Notas sôbre a meiose de pachylis (Hemipt-Coreidae)

Spermatogonial chromosomes of Pachylis laticornis and Pachylis pharaonis begin anaphasic movement with both ends turned toward the same pole, maintaining this form util they reach the poles. This is a proof that they are provided with one kinetochore at each end. Additional proof for a longitudinal division of each longitudinal half of the anaphase chromosomes of the primary sper- matocytes is presented against the idea of a previous end-toend pairing at metaphase. The longitudinal split of the chromosomes of the secondary spermatocytes which used to be considered as tertiary split is therefore a true secondary split. The heterochromosome in both species passes undivided to one pole in the first division of the spermatocyte. In Pachylis laticornis it appears connected with the poles by means of two fibrils detached from each extremity, what may be considered as indicating a rather premature longitudinal spliting. The behavior of the heterochromosome of Pachylis pharaonis is highly interesting and affords one of the most beautiful evidences in favour of the dicentricity of the chromosomes. Really, in metaphase the heterochromosome appears at the equator of the cell with a more or less round shape. In the beginning of anaphase it becomes fusiform. As anaphase proceeds it distends itself between the autosomal plates forming a long fusiform bridge or sends toward the plates a thick chromosomal thread. The bulky part of the heterochromosome as it passes to one side it reincorporates the substance of the thread in this side. The thread in the other side, which becomes generally thiner, is left with its kinetochore in the cell at this side. The heterochromosome therefore becomes terminally monocentric in the first division of the spermatocyte. Some figures, however, suggest that the heterochromossome from time to time may pass with both kinetochores to one of the cells, as ordinarily happens in the case of Pachylis laticornis. Summing up, other things apart the behavior of the heterochromosome in both species studied here puts out of doubt the question of the existence of two terminally located kinetochores.

Nota prévia sôbre a meiose de Corizus (Liorhyssus) hyalinus (Fabr.) (Hemiptera-Corizidae)

The main facts presented in this paper may be summarized as follows: 1) Corizus (Liorhyssus) hyalinus (Fabr.) has primary spermatocytes provided with 6 autosomal tetrads, one pair of microchromosomes and one sex chromosome. 2) The two microchromosomes present in this species sometimes appear at the primary metaphase as an unequal pair of minute elements. In the secondary spermatocytes the unique microchromosome present may be in the limit of visibility or entirely invisible. This invisibility may be partly due to a loss of colourability. 3) The sex chromosome divides transversely in the first division of the spermatocyte, passing undivided to one pole in the second one. In the latter it becomes fusiform in the beginning of anaphase revealing in this manner its dicentricity. In late anaphase it finishes by passing to one pole leaving in the other pole one of its kinetochores sometimes accompanied by a chromosomal fragment. 4) All the chromosomes divide transversely in both divisions, a diagram being enclosed to elucidate the question. 5) Spermatogonial chromosomes are provided with one kinetochore at each end, being curved toward the poles since the most beginning anaphase. 6) The following hypothesis is presented as an essay to explain the origin of microchromosomes: Since microchromosomes parallel sex chromosomes in most respects, as for instances in heteropycnosis and pairing modus, it seems highly probable that they originate from sex chromosomes. One may suppose that the ancestral form of a given species had a sex chromosome which used to lose a small centric fragment when it divided during meiosis. This fragment might well be at first an unstable one. Later, to compensate the effects of such a deficiency a mechanism arose through evolution which produced two useful results : a) the establishment of the fragment as a permanent structure of the cell nucleus and b) the acquirement by the sex chromosome of the faculty of passing to one pole without losing any of its ends.

