André Picard

DNA libraries for sequencing the genome of Ostreococcus tauri (Chlorophyta, prasinophyceae): The smallest free-living eukaryotic cell

Ostreococcus tauri is a marine photosynthetic picoeukaryote presenting a minimal cellular organization with one nucleus, one chloroplast, and one mitochondrion. It has the smallest genome described among free‐living eukaryotic cells, and we showed by pulsed‐field gel electrophoresis (PFGE) that it is divided between 15 bands ranging from 1.2 to 0.15 Mb, giving a total size of 9.7 Mb. A Bacterial Artificial Chromosome (BAC) library was prepared from genomic DNA extracted from a culture of O. tauri. A total of 2457 clones was obtained with an average insert size of around 70 kb, representing an 18‐fold coverage of the genome. The library was spotted on high density filters, and several probes of coding sequences were hybridized to both the high density BAC library filters and directly to the dried PFGE gels of the O. tauri genomic DNA. These hybridizations allowed a preliminary organization of the library and the localization of several markers on the chromosomes. Randomly selected fragments were also sequenced, representing 12% of the O. tauri genome. Many sequences showed significant similarities in data banks, mainly with plant and algae sequences. About 1000 coding sequences could be identified. These data confirmed the position of O. tauri in the green lineage and the hypothesis of a very compact organization of its genome.

The first green lineage cdc25 dual-specificity phosphatase

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G2/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G2/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been co-evolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages. Link to supplemental material: http://www.landesbioscience.com/journals/cc/khadarooCC3-4-sup.pdf

Cell cycle regulation of the primitive dinoflagellate Crypthecodinium cohnii Biecheler: Evidence for the presence of an homolog of cyclin B

Summry— Despite their early emergence in evolution, the protist dinoflagellates present similarities in cell cycle regulation with higher eukaryotes and yeasts. Using antibody directed against the p56edc13 of the fission yeast, we present evidence for the presence of a cyclin B homolog in the dinoflagellate Crypthecodinium cohnii. Biochemistry, immunofluorescence and electron microscopy show the continuous presence of the cyclin B homolog during the C cohnii cell cycle progression. Cyclin B, which appears to be exclusively cytoplasmic, is associated with a specific H1 kinase activity more active in M phase.

Subcellular distribution of aster‐nucleated microtubule length: A more or less mitotic status of cytoplasmic areas during meiosis I of starfish oocytes

Summary— Shortening of aster‐nucleated microtubules is a mitotic feature, which is proportional to the M‐phase promoting factor kinase activity. We have taken advantage of this characteristic to appraise the mitotic status of cytoplasmic territories in polyspermic starfish oocytes. In early prometaphase, asters are small in the nuclear area, where cyclin B accumulated before GVBD, while in the remaining of the oocytes asters are large. In metaphase all asters are small. This coincides with a change in behaviour of cyclin B, whose association with microtubules increases in late prometaphase. We discuss the possibility that uneven distribution of cyclin B‐dependent kinase is a general feature of mitosis and that it allows the cell to build the convenient spindle at the right place.

Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs

Polyphosphoinositides were quantified in metaphase II‐arrested eggs of the amphibian Xenopus laevis, and 8–10 min later in eggs activated by pricking. The content of phosphatidylinositol 4,5‐biphosphate (PIP2) was remarkably high in metaphase II‐arrested eggs with respect to that of phosphatidylinositol 4‐phosphate (PIP). It was found to drop dramatically at activation. In contrast PIP content did not change significantly.

Antipain microinjection prevents progesterone to inhibit adenyl cyclase in Xenopus oocytes

Microinjection of antipain, an inhibitor of thiol and Ca2+-dependent proteases, in immature Xenopus oocytes inhibited meiotic maturation induced by progesterone, but not by transfer of cytoplasm taken from maturing oocytes. Oocytes could be released from antipain inhibition by increasing progesterone concentration. alpha-32P-ATP was microinjected to study adenylcyclase in ovo. As already reported, neosynthesis of cAMP was decreased following progesterone application. This decrease was not observed, or it was considerably reduced, in oocytes previously injected with antipain. In amphibian, full-grown ovarian oocytes are arrested at first meiotic prophase, and have a large nucleus known as the germinal vesicle. Progesterone induces the production of a cytoplasmic maturation-promoting factor (MPF), which itself triggers germinal vesicle breakdown (GVBD), and subsequent events of meiotic maturation (Masui and Markert, 1971; Gerhart et al., 1984). A considerable body of evidences support the view that release from prophase block is due to inactivation of a cAMP-dependent protein kinase (reviewed by Maller, 1983). On the other hand, progesterone has been shown to induce a transient decrease in cAMP level (Speaker and Butcher, 1977; Schorderet-Slatkine et al., 1982; Cicirelli et al., 1985), and this initial drop of cAMP, along with a number of studies indicating a decrease in adenylate cyclase activity (Mulner et al., 1979; Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et al., 1981; Jordana et al., 1981), provided key support to the theory that an early drop in cAMP led to the dephosphorylation of a hypothetical protein which initiates maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

ROLE OF CYCLIN DEPENDENT KINASE IN G1→S TRANSITION AND IN S PHASE OF THE FIRST SEA URCHIN EMBRYONIC CYCLE

BODART Jean-Francois’, FLAMENT..&?phanc’. BROWAEYS Edith’. BERTOUT ~arcl, ROUSSEAU Arlettel, GANNON Julian2 et VILAIN Jean-Pierrel. 1 Centre de Biologie Cellulaire, Unit6 de Dynamique des cellules embryonnaires et cancereuses, Laboratoire de Biologie du Developpcment. EA DRED 1033, UniversitC de Lille 1, SN3, F-59655 Villeneuve d’Ascq cedex, France. 2 Imperial Cancer Research Fund Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD. U.K.