In this study, we show that PB undergoes transposition in the presence of PBase in the fission yeast S. pombe and it can serve as an effective mutagenesis tool for genetic screens.
We developed a PB-based mutagenesis system for S. pombe. In this system, PB transposition is launched from an auxotrophic marker gene locus, thus allowing transposition events to be easily monitored and enriched. A major advantage of our system is that the copy number of the transposon stays at one in most of the mutagenized cells selected by the excision marker, thus avoiding the complication caused by multiple insertions. In addition, we took advantage of the precise nature of PB excision to implement an efficient reversion analysis procedure for verifying the causal relationship between the transposon insertion and the mutant phenotype.
A potential drawback of initiating transposition from a fixed chromosomal locus is the distribution bias caused by local hopping effect. Compared to other transposons used for mutagenesis, PB
is known to have low tendency of local hopping (27
). Our deep sequencing result showed that there was little chromosomal bias of PB
insertions selected by the excision marker. The limited local hopping we observed is not likely to pose a problem for mutagenesis.
As shown for other organisms, TTAA is the preferred target site of PB
in fission yeast while other TTAA-like 4-nt sequences could be targeted occasionally. There are ~110
000 TTAAs across the fission yeast chromosomal genome and ~40% of them are found in annotated genes (39
). More than 98% of the fission yeast genes contain TTAA in their coding sequences and are susceptible to PB
insertion (Supplementary Figure S7
). Thus, it is theoretically possible to utilize PB
to perform exhaustive screens of the fission yeast genome. Experimentally, from a mutant pool of 400
colonies, we detected PB
insertions in 54% of the TTAA-containing ORFs using high-throughput sequencing (2647 out of 4914 genes). Insertions in the essential genes were severely under-represented, most likely due to insertion-caused lethality in haploid cells. For TTAA-containing non-essential ORFs, we found insertions in 66% of them (2306 out of 3468 genes). We believe this ratio is still an underestimate of the level of gene coverage that can be achieved by our system, because not all PB
insertions isolated in our TBZ-resistant screen were detected by deep sequencing. For example, in klp5
, only five of the 12 TTAAs identified as PB
insertion sites in the TBZ screen were identified in sequencing-based profiling. The failure to detect all available insertion sites may be due to lower-than-saturation complexity of the mutant pool used for our sequencing analysis, and/or underrepresentation of certain sites caused by amplification bias of the PCR procedure.
We performed two proof-of-principle genetic screens to validate our system. In the screen for TBZ-resistant mutants, klp5
mutants were found. Our literature search suggested that all the known mutants that can tolerate 40
mg/l of TBZ affect one of these three genes (33
). Thus, our screen has identified all the expected genes. The number of Arg+
cells screened (2
) was about 20 times of the number of TTAA sites in the fission yeast genome. However, the numbers of TBZ-resistant isolates of the three hit genes were not as high as 20 times of the numbers of TTAA sites in these genes. Instead, for klp5
, the numbers of isolates were 2.4, 1.3 and 3 times of the numbers of TTAA sites in their coding regions, respectively. This lower-than-expected recovery rate may be due to a PB
insertion bias. We also observed uneven utilization of the TTAA sites within the same gene. For example, 20 insertion events were mapped to one TTAA site in klp5
whereas no insertion was mapped to eight other TTAA sites in klp5
. Most likely, there are both ‘hot spots’ and ‘cold spots’ among the TTAA sites in the same gene. To cover as many genes as possible, especially for the genes with fewer TTAA sites, it would be advisable to screen at least 106
cells using our system.
Based on current annotation, ~5000 genes are encoded by the S. pombe
). A genome-wide haploid deletion library is commercially available from the Bioneer Corporation (http://pombe.bioneer.co.kr/
). This library covers ~80% of non-essential genes and each mutant has two unique barcodes that can be monitored by microarray or high-throughput sequencing technique (31
). This reverse genetics tool makes it possible to quickly identify genes responsible for a desired phenotype. However, a deletion library-based screen is not without limitations. For example, it would be difficult to carry out a screen in a genetic background different from that of the library because such an undertaking involves crossing the desired background into every single deletion strain in the library. In comparison, it is much easier to introduce the transposon elements into a specific genetic background to obtain a strain from which a transposon-based screen is initiated. Besides, all the mutations in the haploid deletion library are null alleles of non-essential genes, whereas different types of alleles can be expected from insertional mutagenesis, including hypermorphic alleles like the dam1
mutants reported here as well as hypomorphic alleles of essential genes.