Notas sobre cromossômios de alguns escorpiões brasileiros

Three species of Scorpions beloging to two different families were studied cytologically: a) Tityus mattogrossensis Borelli (Fam. Buthidae), - This species presents spermatogonia provided with 20 short chromosomes which orient at metaphase with their axis parallelly to the plane of the equator and move toward the poles without changing this position, from the stage pachytene to metaphase the bivalents become, as in Tityus bahiensis, progressivery shorter and thicker, without showing that chiasmata occured at any time. The paired chromosomes never open themselves, out to form loops as in orthodox meioses. As in Tityus bahiensis the bivalents are inserted In the spindle before reaching their maxim contraction. No diakinesis has been observed. The primary spermatocyte metaphases are provided, with 10 pairs of chromosones, two of which are larger and two smaller than the rest. The bivalents orient as in Tityus bahiensis with their length in the plane of the equator and separate parallelly. Spindle fibres are seen alongst their entire body. While, in Tityus bahiensis the ends of the chromosomes are pronouncedly turned to opposite poles at metaphase, nothing like this was observed in the present species. Only late in anaphase the chromosomes of Tityus mattogrossensis show a bending to the poles. The secondary spermatocytes present 10 short chromosomes, two being larger than, the others. Here, on the contrary, the chromosomes are strongly curved toward the poles since the beginning of anaphase. Some chromosomal anomalies have been noticed. Primary spermatocytes with 14 bivalents, some of which representing probably free fragments, were observed. Primary spermatocytes with 8 bivalents and one cross of 4 chromosomes were interpreted as resulting from breakages followed by translocations Primary spermatocytes with 9 bivalents, one of which being much longer than the longst of the normal plates, show that fusion by the extremities of two non homologous chromosomes on the onde side, and of their respective homologous in the same way on tre other, have occured. Orientation of bivalents with their body parallelly to the spindle axis and anaphasic bridges have been encountered. All in all points to the conclusion that the chromosomes of Tityus mattogrossesis, like those of Tityus bahiensia are provided with one kinetochore at each end. Ananteris balzani Thorell - (Fam. Buthidae). - This species which belongs to the same family as Tityus, is provided with 12 chromosomes (diploid). These studied in embryonic tissues, showed the same behavior as the somatic chromosomes of Tityus bahiensis. Bothrirus sp. (Bothriuridae). - Only spermatogonia were found in the testis, of the single male hitherto investigated. The chromosomes, in number of 36, are of different sizes but small and provided, as ordinarily, with a single kinetochore. They behave therefore in an orthodox manner in mitosis.

Comportamento dos cromossômios no gênero Hypselonotus (Hemiptera -Coreidae)

The three species studied have 19 chromosomes, being one heterochromosome, one pair of microchromosomes and 8 pairs of autosomes. The microchromosomes of Hypselonotus fulvus are amongst the largest we know. During the synizesis, in Hypselonotus fulvus, we can see in several strands that scape from the chromatic knot a place in which they are widley open. As, in that phase the chromosomes have both ends converging to the same place, the openings suggest a side-to-side pairing of the chromosomal threads. The tetrads are like that studied by Piza (1945-1946). The bivalents are united side by side at their entire length. The unpaired part at the midle of the bivalents gives origin to the arms of the cross-shapede tetrads. The chromosomes have a kinetochore at each end. The bivalents sometimes unite their extremities to form ring-shaped figures, which open themselves out before metaphase. The tetrads are oriented parallelly to the spindle axis. At telophase the kinetochores repeli one another, the chiasmata, if present, slip toward the acentric extremities and the chromosomes rotate in order to arrange themselves parallelly to the axis of the new spindle. Separation is therefore through the pairing plane. In the spermatogonial anaphase of Hypselonotus subterpunctatus the chromosomes are curved to the poles, like those described by PIZA (1946) and PIZA and ZAMITH (1946). The sex chromosomes in Hypselonotus interruptus and Hypselonotus fulvus appears longitudinally divided. It is oriented with the ends in the plane of the equator and its chomatids separate by the plane of division. In the second division the sex chromosome, provided as it is with an actve klnetochore at each end, orients itself with its length parallelly to the spindle axis and passes undivided to one pole. Sometimes it is distended between the poles. This corresponds to case (a) established by PIZA (1946) for the sex chromosomes of Hemiptera In Hypselonotus subterpunctatus the sex chromosome, in the first division of the spermatocytes, orients like the tetrads and divides transversaly. In the second division, as its kinetochore becomes inactive, it remans monocentric, does not orient in the spindle, and is finally enclosed in the nearer nucleus. In the secondary telophase it recuperates its dicentricity like the autosomal chromatids. This behavior corresponds to case (c) of PIZA (1946).

