Search results for "microinjection"

Enhancer, chromatin insulator, non-coding RNA and α-histone gene expression during embryogenesis of the sea urchin Paracentrotus lividus.

2009

Core promoters and chromatin insulators (ins) may direct a transcriptional enhancer (enh) to prefer a specific promoter in complex genetic loci. Enh and ins flank the sea urchin Paracentrotus lividus α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. In vivo competition assays of enh and ins functions reveal that the H2A enh-bound MBF-1 activator participates also in the expression of the H3 gene and that the sns5 ins buffers the downstream H1 promoter from the H2A enh. These results suggest that both the H2A enh and the sns5 ins may account for the diverse accumulation of the linker vs core nucleosomal histones during early development of the s…

Functional studies of regulatory genes in the sea urchin embryo.

2009

The sea urchin histone H2A enhancer-binding protein MBF-1 is needed for maximal expression also for the H3 gene, while is buffered by the sns5 insula…

2009

Enhancers are DNA elements which increase the transcription of associated gene in a position and distance independent manner relative to the transcription initiation site. Molecular mechanisms must operate to direct enhancers to specific promoters in complex genetic loci. The sea urchin a-histone genes are organized in several hundred tandem repeated units, each containing one copy of the five histone genes in the order 5’-H4-H2B-H3-H2A-H1-3’. Transcription is limited to the early cleavage and reaches its maximum at morula stage. After hatching these genes are repressed and maintained in the silenced state for whole life cycle of the animal. In Paracentrotus lividus, the MBF-1 activator bin…

A compact cis-regulatory module defines the exclusive expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm

2009

In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. We have explored the cis-regulatory apparatus of the gene and found that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined Aboral Ectoderm Module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for …

Constitutive hsp70 is essential to mitosis during early cleavage of Paracentrotus lividus embryos: The blockage of constitutive hsp70 impairs mitosis

1999

Localization of constitutive hsp70 in eggs and early embryos of sea urchin Paracentrotus lividus is shown by means of in situ immunostaining. An accumulation of this protein is shown in the mitotic structures (asters, spindles and centrosomes). Microinjection of anti-hsp70 antibodies into eggs causes impairment of formation of mitotic structures and of cell division. This impairment goes from a complete mitotic block, to irregular mitotic apparatus formation with irregular cleavage, depending upon the antibody concentration. The localization of hsp70 after antibody microinjection is also described. Blockage of mitotic apparatus formation by nocodazole also blocks the concentration of hsp70 …

Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 a-histone genes is modulated by enhancer and chromat…

2009

Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) alpha-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence su…

Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end

1999

Abstract Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the mod…

UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii.

1996

Abstract We have previously isolated a fibroblast mutant cell with high resistance to the two Rho-modifying glucosyltransferase toxins A and B of Clostridium difficile. We demonstrate here a low level of UDP-glucose in the mutant, which explains its toxin resistance since: (i) to obtain a detectable toxin B-mediated Rho modification in lysates of mutant cells, addition of UDP-glucose was required, and it promoted the Rho modification dose-dependently; (ii) high pressure liquid chromatography analysis of nucleotide extracts of cells indicated that the level of UDP-glucose in the mutant (0.8 nmol/106 cells) was lower than in the wild type (3.7 nmol/106 cells); and (iii) sensitivity to toxin B…

Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells.

2005

Abstract PlOtp (Orthopedia), a homeodomain-containing transcription factor, has been recently characterized as a key regulator of the morphogenesis of the skeletal system in the embryo of the sea urchin Paracentrotus lividus . Otp acts as a positive regulator in a subset of oral ectodermal cells which transmit short-range signals to the underlying primary mesenchyme cells where skeletal synthesis is initiated. To shed some light on the molecular mechanisms involved in such a process, we begun a functional analysis of the cis -regulatory sequences of the Otp gene. Congruent with the spatial expression profile of the endogenous Otp gene, we found that while a DNA region from −494 to +358 is s…

Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three esse…

2000

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, th…