome are transpositionally competent, two elements were fused to the GALI promoter and tested for their ability to transpose. These native elements, Tyl-588 and Ty2-117, transposed at high levels when the GAL) promoter was induced. Three Ty's identified as spontaneous transpositions in specific target genes were also tested. Of these three, Ty2-917 and the previously characterized element Tyl-H3 were shown to be transpositionafly competent. The third element, Tyl-Hi, was transposition defective. In addition, we marked the chromosomal copy of Tyl-588 with the NEO gene and demonstrated that Tyl-588NEO was actively transcribed in yeast cells. Tyl-588NEO transcription was regulated by the SPT3 and MAT loci in the same manner as that observed for Ty's collectively. These results indicate that the yeast genome contains functional Ty elements. The presence of a transpositionally competent, actively transcribed element suggests that regulation of Ty transposition occurs at a posttranscriptional level.

We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.

Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element is transcribed into RNA, and this RNA is reverse transcribed into a DNA copy capable of insertion into many different chromosomal locations. Maturation of proteins and reverse transcription take place within noninfectious intracellular viruslike particles. We have studied the element Ty, which is found dispersed in the genome of the yeast Saccharomyces cerevisiae. The frequency of Ty element transposition is normally quite low but can be greatly increased by expressing an element from a strong promoter. We have used the ability to control the level of Ty transposition to investigate the functions of Ty proteins, the regulation of Ty transposition, and the exploitation of Ty elements as insertional mutagens in yeast. The information gained from these experiments should be applicable to the study of retrotransposons found in multicellular organisms.