Sobre o comportamento mitótico e meiótico de cromossomas policêntricos ou com ponto de inserção difuso

Material: Studies were made mainly with Ascaris megalocephála Cloq. univalens and bivalens, and also with Tityus bahiensis Perty. 1) Somatic pairing of heterochromatic regions. The heterochromatic ends of the somatic chromosomes in Ascaris show a very strong tendency for unspecifical somatic pairing which may occur between parts of different chromosomes (Figs. 1, 2, 3, 7, 10, 11, 12, 13, 14, 16, 18,), between the two ends of the same chromosome either directly (Figs. 4, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18) or inversely (Fig. 8, in the arrow) and also within a same chromosomal arm (Fig. 6). 2) During the early first cleavage division the chomosomes are an isodiametric cylinder (Figs. 6, 9, 11, 13, 14). But in later metaphase the ends become club shaped (Figs. 1, 2, 3, 4, 5, 7, 10) which is interpreted as the beginning of migration of chromatic substance from the central euchromatic region towards the heterochromatic regions. This migration becomes more and accentuated in anaphase (Figs. 19, 22, 23) and in the vegetative cells where euchromatic region looses more and more staing power, especially in the intersititial zones between the individual small spherical chromosomes into which the euchromatic region desintegrates. The emigrated chromatin material is finally eliminated with the heterochromatic chromosome ends (Fig. 23 and 24). 3) It seems a general rule that during mitotic anaphase all chromosomes with diffuse or multiple spindle fiber attachement (Ascaris, Tityus, Luzula, Steatococcus, Homoptera and Heteroptera in general) move to the poles in the form of an U with precedence of the chromosomal ends. In Ascaris, the heterocromatic regions are pulled passively towards the poles and only the euchromatic central portion may be U-shaped (Fig. 19, 22, 25). While in the other species this U-shape is perfect since the beginning of anaphase, giving the impression that movement towards the poles begins at both ends of a chromosome simultaneously, this is not the case in Ascaris. There the euchromatic region is at first U-shaped, passing then to form a straight or zig-zag line and becoming again U-shaped during late anaphase. This is explained by the fact that the ends of the euchromatic regions have to pull the weight of the passive heterochromatic portions. 4) While it is generally accepted that, during first meio-tic division untill second anaphase, all attachement regions remain either undivided or at least united closely, this is not the case in chromosomes with diffused or multiple attachment. Here one clearly sees in all cases so far studied four parallel chromatids at first metaphase. In Luzula and Tityus (for Tityus all figs. 26 to 31) this division is allready quite clear in paraphase (pro-metaphase) and it cannot be said wether in other species the division in sister chromatids is allready present, but not visible at this stage. During first anaphase the sister chromatids of Titbits remain more or less in contact, while in Luzula and especially in Ascaris they are quite separated. Thus one can count in late anaphase or telophase of Ascaris megalocephala bivalens, nearly allways, four separate chromosomes near each pole, or a total of eight chromatids per division figure (Figs. 35, 36, 37, 38, 39, 40, 41).

Influência do propianato de testosterona e do "Diffusing factor" sôbre a cicatrização cutânea de lesões experimentais

The effect of testosterone propionate in different treatments was tested in adult male rats (250 g.) with mechanical skin experimental lesions. The whole period of cicatrization was investigated in normals, castrated and testosterone treated animals. We could not detect any alteration in the regeneration process in both treated and untreated rats (normals and castrated). Diffusing factor obtained from homologous testis, directly applied upon the lesions also do not change the healing period. Related to the course of the healing process, little evidence is presented by variance analysis that significative differences could be detected in the first periods, in both castrated and testosterone treated groups; however new well planed experiments should be carried to test this point.

Trypanosoma cruzi: circulating antigens

Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.

Anti-schistosomal drugs: observations on the mechanism of drug resistance to hycanthone, and on the involvement of host antibodies in the mode of action of praziquantel

This paper reports recent observations from our laboratory dealing with the anti-schistosome drugs hycanthone (HC) and praziquantel (PZQ). In particular, we discuss a laboratory model of drug resistance to HC in Schistosoma mansoni and show that drug sensitive and resistant lines of the parasite can be differentiated on the basis of restriction fragment length polymorphisms using homologous ribosomal gene probes. In addition, we summarize data demonstrating that effective chemotherapy of S. mansoni infection with PZQ in mice requires the presence of host anti-parasite antibodies. These antibodies bind to PZQ treated worms and may be involved in an antibody-dependent cellular cytotoxicity reactions which result in the clearance of worms from the vasculature.

