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Initial mechanisms of spatial gene regulation in the sea urchin embryo
20
Cell Biology International
Ex0cYToS1sIN
PARAMEC IUM CELLS Helmut Plattner, Christine J. Lump&, Gerd Knoll, Roland Kissmehl, Barbara HGhne, Massoud Momayezi, Faculty of Biology, University of Konstanz, POB 5560, D-7750 Konstanz, Fed. Rep. Germany The ciliated protozoan Paramecium tetraurelia possesses > loo0 secretory organelles (trichocysts) docked at the cell membrane for immediate release. Exocytosis sites display a distinct freeze -fracture morphology. Synchronous (X 1s) release of trichocysts can be trig gered by aminoethyldextran (AED). The cell surface complex (cortces) can be isolated for in vitro studies on exocytosis. In addition microinjection studies (nucleotides, antibodies, enzymes etc.) are also possible. A new approach is a new quenched-flow procedure (combined with fast freezing) applicable to this synchronous exocytosis system to obtain defined functional stages (f10 ms) for parallel ultrastructural (freeze -fracture: membrane fusion stages, freeze substitution, e.g., for Ca*+ localisation by electron energy loss spectroscopic imaging) and biochemical analyses (de - re - phosphorylation of the exocytosis - sensitive phosphoprotein “PP63”, PI turnover). By this combined methodology we could ascertain some important aspects of stimulus-secretion coupling in Paramecium cells ATP primes exocytosis sites. Membrane fusion is facililated by GTP by a left -shift of the Ca*+ sensitivity CUIVC. A subplasmalemmal corn partment (“alveolar sacs”) stores Ca*+. IP, is generated during exocytosis. (It is open so far whether GTP or IP, might mobiise internal Ca2+). During the period of membrane fusion (SO ms) PP63 is dephosphorylated; we assume this to occur by a calmodulin stimulated (“calcineurin” -like) phosphatase (with the two charac teristic bands of a small and large subunit, immunologically crossreacting with anti-bovine -calcineurin antibodies on Western blots from cell cortices). Membrane fusion is focal and can occur without extracellular Cap+ (which, however, is required for contents extrusion). Membrane resealing after contents release is accomplished within NO ms. The synchrony of the system used, combined with new methods, allows us to analyse different controversial aspects of exo - endocyto sis regulation. - Supported by SFB 156.
S61
TRANSCRIPTIONAL ELEMENTS OF EARLY SUBTYPE SEA URCHIN HISTONE GENES Giovanni Spinelli, Franc0 Palla*,Caterina Casano, Letizia Anello*,Celestino Bonura, Roberta Russo. Department of Cellular and Developmental Biology, and *Institute of Developmental Biology, CNR, via Archirafi 20 - 90123 Palermo - Italy To delucidate the molecular mechanisms involved in the regulation of histone gene transcription during early embryogenesis, we tested, by microinjection into Xenopus oocytes, the activity of constructs containing the CAT gene fused to the promoter region of the early subtype HZA gene. The results showed that the modulator potentiates transcription in the physiological orientation. Furthermore, DNaseI footprinting revealed that the H2A modulator contains two binding sites, USE1 and USEZ, for nuclear factors extracted from sea urchin embryos actively transcribing the early histone genes. Cloned oligonucleotides containing USE1 and USE2 sequences respectively, competed, although with a slightly different efficiency, with the H2A modulator to prevent the enhancementof transcription of the reporter gene. Gel shift analysis conducted with oligo USE1 and USE2 respectively, suggested that the protein factors are more abundant at early blastula when the early subtype histone stagethanatgastrula, Interestingly, the USE1 sequence genes are silent. is also present in the promoter region of H3 and Hl the hypothesis that this transcgenes, suggesting riptional element is involved in the coordinate expression of this set of genes.
