High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome.

BACKGROUND: Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate. RESULTS: Here, we report the genome sequence and analysis of Saccharomyces arboricolus, a yeast species recently isolated in China, that is closely related to S. cerevisiae. We obtained high quality de novo sequence and assemblies using a combination of next generation sequencing technologies, established the phylogenetic position of this species and considered its phenotypic profile under multiple environmental conditions in the light of its gene content and phylogeny. CONCLUSIONS: We suggest that the genome of S. arboricolus will be useful in future comparative genomics analysis of the Saccharomyces sensu stricto yeasts.

Unsuccessful attempt at gene-editing by homologous recombination in the zebrafish germ line using the approach of "Rong and Golic".

We have investigated the practicality of implementing a strategy for site-specific editing by homologous recombination in zebrafish analogous to that developed by Rong and Golic (Rong and Golic in Genetics 157:1307-1312, 2001) in Drosophila melanogaster. We analysed approximately 7,300 offspring from 22 crosses and demonstrated successful excision of the gene editing construct but failed to detect either gene editing or the random integration of the intact editing construct subsequent to excision. The clustering of events in our data set demonstrates that the excision events are not occurring independently and emphasise that a promoter driving high level, tissue-specific transcription in meiotic cells is likely to be necessary if this general approach to site-specific editing by homologous recombination is to fulfil its potential.

Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage phiBT1.

We have established the integrase of the Streptomyces phage phiBT1 as a tool for eukaryotic genome manipulation. We show that the phiBT1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in Schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. We show that the phiBT1 integrase can be used in conjunction with Cre recombinase to promote the iterative integration of transgenic DNA. We describe five cycles of iterative integration of a candidate mouse centromeric sequence 80 kb in length into a human mini-chromosome within a human-Chinese hamster hybrid cell line. These results establish the generality of the iterative site-specific integration technique.

Chromosome engineering in DT40 cells and mammalian centromere function.

Chromosome engineering is the term given to procedures which modify the long range structure of a chromosome by homologous and site specific recombination or by telomere directed chromosome breakage. DT40 cells are uniquely powerful for chromosome engineering because mammalian chromosomes may be moved into them, efficiently modified and then moved back into a mammalian cell lines (Dieken et al., 1996). The high rate of sequence targeting seen in DT40 cells carrying human chromosomes is necessary but not sufficient for chromosome engineering. The ability to either delete or introduce long tracts of DNA subsequent to a sequence targeting reaction depends upon the use of site specific recombinases. We have made important progress in the development of this technology in the past few years and much of this review will be used to describe this work.

Iterative in vivo assembly of large and complex transgenes by combining the activities of phiC31 integrase and Cre recombinase.

We have used the phiC31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than approximately 150 kb we have established a method that we call; 'Iterative Site Specific Integration' (ISSI). ISSI combines the activities of phiC31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.

Rearranging the centromere of the human Y chromosome with phiC31 integrase.

We have investigated the ability of the integrase from the Streptomyces phiC31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phiC31 integrase attB site and the chicken genome.