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

Characterization of a Trypanosoma cruzi genomic fragment complementary to several species-specific mRNAs but different from the spliced leader sequence

We have isolated a clone of Trypanosoma cruzi genimic DNA, lambda 3b2-5, which contains sequences that are reiterated in the genome. Northtern blot analysis showed that clone 3b2-5 hybridizes to 1,200-5,000 bases different mRNA species. The number of mRNAs species hybridized to clone 3b2-5 exceeds its coding capacity showing that this clone carries sequences that are common to several mRNAs species and conserved in the poly A(+) RNA. These sequences are not homologous to the T. cruzi spliced leader sequence, since clone 3b2-5 hybridize to a synthetic 20 nucleotice complementary to the spliced leader sequence. Clone 3b2-5 does not hybridize to DNA and RNA from several genera of Trypanosomatidae and other Trypanosoma species indicating that it carries T. cruzi species-specific sequences.

Characterisation of heteromers and oligomers in the TNF family of ligands and receptors

TNF family ligands and receptors fulfill a number of functions, mainly in the immune system. For example, the ligands BAFF and APRIL control growth and survival of mature Β cells at various stages of differentiation. TNF family ligands usually form homotrimers, but heteromers have also been described for lymphotoxin α1β2 and for BAFF and APRIL. Interestingly, twenty BAFF homotrimers can assemble into virus-like particles coined BAFF 60-mer, which are superior to BAFF 3-mer regarding their ability to signal in primary Β cells. A screen was performed in 293T cells, by co-transfecting differently tagged ligands, to identify six novel heteromers. The specificity of these novel heteromers, however, did not correspond to that of orphan receptors in the TNFR family. Little is known about heteromers of BAFF and APRIL, in particular their receptor-binding specificity and their ability to signal. A method to produce and purify heteromers of defined stoechiometry was developed, and the resulting reagents were used to demonstrate that BAFF2APRIL, like BAFF, binds to all BAFF receptors - namely BAFFR, TACI and Β CM A -, while APRIL2BAFF and APRIL only binds to TACI and BCMA. Heteromers could signal via their cognate receptors, sometimes as potently and sometimes less potently than homomers, depending on the receptors. A promising system to measure the activity of single-chain homo- and heteromers in vivo was set up: it measures mature Β cell rescue upon administration of single-chain ligands into BAFF-ko mice. To tackle the question of the physiological importance of BAFF 60-mer, a point mutation that prevents assembly of mouse BAFF into 60-mer while retaining its ability to form trimers was identified. This mutation (E247K) was introduced by homologous recombination into mouse embryonic stem cells that are now being used to generate knock-in mice. Results obtained in this work will help to better understand the role of various BAFF and APRIL forms that are elevated in a several autoimmune diseases. - Les ligands et récepteurs de la famille du TNF joue un rôle prédominant dans le système immunitaire. Par exemple, les ligands BAFF et APRIL contrôlent la croissance et la survie des cellules Β matures à différents stades de différenciation. Ces ligands existent souvent sous forme d'homotrimères (3-mer), bien que des héteromères aient été décrits pour la lymphotoxine α1β2 et pour BAFF et APRIL. Dans le cas de BAFF, vingt trimères peuvent, telle une particule virale, s'assembler en 60-mer qui surpasse le 3-mer pour signaler dans des cellules Β primaires. Un crible effectué dans des cellules 293T, par co-transfection de ligands différemment marqués, a permis d'identifier six nouveaux heteromères dont la spécificité n'a, hélas, pas correspondu à celle d'un récepteur orphelin de la famille du TNFR. Les connaissances sur la spécificité de liaison aux récepteurs et la capacité à signaler des heteromères de BAFF et d'APRIL sont fragmentaires. Une méthode pour produire et purifier des heteromères "simple chaîne" de stoechiométrie déterminée a été mise au point, et les réactifs ainsi obtenus utilisés pour démontrer que BAFF2APRIL, comme BAFF, lie tous les récepteurs de BAFF - c'est-à-dire BAFFR, TACI et BCMA -, alors qu'APRIL2BAFF et APRIL ne lient que TACI et BCMA. Les héteromères peuvent transmettre des signaux, parfois aussi bien et parfois plus faiblement que les homomères, selon les récepteurs. Un système prometteur pour mesurer l'activité des ligands simple chaîne in vivo a été mis au point. Il mesure la réapparition de cellules Β matures dans des souris déficientes pour BAFF après administration des ligands. Pour s'attaquer à la question de l'importance physiologique du 60-mer de BAFF, ime mutation empêchant l'assemblage en 60-mer sans affecter la capacité à former des trimères a été identifiée. Cette mutation (E247K) a été introduite par recombinaison homologue dans des cellules souches embryonnaires de souris qui sont utilisées pour obtenir des souris déficientes en BAFF 60-mer. Les résultats de ces travaux contribueront à mieux cerner le rôle des différentes formes de BAFF et d'APRIL produites en excès dans plusieurs maladies auto-immunes.