S64
Reports, Vol. 14, Abstracts Supplement
7990
INITIAL MECHANISMS OF SPATIAL GENE REGULATION IN THE SEA URCHIN EMBRYO E. H. Davidson. Division of Biology, California Institute of Technology, Pasadena, CA 91125. This presentation will concern the molecular basis of the process by which differential patterns of regional gene activity are instituted in the embryo. The cytoskeletal actin gene CyIIIa provides an excellent molecular marker for the zygotic program of gene expression characteristic of the embryonic aboral ectoderm. According to our recent lineage tracer studies the aboral ectoderm derives clonally from I1 specific cleavage stage founder cells. However, the egg is initially radially symmetric, and interblastomere interaction as well as an early cytoplasmic polarization are probably required for aboral ectoderm specification. Regulatory sequences of the CyIIIa gene have now been shown to accurately direct spatial and temporal expression of exogenous reporter genes, after injection of the appropriate fusion gene constructs into sea urchin eggs. By using this method of gene transfer the cis regulatory domain of the CyIIIa gene has been delimited. Coinjection of excess quantities of DNA fragments containing subregions of the regulatory domain results in in viva competition, and thus it is possible to analyze significance of individual the functional DNA-protein binding studies regulatory elements. in vitro demonstrate at least 20 sites where highly specific interactions occur within this domain (104-10 fold preference for CyIIIa site vs. random DNA sequence). Some of the factors that bind to these sites are probably zygotic gene products, since their concentration increases as development proceeds and they cannot be detected in extracts of unfertilized eggs. Analysis of the origin, the cytological distribution and possibly the activation of certain of these CyIIIa regulatory factors in the egg and early embryo, considered in conjunction with the lineage and location of the aboral ectoderm precursors, will provide a molecular interpretation of how the CyIIIa gene becomes differentially expressed as the aboral ectoderm is formed. An interesting result follows from introduction of the CyIIIa regulatory sequences into eggs of a different sea urchin species. Though the exogenous fusion construct is regulated temporally in a correct manner, spatial regulation is wholly deranged. The same pattern of ectopic expression is observed in in vivo competition experiments, when two specific cis-trans interactions In the CyIIIa regulatory domain are prevented. These are mediated by factors P3A2 and P711. P3A2 is cloned, as are a number of other factors involved in early regulation of this and other lineagespecific genes in the S. purpuratus embryo. The P3A2 mRNA is a rare maternal species, but calculations show that its prevalence suffices to generate functional levels of P3A2 factor in blastomere nuclei during the process of specification. By immunocytology we also show that P3A2 is localized to the cell types where CyIIIa transcription is repressed, in accord with its known role as a negative spatial regulator. An interpretation of sea urchin embryogenesis integrating classical and molecular evidence will be presented.
S62
S63
STRUCTURAL-FUNCTIONAL ORGANIZATION OF DNA DOMAINS IN EUKARYOTES G.P. Georgiev, Institute of Molecular Biology, Academy of Sciences of the USSR, Vavilov Street 32, 117984 Moscow B334, USSR
Cell Biology International
Ex0cYToS1sIN
PARAMEC IUM CELLS Helmut Plattner, Christine J. Lump&, Gerd Knoll, Roland Kissmehl, Barbara HGhne, Massoud Momayezi, Faculty of Biology, University of Konstanz, POB 5560, D-7750 Konstanz, Fed. Rep. Germany The ciliated protozoan Paramecium tetraurelia possesses > loo0 secretory organelles (trichocysts) docked at the cell membrane for immediate release. Exocytosis sites display a distinct freeze -fracture morphology. Synchronous (X 1s) release of trichocysts can be trig gered by aminoethyldextran (AED). The cell surface complex (cortces) can be isolated for in vitro studies on exocytosis. In addition microinjection studies (nucleotides, antibodies, enzymes etc.) are also possible. A new approach is a new quenched-flow procedure (combined with fast freezing) applicable to this synchronous exocytosis system to obtain defined functional stages (f10 ms) for parallel ultrastructural (freeze -fracture: membrane fusion stages, freeze substitution, e.g., for Ca*+ localisation by electron energy loss spectroscopic imaging) and biochemical analyses (de - re - phosphorylation of the exocytosis - sensitive phosphoprotein “PP63”, PI turnover). By this combined methodology we could ascertain some important aspects of stimulus-secretion coupling in Paramecium cells ATP primes exocytosis sites. Membrane fusion is facililated by GTP by a left -shift of the Ca*+ sensitivity CUIVC. A subplasmalemmal corn partment (“alveolar sacs”) stores Ca*+. IP, is generated during exocytosis. (It is open so far whether GTP or IP, might mobiise internal Ca2+). During the period of membrane fusion (SO ms) PP63 is dephosphorylated; we assume this to occur by a calmodulin stimulated (“calcineurin” -like) phosphatase (with the two charac teristic bands of a small and large subunit, immunologically crossreacting with anti-bovine -calcineurin antibodies on Western blots from cell cortices). Membrane fusion is focal and can occur without extracellular Cap+ (which, however, is required for contents extrusion). Membrane resealing after contents release is accomplished within NO ms. The synchrony of the system used, combined with new methods, allows us to analyse different controversial aspects of exo - endocyto sis regulation. - Supported by SFB 156.
