Translational repression (TR) of mRNAs plays an important role in sexual differentiation and gametogenesis in multicellular eukaryotes. We show here that TR and mRNA turnover are key influences on stage specific gene expression in the protozoan Plasmodium. The DDX6 class RNA helicase, DOZI (development of zygote inhibited), is found in cytoplasmic bodies of female, blood stage gametocytes in a complex with mRNA species known to be translationally repressed. Genetic disruption of pbdozi inhibits the formation of translationally quiescent mRNPs and targets these and other (>350 different) transcripts to the degradation pathway rather than directing them to translating polysomes, preventing zygote development prior to meiosis in the mosquito. Thus TR is essential in malaria parasite development. A full catalogue of the proteins and processes associated with DOZI (the first described malaria parasite TR-effector protein) might lead to novel approaches to prevent parasite development.Translational repression (TR) of mRNAs in higher eukaryotes controls temporal expression of specific protein cascades or directs the location of translation within a cell, and is prominent after gamete fertilisation (zygote formation) in the early embryo when de novo transcription of mRNA is restricted (1-5). The hallmark of repression is the assembly of certain mRNAs together with proteins into quiescent messenger ribonucleoprotein particles (mRNPs) where these transcripts are stored for translation at a later time. The DDX6-family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the * To whom correspondence should be addressed waters@lumc.nl. Post-fertilsation development of Plasmodium zygotes in the mosquito relys on the formation of translationally quiescent mRNPs in blood-stage, female sexual precursor cells Supporting Online Material www.sceincemag.org Materials and Methods Figs. S1 to S4 Tables S1 to S4 References

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.

It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Translocations are the best-studied nonrandom chromosomal aberrations associated with specific subtypes of leukemia. As a result of a translocation, an oncogene can be activated through alterations in regulatory DNA sequences that leave the encoded protein intact (e.g., myc) or through formation of a fusion gene, encoding a chimeric protein (e.g., bcr-abl). The t(9;22) associated with chronic myeloid leukemia, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (29) results in the expression of a chimeric BCR-ABL protein with enhanced tyrosine kinase activity (16,19,27,38,45). Pendergast et al. showed that defined sequences encoded by the first exon of bcr interact with the SH2 domain of ABL (33). This interaction is essential for the activation of the ABL tyrosine kinase activity and for the transforming capacity of BCR-ABL. More recently, other fusion genes have been isolated. t(1;19), occurring in childhood pre-B-cell acute leukemia, fuses the E2a gene, encoding transcription factors E12 and E47, to a novel homeobox gene, PBX1 (26, 32). t(15;17), strongly associated with acute promyelocytic leukemia, fuses part of the retinoic acid receptor type a gene (RARt) to a novel gene on chromosome 15 named PML, which is predicted to be a transcription factor (9, 25). bcr-abl, E2A-pbx, andpml-RARao seem to be highly consistent partners.Previously we reported the cloning of t(6;9) breakpoints (43). t(6;9) is the hallmark of a specific subtype of AML characterized by a poor prognosis and a young age of onset. It is classified in the French-American-British system mostly as M2/M4 and rarely as Ml or refractive anemia with excess of blast cells (RAEB) (2,36,39). On chromosome 9, break-* Corresponding author.points take place in a specific intron, icb-9, of a large gene (>140 kb) named Cain (can) (43). On chromosome 6, breakpoints also occur in a single intron, icb-6, of a gene named dek (42). The result of t(6;9) is the formation of a dek-can fusion gene on chromosome 6p-, which is transcribed into an invariable, 5.5-kb, leukemia-specific dek-can mRNA (39). The fusion transcript encodes a 165-kDa chimeric protein, which derives from the in-frame fusion of dek and can open reading frames (ORFs). Sequence comparison of DEK and CAN with entries in the EMBL data base shows no homology to any known protein sequences. CAN contains several putative dimerization motifs, and the C-terminal part may function as an ancillar...

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We describe a detection principle for indirect fluorescence in situ hybridization (FISH) methods that with only one or two antibody layers dramatically improves FISH signal intensities. The method uses as a first layer an anti-hapten immunoglobulin [or (strept)avidin] conjugated to peroxidase. The quintessence of the method is the use of fluorochrome- or biotin labelled tyramides as peroxidase substrates to generate and deposit many fluorochrome or biotin molecules close to the in situ bound peroxidase. These may either be directly evaluated under the fluorescence microscope or after another incubation with fluorochrome-labelled (strept)avidin.

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