Origine des populations de rainette verte (Hyla spp.) de l'ouest de la Suisse

DNA-based techniques are important tools for species assignment, in particular when identification with morphological criteria is difficult. The aim of this study was to genetically determine the species identity of tree frogs (Hyla spp.) populations from western and northern Switzerland (Swiss Plateau), this area being frequently subjected to introductions of species or sub-species from south of the Alps. We sequenced 261 base pairs of the mitochondrial DNA cytochrome b gene from 24 samples of tree frogs from the Swiss Plateau, Ticino (southern Switzerland) and the Dombes region (Ain, France), and compared them with homologous sequences retrieved from DNA databases. The phylogenetic analyses revealed two distinct clades. The first one is represented by samples of Green tree frog (Hyla arborea) from the Swiss Plateau, France, Germany and Greece, confirming the current knowledge about the species' distribution. The second clade includes samples belonging to the Italian tree frog (Hyla intermedia) from south of the Alps (Ticino and Italy), and unexpectedly from the Grangettes site in western Switzerland. These results suggest the introduction of the Italian tree frog H. intermedia north of the Alps, and raise questions about the management of the Grangettes protected area.

No evidence for immune priming in ants exposed to a fungal pathogen.

There is accumulating evidence that invertebrates can acquire long-term protection against pathogens through immune priming. However, the range of pathogens eliciting immune priming and the specificity of the response remain unclear. Here, we tested if the exposure to a natural fungal pathogen elicited immune priming in ants. We found no evidence for immune priming in Formica selysi workers exposed to Beauveria bassiana. The initial exposure of ants to the fungus did not alter their resistance in a subsequent challenge with the same fungus. There was no sign of priming when using homologous and heterologous combinations of fungal strains for exposure and subsequent challenges at two time intervals. Hence, within the range of conditions tested, the immune response of this social insect to the fungal pathogen appears to lack memory and strain-specificity. These results show that immune priming is not ubiquitous across pathogens, hosts and conditions, possibly because of immune evasion by the pathogen or efficient social defences by the host.

CTCF-T, a paralogue of CTCF, directs sequence specific DNA methylation of the imprinting control region of Igf2/H19

