pGATA: a positive selection vector based on the toxicity of the transcription factor GATA-1 to bacteria.

The transcription factor GATA-1 is a zinc finger DNA-binding protein essential for the development of red blood cells. When we expressed different regions of the zinc finger domain in bacteria using an isopropyl-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria harboring the active DNA-binding domain of GATA-1 was rapidly inhibited upon IPTG induction. The growth inhibition pattern suggested it may be occurring at the level of the initiation of replication, and GATA-1 was found to bind to three of the four DNA A protein-binding sites in the origin of replication. This toxicity was used to develop a positive selection vector system in which cloned DNA fragments interfered with the production of the GATA-1 DNA-binding domain. Thus, vector molecules containing the insert of interest are selected for when bacteria are grown in the presence of IPTG. With this system, the vector does not need to be dephosphorylated, purified or completely digested with a restriction enzyme for the efficient cloning of DNA fragments even when the vector-to-insert DNA molar ratio in ligation reactions is 10 to 1. Moreover, no special strain of Escherichia coli is required, and the selection might also be applicable to other species of bacteria if the toxicity of GATA-1 relates to inhibition of the DNA A protein.

Murine erythroleukemia cells contain two distinct GATA-binding proteins that have different patterns of expression during cellular differentiation.

GATA-1 is a major transcription factor of the erythroid lineage that has been implicated in the induced expression of a variety of red cell-specific genes during terminal differentiation of murine erythroleukemia cells. Although the GATA-1 protein is present at nearly equal levels before and after differentiation of murine erythroleukemia cells, in this study it was found that in the early commitment stages of the differentiation program there is a transient decrease in the GATA-1 mRNA and DNA binding activity levels due to a temporary block in transcription of the gene. Moreover, using a whole cell extraction procedure it was discovered that murine erythroleukemia cells contain a second GATA binding activity (denoted GATA-rel) which appears to be distinct from the GATA-1 factor based on its non-reactivity to two GATA-1 antisera. This protein has a limited tissue specificity, as it could not be detected in extracts from CHO, NIH 3T3, or COS cells. Similarly to the GATA-1 DNA-binding activity, the GATA-rel activity decreased during the early stages of differentiation. However, unlike GATA-1, GATA-rel activity did not return to pre-induced levels at later times. These results suggest that changes in gene expression during erythroid terminal differentiation may involve an interplay on levels of different GATA-binding factors.

[A qualitative study of the violence in hockey: perceptions of trainers and players]. / Etude qualitative de la violence au hockey: perceptions d'entraîneurs et de joueurs.

During semistructured interviews, coaches and players have expressed their perception of violence in hockey through several game situations. The responses reveal that coaches disapprove and even sanction players receiving too many useless penalties, but occasionally congratulate them for a penalized action executed to save a goal. During matches, verbal intimidation is high and not always criticized, especially when it causes the opponent to lose concentration and take a penalty. Body checks have been identified as a main generator of frustration and lack of discipline among players. Data analysis suggests two interventions in training programmes for coaches: the development of teaching material on body checking and on individual counselling techniques to impart sportsmanship attitudes to young players.

[Analysis of the behavior of the coach in relation to violence in minor league hockey]. / Analyse des comportements de l'entraîneur par rapport à la violence au hockey mineur.

The purpose of this study was to verify if, during games, the behavior of ice hockey coaches at the bantam level tends to incite players to use roughness and to infringe upon the rules of the game, as the Néron report (1977) states. The video recording of 27 games using a split-screen technique made it possible to view simultaneously the players in action as well as the coaches' behavior. Analysis of the videotapes revealed that the coaches (n = 11) at the bantam level often exhort their players to put more intensity in their physical contacts (legal body checking), but they more often encouraged them to control themselves and avoid penalties. In general, the coaches displayed very little behavior that encouraged violent actions from the players.

Characterization of nonconservative homologous junctions in mammalian cells.

Homologous recombination in mammalian cells between extrachromosomal molecules, as well as between episomes and chromosomes, can be mediated by a nonconservative mechanism. It has been proposed that the key steps in this process are the generation (by double-strand cleavage) of overlapping homologous ends, the creation of complementary single-strand ends (either by strand-specific exonuclease degradation or by unwinding of the DNA helix), and finally the creation of heteroduplex DNA by the annealing of the single-strand ends. We have analyzed in detail the structure of nonconservative homologous junctions and determined the contribution of each end to the formation of the junction. We have also analyzed multiple descendants from single recombination events. Two types of junctions were found. The majority (90%) of the junctions were characterized by a single crossover site. These crossover sites were distributed randomly throughout the junction. The remaining 10% of the junctions had mosaic patterns of parental markers. Furthermore, in 9 of 10 cases, multiple descendants from a single recombination event were identical. Thus, it appears that in most cases few parental markers were involved in junction formation. This finding suggests that nonconservative homologous junctions are mediated mainly by short heteroduplexes of a few hundred base pairs or less. These results are discussed in terms of the current models of nonconservative homologous recombination.

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation.

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.

Modification of the ultrastructure of Entamoeba histolytica after phagocytosis of liposomes loaded with phalloidin.

The specific actin-interacting drug phalloidin has been introduced into the cytoplasm of a highly motile amoeba, Entamoeba histolytica, by a new technique: the phagocytosis of liposomes containing phalloidin. After ingestion of these liposomes, two important modifications of the ultrastructure of the amoeba were observed. First, large nodules of densely packed fine filaments are formed, which may be due to the polymerization of actin induced by the release of phalloidin within the cell's cytoplasm. Second, phalloidin induces the proliferation of ribosome crystals known as chromatoid bodies in encysted cells. This formation could be the direct consequence of the action of phalloidin on actin, where filaments form and ribosomes detach from the original oligo or polymers. However, it could also result from an unspecific toxic effect on the amoeba which, under physiological stress, starts to encyst and show multiplication of these chromatoid bodies upon encystment.

[Endocytosis of liposomes by Entamoeba histolytica and transport of encapsulated molecules toward the cytoplasm]. / Endocytose des liposomes par Entamoeba histolytica et transfert des molécules encapsulées vers le cytoplasme.

We have examined the interaction of a highly phagocytosing cell: Entamoeba histolytica with liposomes of different lipid compositions, and followed, by a semi-quantitative method, the intracellular fate of the entrapped molecules. Liposomes containing a small molecule, 6-carboxyfluorescein, are first phagocytosed. Then the encapsulated compound migrates from the vacuoles to the cytoplasm. Liposomes containing macromolecular substances, such as fluorescent albumin or ferritin, are also phagocytosed, but the encapsulated molecules remain within the vacuoles. We conclude that the transfer of carboxyfluorescein does not involve a fusion between liposomes and vacuoles, but more likely occurs via diffusion through membranes. The lipid composition of the liposomes does not affect phagocytosis of liposomes. In contrast, oleic acid greatly increases the transfer of carboxyfluorescein from vacuole to cytoplasm.