Integration Profiling of Gene Function With Dense Maps of Transposon Integration

Abstract

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.T HE accelerated rate of gene discovery in an increasing number of species has challenged the existing methods for determining the functions of genes. Traditional approaches for characterizing the function of genes rely on obtaining mutant alleles and testing them in individual experiments for phenotypes. Direct and systematic methods for evaluating gene function have been developed. Genomewide RNAi screens of cultured cells require the synthesis, validation, and refinement of large libraries of double stranded RNAs or vectors that express double stranded RNAs. While RNAi screens have successfully identified many genes that may contribute to key functions such as the replication of human immunodeficiency virus (Brass et al. 2008;Konig et al. 2008;Zhou et al. 2008), the production of RNAi libraries is resource intensive and substantial complications exist, such as off-target effects and incomplete mRNA knockdown.An alternate approach for characterizing gene function in haploid cells relies on targeted gene deletions. For Saccharomyces cerevisiae and Schizosaccharomyces pombe, collections of strains have been created that contain systematic deletions of the predicted coding sequences (Winzeler et al. 1999;Kim et al. 2010). These comprehensive collections of strains can be readily screened under a variety of conditions to probe the contributions of individual genes to specific processes. However, considerable effort and resources are required to generate deletion sets and once generated, it is difficult to study the deletions in combination with other mutations. Another limitation with deletion collec...

Functional mapping of yeast genomes by saturated transposition

“…Because the approach only necessitates a transposon and a transposase, it should not only be feasible in S. cerevisiae and S. pombe (Guo et al, 2013), but also amenable to other industrially-, medically- or scientifically-relevant haploid fungi, such as  Y. lipolytica ,  C. glabrata, K. lactis  and  P. pastoris .…”

Mechanisms of DNA Transposition

“…Other RNase H-like transposases have preferred sites of integration. These involve distinct and often palindromic (127) patterns of base pairs (128)(129)(130)(131)(132)(133)(134)(135)(136)(137), suggesting that these target site preferences might reflect some other property other than sequence specificity, for example perhaps DNA bendability. Indeed, target DNA has been repeatedly observed to be bent when bound by transpososomes (e.g., for Tn7 (138), Tn10 (139), Mos1 (140), MuA (93)), and such bending has been proposed to be an effective mechanism to ensure the directionality of the reaction (93,141).…”

Shortening distance of forward and reverse primers for nucleic acid isothermal amplification

“…We infer that there are more complex linking structures in DNA, and that just via these 'trivial' DNA fragments (see the yellow and red parts in Figure 5) the primers have realized 'jump' amplification. Here we define these 'trivial' DNA fragments as medium genes that may be a transposon of the DNA (Guo et al, 2013;Katter et al, 2013;Martienssen and Chandler, 2013;van Opijnen, and Camilli, 2013), or linkages in DNA or linkages between one DNA and another DNA. The role of the medium genes in DNA structure is not yet known to us.…”