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Translational repression of messenger RNAs (mRNAs) plays an important role in sexual differentiation and gametogenesis in multicellular eukaryotes. Translational repression and mRNA turnover were shown to influence stage-specific gene expression in the protozoan PLASMODIUM: The DDX6-class RNA helicase, DOZI (development of zygote inhibited), is found in a complex with mRNA species in cytoplasmic bodies of female, blood-stage gametocytes. These translationally repressed complexes are normally stored for translation after fertilization. Genetic disruption of pbdozi inhibits the formation of the ribonucleoprotein complexes, and instead, at least 370 transcripts are diverted to a degradation pathway.

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.

The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.

The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.