SUMMARY Genomic imprinting is an epigenetic mechanism of transcriptional regulation that ensures restriction of expression of a subset of mammalian genes to a single parental allele. The best studied example of imprinted gene regulation is the Igf2/H19 locus, which is also the most commonly altered by loss of imprinting (LOT) in cancer. LOT is associated with numerous hereditary diseases and several childhood, and adult cancers. Differential expression of reciprocal H19 and 1gf2 alleles in somatic cells depends on the methylation status of the imprinting control region (ICR) which regulates binding of CTCF, an ubiquitously expressed 11-zinc finger protein that binds specifically to non-methylated maternal ICR and thereby attenuates expression of Igf2, while it does not bind to methylated paternal ICR, which enables Igf2 expression. Initial ICR methylation occurs during gametogenesis by an as yet unknown mechanism. The accepted hypothesis is that the event of differential maternal and paternal DNA methylation depends on germ-line specific proteins. Our Laboratory identified a novel 11-zinc-finger protein CTCF-T (also known as CTCFL and BORIS) that is uniquely expressed in the male germ-line and is highly homologous within its zinc-finger region with CTCF. The amino-acid sequences flanking the zinc-finger regions of CTCF and CTCF-T have widely diverged, suggesting that though they could bind to the same DNA targets (ICRs) they are likely to have different functions. Interestingly, expression of CTCF-T and CTCF is mutually exclusive; CTCF-T-positive (CTCF-negative) cells occur in the stage of spermatogenesis that coincides with epigenetic reprogramming, including de novo DNA methylation. In our study we demonstrate the role that CTCF-T plays in genomic imprinting. Here we show that CTCF-T binds in vivo to the ICRs of Igf2/H19 and Dlk/Gt12 imprinted genes. In addition, we identified two novel proteins interacting with CTCF-T: a protein arginine methyltransferase PRMT7 and an arginine-rich histone H2A variant that we named trH2A. These interactions were confirmed and show that the two proteins interact with the amino-teiminal region of CTCF-T. Additionally, we show interaction of the amino- terminal region of CTCF-T with histones H1, H2A and H3. These results suggest that CTCF-T is a sequence-specific DNA (ICR) binding protein that associates with histones and recruits PRMT7. Interestingly, PRMT7 has a histone-methyltransferase activity. It has been shown that histone methylation can mark chromatin regions thereby directing DNA-methylation; thus, our hypothesis is that the CTCF-T protein-scaffold directs PRMT7 to methylate histone(s) assembled on ICRs, which marks chromatin for the recruitment of the de novo DNA methyltransferases to methylate DNA. To test this hypothesis, we developed an in vivo DNA-methylation assay using Xenopus laevis' oocytes, where H19 ICR and different expression cDNAs, including CTCF-T, PRMT7 and the de novo DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt3L) are microinjected into the nucleus. The methylation status of CpGs within the H19 ICR was analysed 48 or 72 hours after injection. Here we demonstrate that CpGs in the ICR are methylated in the presence of both CTCF-T and PRMT7, while control oocytes injected only with ICR did not show any methylation. Additionally, we showed for the first time that Dnmt3L is crucial for the establishment of the imprinting marks on H19 ICR. Moreover, we confirmed that Dnmt3a and Dnmt3b activities are complementary. Our data indicate that all three Dnmt3s are important for efficient de novo DNA methylation. In conclusion, we propose a mechanism for the establishment of de novo imprinting marks during spermatogenesis: the CTCF-T/PRMT7 protein complex directs histone methylation leading to sequence-specific de novo DNA methylation of H19 ICR. RESUME L'empreinte génomique parentale est un mécanisme épigénétique de régulation transcriptionelle qui se traduit par une expression différentielle des deux allèles de certains gènes, en fonction de leur origine parentale. L'exemple le mieux caractérisé de gènes soumis à l'empreinte génomique parentale est le locus Igf2/H19, qui est aussi le plus fréquemment altéré par relaxation d'empreinte (en anglais: loss of imprinting, LOI) dans les cancers. Cette relaxation d'empreinte est aussi associée à de nombreuses maladies héréditaires, ainsi qu'à de nombreux cancers chez l'enfant et l'adulte. Dans les cellules somatiques, les différences d'expression des allèles réciproques H19 et Ig12 est sous le contrôle d'une région ICR (Imprinting