Team Leader - Gerald Marsischky

Introduction

For the past several decades, the yeast Saccharomyces cerevisiae has been the archetype genetic system for eukaryotic model organisms. The ease and power with which its genes can be manipulated have lead to many insights and fundamental discoveries that have impacted all of biology. Given its preeminence as a genetic model system, the relative availability of molecular biology tools for S. cerevisiae is surprisingly less well developed. Nevertheless, it is uniquely positioned for the development and utilization of genome-scale tools: its genome has been fully sequenced and is well annotated, it has a limited repertoire of genes, there are excellent tools for introducing and regulating the expression of cloned genes, and the organism is easy to grow and process in high-throughput experiments.

In order to create a genome-scale toolset for S. cerevisiae, the Yeast Group at HIP together with our collaborators (Richard Kolodner’s group at the Ludwig Institute for Cancer Research at the University of California, San Diego, and Andrew Simpson’s group in the Laboratory of Cancer Genetics at the Ludwig Institute for Cancer Research in Sao Paolo, Brazil) are creating the Yeast FLEXGene Collection, a comprehensive, sequence-verified collection of S. cerevisiae genes in a recombinational cloning format. Once the Collection has been created, it will be made available to the public and private research communities at the lowest possible cost.

At the time of the inception of the Yeast FLEXGene project, the Gateway cloning system from Life Technologies (now owned by Invitrogen) was the best-established recombinational cloning system, and as the only practical recombinational cloning system, was the obvious choice for the project. The strength of the recombinational cloning approach is that a single validated gene collection can be used to create many different gene expression libraries for any number of experiments. The Yeast FLEXGene Collection thus has the potential to become an invaluable tool for basic research. It should also be noted that the yeast FLEXGene project also serves as a prototype for the ongoing human FLEXGene project.

Progress Report

Cloning Effort

Gene specific PCR primers for the 6277 S. cerevisiae ORFs (Research Genetics) were used for high fidelity PCR to amplify all of the ORFs from genomic DNA utilizing a two-step PCR process resulting in the addition of Gateway recombinational cloning sequences to each PCR product.

Figure1. Two-Stage PCR for Gateway Cloning  Enlarge

These PCR products were then gel purified, recombined into the Gateway donor vector, pDONR201, and transformed into bacteria in order to create Gateway Entry plasmids with an apparent overall cloning success rate of approximately 90%.

Figure 2. Strategy for Yeast Clone Assembly  Enlarge

The use of a PCR step in the production of the clones suggested the potential for the introduction of errors into the clones. To improve the chances of acquiring an error-free clone, 4 separate isolates for each gene were selected and processed into plasmid DNA, resulting in a total of 23,252 clones for 5813 genes. An unanticipated obstacle arising during the development of this project was the discovery that the recombination sites in the Entry vector prevent facile DNA sequencing from this vector. For this reason all 23,252 of the genes were subsequently transferred into a plasmid that was easier to sequence. As the vector chosen was a Gateway-modified galactose inducible yeast expression vector [Figure 3], this step also created yeast expression clones ready for immediate use in experiments.

Figure 3. YeastExpression Plasmid pBY011 Enlarge

Sequencing Effort

The Yeast FLEXGene group at Ludwig Brazil has established an optimized high-throughput DNA sequencing method for the yeast expression clones produced by HIP. In the first round of sequence analysis, clones have been sequenced by end reads only. In the next round of sequencing, internal reads to determine full-length sequences for clones will be done with the 2 best clones identified from analysis of the end reads.

Ludwig Brazil has created a set of programs that analyze DNA sequence reads and establish whether the sequenced clones match the expected sequence for each gene. The results of this analysis are stored in a database created by Ludwig Brazil for this purpose. Ludwig Brazil has also created a web interface that allows other members of the Working Group to access the results of the sequence analysis. This reporting allows, for instance, HIP to re-array clones into 2 groups: fully sequenced clones (ready for use in experiments), and clones that require more sequencing. It also identifies the genes for which there is no acceptable clone.

Sequencing Results

Table 1. Summary of Sequencing Results from First Round

At this time, approximately 68% of the yeast clones (representing an expected 4248 genes) have been sequenced and analyzed. From this analysis, clones that were fully sequenced and had no changes from the expected sequence were identified for 930 yeast genes (22%). Clones representing an additional 2046 yeast genes (48%) requiring further sequencing were also identified. No acceptable clones were identified for 1272 genes (30%); the majority (93%) of these failed clones represent false, or empty clones mostly from genes larger than 2000 nucleotides.

The projected totals after completion of the end read sequencing phase are 1374 fully sequenced clones with no sequence changes and 3023 partially sequenced genes. It is expected that 1880 genes will fail to produce acceptable clones in this round. However, as some of the partially sequenced clones have sequence changes, it is expected that for a fraction of the genes represented by this group there will be no acceptable clones. It would be reasonable to assume that an additional 500 genes might be lost in this way, resulting in the need to re-clone as many as 2500 genes. These genes will be re-cloned with improved methods in the finishing phase of the project. At this point, we have rearrayed 930 fully sequenced ORFs, and 1888 partially sequenced ORFs, for distribution to our collaborators as well as for experiments at HIP.

Finishing the Yeast FLEXGene Collection

Future work on the Yeast FLEXGene project will be divided into three phases: 1) a finishing phase of sequencing the Round 1 clones, 2) a finishing round of cloning, and 3) sequencing the finishing round clones. A final finishing round of cloning will, in all likelihood, be needed for the handful of genes that fail cloning a second time.