With the introduction of new deep sequencing technology it is now possible to sequence many millions of transposon insertions in a single experiment. We tested whether Illumina sequencing could be used to generate a dense profile of transposon insertions that would reveal which genes are required for cell division. For this experiment we used a haploid strain of S. pombe and Hermes, a DNA transposon from the housefly. In previous work we found that the Hermes transposon was highly active in S. pombe and that a large fraction of the insertions occurred in ORFs. We predicted that in actively growing cultures, Hermes insertions would not be tolerated in essential ORFs. We induced Hermes transposition in a large culture S. pombe that was grown for 80 generations. With ligation mediated PCR and Illumina sequencing we were able to sequence 360,513 independent insertion events. On average, this represented one insertion for every 29 bp of the S. pombe genome. An analysis of integration density revealed that the ORFs largely separated into two classes, one with high numbers of insertions and another with much lower numbers. In collaboration with a group that deleted each of the genes of S. pombe, we found the ORFs with low numbers of Hermes insertion corresponded to the essential genes. The ORFs with higher integration densities were in genes classified as nonessential. These results validated integration profiling as a new method for identifying genes with essential function. Importantly, by applying specific conditions of selection during growth, this method can be adopted to identify genes that contribute to a wide variety of functions. Recently we have pursued the use of Hermes integration profiles to identify the complement of genes that contribute to heterochromatin. Since heterochromatin is a complex process that silences genes and because S. pombe is an excellent model for the study of heterochromatin, we generated independent libraries containing 1 million integration events. The density of these libraries is substantially greater then previously generated and average one insertion per 10 bp of the S. pombe genome. This year our analysis of these integration sites generated a list of approximately 150 genes that may contribute to hetrochromatin formation. Importantly, many of these candidates are novel and our study of mutations in several of these genes indicates they do play a role in hetrochromatin formation.
| Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K et al. (2016) Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe. Methods Mol Biol 1400:117-30 |
