Heterochromatin DNA replication and Rif1.

Constitutive heterochromatin is essential for chromosome maintenance in all eukaryotes. However, the repetitive nature of the underlying DNA, the presence of very stable protein-DNA complexes and the highly compacted nature of this type of chromatin represent a challenge for the DNA replication machinery. Data collected from different model organisms suggest that at least some of the components of the DNA replication checkpoint could be essential for ensuring the completion of DNA replication in the context of heterochromatin. I review and discuss the literature that directly or indirectly contributes to the formulation of this hypothesis. In particular, I focus my attention on Rif1, a newly discovered member of the DNA replication checkpoint. Recent data generated in mammalian cells highlight the spatial and temporal relation between Rif1, pericentromeric heterochromatin and S-phase. I review these recent and the previous data coming from studies performed in yeast in order to highlight the possible evolutionary conserved links and propose a molecular model for Rif1 role in heterochromatin replication.

Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Sccl, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis i.

The orderly reduction in chromosome number that occurs during meiosis depends on two aspects of chromosome behavior specific to the first meiotic division. These are the retention of cohesion between sister centromeres and their attachment to microtubules that extend to the same pole (monopolar attachment). By deleting genes that are upregulated during meiosis, we identified in Saccharomyces cerevisiae a kinetochore associated protein, Mam1 (Monopolin), which is essential for monopolar attachment. We also show that the meiosis-specific cohesin, Rec8, is essential for maintaining cohesion between sister centromeres but not for monopolar attachment. We conclude that monopolar attachment during meiosis I requires at least one meiosis-specific protein and is independent of the process that protects sister centromere cohesion.

The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element.

Small nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus.

A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis.

A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis. Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II. The eukaryotic cell's cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process.

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation.

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1] [2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.

Enzymatic profile of Cryptococcus neoformans strains by using the API-ZYM system.

Determination of the enzymatic profile of 41 Cryptococcus neoformans strains, 20 isolated from AIDS patients and 21 from bird droppings, was performed by using the API-ZYM commercial kit system (Bio-Mérieux, France), which tests 19 different kinds of enzymes. All the strains showed positive enzymatic activity to the esterase (C4) (n. 3). On the contrary, alkaline phosphatase (n. 2), cystine arylamidase (n. 8), trypsin (n. 9), chymotripsin (n. 10), alpha-galactosidase (n. 13), beta-glucuronidase (n. 15), alpha-mannosidase (n. 19), alpha-fucosidase (n. 20) were negative in all the strains. The other 10 enzymes (n. 4,5,6,7,11,12,14,16,17,18) were distributed among the strains in different positive percentages. From the results of each enzymatic profile obtained, the 20 AIDS strains were grouped into 15 types, while the 21 bird dropping strains were grouped into 14 types. Interestingly, only one enzyme profile type occurred in the strains isolated from the AIDS patients and from the bird droppings. These results suggest that the API ZYM system is useful in discriminating between the AIDS strains and the bird dropping strains.

Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes.

The in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed.

[Cervico-facial non-Hodgkin's lymphomas. Apropos of a case of orbital localization]. / Les lymphomes non-hodgkiniens cervico-faciaux. A propos d'une localisation orbitaire.

The orbital localization of the non-Hodgkin's lymphoma is not the more common site of presentation. The most classical sites in the head and neck are the cervical lymph nodes and Waldeyer's ring. The treatment is depending on the Staging and includes radiation therapy, chemotherapy and surgery or a combination of these modalities. We describe here an orbital localization of intermediate-grade. The clinical and pathological features are discussed, and the literature is summarized.

Study of focal epilepsy by multichannel neuromagnetic measurements.

A systematic investigation of several cases of focal epilepsy has been performed in an unshielded environment using a 4-channel neuromagnetic sensor. The localizations provided by the magnetic measurements have been compared with clinical evidence and confirmed by X-ray findings, and in one case also by intracranial surgery. The results show the importance of simultaneous detection of magnetic fields at different sites of the scalp in order to get a dynamic view of the epileptic activity and to detect multifocal activity unsuspected on the basis of the EEG investigation.