Mutation and selection explain why many eukaryotic centromeric DNA sequences are often A + T rich.

We have used chromosome engineering to replace native centromeric DNA with different test sequences at native centromeres in two different strains of the fission yeast Schizosaccharomyces pombe and have discovered that A + T rich DNA, whether synthetic or of bacterial origin, will function as a centromere in this species. Using genome size as a surrogate for the inverse of effective population size (Ne) we also show that the relative A + T content of centromeric DNA scales with Ne across 43 animal, fungal and yeast (Opisthokonta) species. This suggests that in most of these species the A + T content of the centromeric DNA is determined by a balance between selection and mutation.

Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae.

Background: Phage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology.

Results: The activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S.

Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe.

Kinetochores in multicellular eukaryotes are usually associated with heterochromatin. Whether this heterochromatin simply promotes the cohesion necessary for accurate chromosome segregation at cell division or whether it also has a role in kinetochore assembly is unclear. Schizosaccharomyces pombe is an important experimental system for investigating centromere function, but all of the previous work with this species has exploited a single strain or its derivatives.

A Geographically Diverse Collection of Schizosaccharomyces pombe Isolates Shows Limited Phenotypic Variation but Extensive Karyotypic Diversity.

The fission yeast Schizosaccharomyces pombe has been widely used to study eukaryotic cell biology, but almost all of this work has used derivatives of a single strain. We have studied 81 independent natural isolates and 3 designated laboratory strains of Schizosaccharomyces pombe. Schizosaccharomyces pombe varies significantly in size but shows only limited variation in proliferation in different environments compared with Saccharomyces cerevisiae.

Serine recombinases as tools for genome engineering.

The serine recombinases differ mechanistically from the tyrosine recombinases and include proteins such as ϕC31 integrase which, unlike Cre and Flp, promote unidirectional reactions. The serine recombinase family is large and includes many other proteins besides ϕC31 integrase with the potential to be widely used in genome engineering. Here we review the details of the mechanism of the reactions promoted by the serine recombinases and discuss how these not only limit the utility of this class of recombinase but also creates opportunities for the engineering of new enzymes.

Site-specific recombination by phiC31 integrase and other large serine recombinases.

Most temperate phages encode an integrase for integration and excision of the prophage. Integrases belong either to the lambda Int family of tyrosine recombinases or to a subgroup of the serine recombinases, the large serine recombinases. Integration by purified serine integrases occurs efficiently in vitro in the presence of their cognate (~50 bp) phage and host attachment sites, attP and attB respectively.

The 'kinetochore maintenance loop': the mark of regulation?

Kinetochores can form and be maintained on DNA sequences that are normally non-centromeric. The existence of these so-called neo-centromeres has posed the problem as to the nature of the epigenetic mechanisms that maintain the centromere. Here we highlight results that indicate that the amount of CENP-A at human centromeres is tightly regulated.

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.

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).

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).

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.

Comparison of Dam tagging and chromatin immunoprecipitation as tools for the identification of the binding sites for S. pombe CENP-C.

We have established the identity of the Schizosaccharomyces pombe homologue of vertebrate CENP-C and Saccharomyces cerevisiae MIF2p and have used it to compare Dam tagging and chromatin immunoprecipitation (ChiP)as tools for the mapping of protein binding sites on DNA. ChiP shows that S. pombe CENP-C binds to the central core and inner repeats of the S.

Transcriptome analysis for the chicken based on 19,626 finished cDNA sequences and 485,337 expressed sequence tags.

We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set.

An unpaired mouse centromere passes consistently through male meiosis and does not significantly compromise spermatogenesis.

ST1 is an artificial mini-chromosome approximately 4.5 Mb in size containing mouse minor and major satellite DNA, human alphoid DNA and sequences derived from interval 5 of the human Y chromosome. Here we have measured the mitotic and meiotic transmission of ST1 and have used the mini-chromosome to define the ability of mice to monitor the presence of unpaired centromeres during meiosis.

A comprehensive collection of chicken cDNAs.

Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank.