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. Combining the experimental results and the evolutionary analyses allows us to conclude that A + T rich DNA of almost any sequence will function as a centromere in most Opisthokonta species. The fact that many G/C to A/T substitutions are unlikely to be selected against may contribute to the rapid evolution of centromeric DNA. We also show that a neo-centromere sequence is not simply a weak version of native centromeric DNA and suggest that neo-centromeres require factors either for their propagation or establishment in addition to those required by native centromeres.

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. cerevisiae and in mammalian cells suggested that the major determinant of the activity of an integrase is the enzyme itself and not the cell in which it is working. We used an inducible promoter to show that six integrases were toxic as judged by their effects upon the proliferative ability of transformed yeast. We show that in general the active phage-encoded serine integrases were an order of magnitude more efficient in promoting genome integration reactions than a simple homologous recombination. CONCLUSIONS: The results of our study allow us to identify the integrases of the phage ϕBT1, TP901 ~ nls, R4, Bxb1, MR11, A118, ϕK38, ϕC31 ~ nls, Wß and SPBC ~ nls as active in S. cerevisiae and indicate that vertebrate cells are more restricted than yeast in terms of which integrases are active.

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.

BACKGROUND: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. RESULTS: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. CONCLUSIONS: We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.

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.

A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

Use of a selective inhibitor to define the chemotherapeutic potential of the plasmodial hexose transporter in different stages of the parasite's life cycle.

During blood infection, malarial parasites use D-glucose as their main energy source. The Plasmodium falciparum hexose transporter (PfHT), which mediates the uptake of D-glucose into parasites, is essential for survival of asexual blood-stage parasites. Recently, genetic studies in the rodent malaria model, Plasmodium berghei, found that the orthologous hexose transporter (PbHT) is expressed throughout the parasite's development within the mosquito vector, in addition to being essential during intraerythrocytic development. Here, using a D-glucose-derived specific inhibitor of plasmodial hexose transporters, compound 3361, we have investigated the importance of D-glucose uptake during liver and transmission stages of P. berghei. Initially, we confirmed the expression of PbHT during liver stage development, using a green fluorescent protein (GFP) tagging strategy. Compound 3361 inhibited liver-stage parasite development, with a 50% inhibitory concentration (IC50) of 11 µM. This process was insensitive to the external D-glucose concentration. In addition, compound 3361 inhibited ookinete development and microgametogenesis, with IC50s in the region of 250 µM (the latter in a D-glucose-sensitive manner). Consistent with our findings for the effect of compound 3361 on vector parasite stages, 1 mM compound 3361 demonstrated transmission blocking activity. These data indicate that novel chemotherapeutic interventions that target PfHT may be active against liver and, to a lesser extent, transmission stages, in addition to blood stages.

Targeted disruption of py235ebp-1: invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein.

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

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. We discuss the unanswered questions posed by genome engineering experiments in a variety of systems in which the serine recombinases have been used and finally describe more recently discovered serine recombinases that have the potential to be used in genome engineering.

The Armadillo repeat protein PF16 is essential for flagellar structure and function in Plasmodium male gametes.

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.

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. It is also known that kinetochore assembly requires sister chromatid cohesion at mitosis. We therefore suggest that separation or stretching between the sister chromatids at metaphase reciprocally determines the amount of centromere assembly in the subsequent interphase. This reciprocal relationship forms the basis of a negative feedback loop that could precisely control the amount of CENP-A and faithfully maintain the presence of a kinetochore over many cell divisions. We describe how the feedback loop would work, propose how it could be tested experimentally and suggest possible components of its mechanism.

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.

Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in the endothecium and is essential for anther dehiscence.

The Arabidopsis thaliana MYB26/MALE STERILE35 (MS35) gene is critical for the development of secondary thickening in the anther endothecium and subsequent dehiscence. MYB26 is localized to the nucleus and regulates endothecial development and secondary thickening in a cell-specific manner in the anther. MYB26 expression is seen in anthers and also in the style and nectaries, although there is no effect on female fertility in the ms35 mutant. MYB26 expression in anthers occurs early during endothecial development, with maximal expression during pollen mitosis I and bicellular stages, indicating a regulatory role in specifying early endothecial cell development. Overexpression of MYB26 results in ectopic secondary thickening in both Arabidopsis and tobacco (Nicotiana tabacum) plants, predominantly within the epidermal tissues. MYB26 regulates a number of genes linked to secondary thickening, including IRREGULAR XYLEM1 (IRX1), IRX3, IRX8, and IRX12. Changes in expression were also detected in two NAC domain genes, NAC SECONDARY WALL-PROMOTING FACTOR1 (NST1) and NST2, which have been linked to secondary thickening in the anther endothecium. These data indicate that MYB26 regulates NST1 and NST2 expression and in turn controls the process of secondary thickening. Therefore, MYB26 appears to function in a regulatory role involved in determining endothecial cell development within the anther and acts upstream of the lignin biosynthesis pathway.

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.