S61
TRANSCRIPTIONAL ELEMENTS OF EARLY SUBTYPE SEA URCHIN HISTONE GENES Giovanni Spinelli, Franc0 Palla*,Caterina Casano, Letizia Anello*,Celestino Bonura, Roberta Russo. Department of Cellular and Developmental Biology, and *Institute of Developmental Biology, CNR, via Archirafi 20 - 90123 Palermo - Italy To delucidate the molecular mechanisms involved in the regulation of histone gene transcription during early embryogenesis, we tested, by microinjection into Xenopus oocytes, the activity of constructs containing the CAT gene fused to the promoter region of the early subtype HZA gene. The results showed that the modulator potentiates transcription in the physiological orientation. Furthermore, DNaseI footprinting revealed that the H2A modulator contains two binding sites, USE1 and USEZ, for nuclear factors extracted from sea urchin embryos actively transcribing the early histone genes. Cloned oligonucleotides containing USE1 and USE2 sequences respectively, competed, although with a slightly different efficiency, with the H2A modulator to prevent the enhancementof transcription of the reporter gene. Gel shift analysis conducted with oligo USE1 and USE2 respectively, suggested that the protein factors are more abundant at early blastula when the early subtype histone stagethanatgastrula, Interestingly, the USE1 sequence genes are silent. is also present in the promoter region of H3 and Hl the hypothesis that this transcgenes, suggesting riptional element is involved in the coordinate expression of this set of genes.
S64
Reports, Vol. 14, Abstracts Supplement
7990
INITIAL MECHANISMS OF SPATIAL GENE REGULATION IN THE SEA URCHIN EMBRYO E. H. Davidson. Division of Biology, California Institute of Technology, Pasadena, CA 91125. This presentation will concern the molecular basis of the process by which differential patterns of regional gene activity are instituted in the embryo. The cytoskeletal actin gene CyIIIa provides an excellent molecular marker for the zygotic program of gene expression characteristic of the embryonic aboral ectoderm. According to our recent lineage tracer studies the aboral ectoderm derives clonally from I1 specific cleavage stage founder cells. However, the egg is initially radially symmetric, and interblastomere interaction as well as an early cytoplasmic polarization are probably required for aboral ectoderm specification. Regulatory sequences of the CyIIIa gene have now been shown to accurately direct spatial and temporal expression of exogenous reporter genes, after injection of the appropriate fusion gene constructs into sea urchin eggs. By using this method of gene transfer the cis regulatory domain of the CyIIIa gene has been delimited. Coinjection of excess quantities of DNA fragments containing subregions of the regulatory domain results in in viva competition, and thus it is possible to analyze significance of individual the functional DNA-protein binding studies regulatory elements. in vitro demonstrate at least 20 sites where highly specific interactions occur within this domain (104-10 fold preference for CyIIIa site vs. random DNA sequence). Some of the factors that bind to these sites are probably zygotic gene products, since their concentration increases as development proceeds and they cannot be detected in extracts of unfertilized eggs. Analysis of the origin, the cytological distribution and possibly the activation of certain of these CyIIIa regulatory factors in the egg and early embryo, considered in conjunction with the lineage and location of the aboral ectoderm precursors, will provide a molecular interpretation of how the CyIIIa gene becomes differentially expressed as the aboral ectoderm is formed. An interesting result follows from introduction of the CyIIIa regulatory sequences into eggs of a different sea urchin species. Though the exogenous fusion construct is regulated temporally in a correct manner, spatial regulation is wholly deranged. The same pattern of ectopic expression is observed in in vivo competition experiments, when two specific cis-trans interactions In the CyIIIa regulatory domain are prevented. These are mediated by factors P3A2 and P711. P3A2 is cloned, as are a number of other factors involved in early regulation of this and other lineagespecific genes in the S. purpuratus embryo. The P3A2 mRNA is a rare maternal species, but calculations show that its prevalence suffices to generate functional levels of P3A2 factor in blastomere nuclei during the process of specification. By immunocytology we also show that P3A2 is localized to the cell types where CyIIIa transcription is repressed, in accord with its known role as a negative spatial regulator. An interpretation of sea urchin embryogenesis integrating classical and molecular evidence will be presented.
S62
S63
STRUCTURAL-FUNCTIONAL ORGANIZATION OF DNA DOMAINS IN EUKARYOTES G.P. Georgiev, Institute of Molecular Biology, Academy of Sciences of the USSR, Vavilov Street 32, 117984 Moscow B334, USSR