Control Region). La méthylation de cette région ICR régule l'ancrage de la protéine à douze doigts de zinc CTCF, qui se lie spécifiquement à l'ICR maternel non-méthylé, atténuant ainsi l'expression de Igf2, alors qu'elle ne s'ancre pas à l'ICR paternel méthyle. Le mécanisme qui accompagne la méthylation initiale de la région ICR durant la gamétogenèse n'a toujours pas été élucidé. L'hypothèse actuelle propose que la différence de méthylation entre l'ADN maternel et paternel résulte de l'expression de protéines propres aux zones germinales. Notre laboratoire a récemment identifié une nouvelle protéine à douze doigts de zinc, CTCF-T (aussi dénommée CTCFL et BORRIS), qui est exprimée uniquement dans les cellules germinales mâles, dont la partie à douze doigts de zinc est fortement homologue à la protéine CTCF. La séquence d'acides aminés de part et d'autre de cette région est quant à elle très divergente, ce qui implique que CTCF-T se lie sans doute au même ADN cible que CTCF, mais possède des fonctions différentes. De plus, l'expression de CTCF-T et de CTCF s'oppose mutuellement; l'expression de la protéine CTCF-T (cellules CTCF-T positives, CTCF negatives) qui a lieu pendant la spermatogenèse coïncide avec la reprogrammation épigénétique, notamment la méthylation de novo de l'ADN. La présente étude démontre le rôle essentiel joué par la protéine CTCF-T dans l'acquisition de l'empreinte génomique parentale. Nous montrons ici que CTCF-T s'associe in vivo avec les régions ICR des loci Igf2/H19 et Dlk/Gt12. Nous avons également identifié deux nouvelles protéines qui interagissent avec CTCF-T : une protéine arginine méthyl transférase PRMT7, et un variant de l'histone H2A, riche en arginine, que nous avons dénommé trH2A. Ces interactions ont été analysées plus en détail, et confinnent que ces deux protéines s'associent avec la région N-terminale de CTCF-T. Aussi, nous présentons une interaction de la région N-terminale de CTCF-T avec les histones H1, H2, et H3. Ces résultats suggèrent que CTCF-T est une protéine qui se lie spécifiquement aux régions ICR, qui s'associe avec différents histones et qui recrute PRMT7. PRMT7 possède une activité méthyl-tansférase envers les histones. Il a été montré que la méthylation des histones marque certains endroits de la chromatine, dirigeant ainsi la méthylation de l'ADN. Notre hypothèse est donc la suivante : la protéine CTCF-T sert de base qui dirige la méthylation des histones par PRMT7 dans les régions ICR, ce qui contribue à marquer la chromatine pour le recrutement de nouvelles méthyl transférases pour méthyler l'ADN. Afin de valider cette hypothèse, nous avons développé un système de méthylation de l'ADN in vivo, dans des oeufs de Xenopus laevis, dans le noyau desquels nous avons mico-injecté la région ICR du locus H19, ainsi que différents vecteurs d'expression pour CTCF-T, PRMT7, et les de novo méthyl transférases (Dnmt3a, Dnmt3b et Dnmt3L). Les CpGs méthyles de la région ICR du locus H19 ont été analysé 48 et 72 heures après l'injection. Cette technique nous a permis de démontrer que les CpGs de la région ICR sont méthyles en présence de CTCF-T et de PRMT7, tandis que les contrôles injectés seulement avec la région ICR ne présentent aucun signe de méthylation. De plus, nous démontrons pour la première fois que la protéine méthyl transférase Dnmt3L est déterminant pour l'établissement de l'empreinte génomique parentale au niveau de la région ICR du locus H19. Aussi, nous confirmons que les activités méthyl transférases de Dnmt3a et Dnmt3b sont complémentaires. Nos données indiquent que les trois protéines Dnmt3 sont impliquées dans la méthylation de l'ADN. En conclusion, nous proposons un mécanisme responsable de la mise en place de nouvelles empreintes génomiques pendant la spermatogenèse : le complexe protéique CTCF-T/PRMT7 dirige la méthylation des histones aboutissant à la méthylation de novo de l'ADN au locus H19.

Trypanosomatidae codon usage and GC distribution

A study od Typanosomatidae GC distribution and codon usage is presented. The codon usage patterns in coincidence with the phylogenetical data are similar in Crithidia and Leishmania, whereas they are more divergent in Trypanosoma brucei and T. cruzi. The analyisis of the GC mutational pressure in these organisms reveals that T. brucei, and to a lesser extent T. cruzi, have envolved towards a more balanced use of all bases, whereas Leishmania and Crithidia retain features of a primeval genetic apparatus. Tables with approximated GC mutational pressure in homologous genes, and codon usage in Trypanosomatidae are presented.