Colloque SBCF-ATIPEICNRS Biologie du D&eloppement, mars 1995 91

TRANSCRIPTIONAL CONTROL OF CDC2 GENE DURING THE DEVELOPMENT OF THE QUAIL NEURORETINA

&&Jb!& Xa iff, NORTH Sophie, BRUN Gilbert and GILLET Germain. CNRS-VMRv49. Ecole Normale SuOerieure de Lyon. 46. al&e d’lralie,

69364 LYdN cedex 07 -

During the development of the quail neuroretina, we have observed a transcription arrest of cdc2 gene between embryonic days E7 and E8. This event seems to be correlated with the onset of differentiation in this organ.

To study the molecular mechanisms involved in the down regulation of cdc2, we have cloned the quail promoter region of this gene. It is a TATA- less promoter possessing two CAAT boxes and an EzF-1 binding site. This promoter works in human cells (HeLa) and in avian cells (QT6). CAT- ELBA and gel retardation experiments have shown that EzF-1 transactivates the promoter of cdc2 up to 40 fold. A constitutively active mutant of pl05Rb abolishes in a dosedependent manner the transactivation mediated by EzF- 1. Gel retardation exueriments carried out with nuclear exuacts from embrvonic retina harvested ai different stages of development have shown that &F-l DNA binding activity diSaDDeaTS between E9 and ElO. This disaDDearance is correlated wgh two e%ents:‘the transition from the inactive to th;z&tive form of plO5Rb and the diSaDDCaranCe. of E?F-I itself due to the arrest of its tr&scription. Since cdc2’a turned off two days before EzF- 1, we think that the disappearance of E2F-1 might be necessary to maintain the long term transcr$bnal inactivati& of cd.?2 in the matureneuroretina.

Cdc2 transcription being under the control of E2F-1 which is itself negatively regulated during development, we have cloned the promoter of the quail &F-I gene. Like the promoter of cdc2, it is a TATA-less promoter with two CAAT boxes. It possesses one single E2F-1 binding sites whereas the human promoter has two E2F-1 sites (JOHNSON D.. OHTANI K. and NEVINS J. (1994). Genes & Dev., 8, 1X4-1525). However, this single site is sufficient to allow the transactivation by its own product.

In the near future, we intend to characterize by DNase foot printing additional transcription factors that participate to the control of cdc2 and EzF- I expression during development.

TRANSCRIPTIONAL INDUCTION OF THE HUMAN RENIN GENE BY CAMP IN CULTURED CHORIONIC CELLS. GERMAIN Sttphane, KONOSHITA Tadashi, PHILIPPE Josette, CORVOL Pierre et PINET Florence. INSERM V36, Colkge de France, 3 rue d’Vlm, 7.5005 Paris.

Renin plays a major role in blood pressure regulation and fluid and electrolytic homeostasis. Human renin is expressed in many cell types but its expression is particularly high in juxtaglomerular and chorionic cells. The mechanisms of human renin gene transcription have been poorly studied because of the lack of cell lines expressing renin. Chorionic cells were used to determine the cis-regulatory elements and the rrans-activating factors involved in human renin gene transcription. DNase I footprinting performed in previous studies have shown a footprint in the human renin promoter (-234 to -214): TAGCGTCA which shares strong homology to the CAMP-responsive element (CRE) binding site. To delineate the cis-acting elements responsible for CAMP induced human renin gene transcription, 5’ flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected transiently in chorionic ells r,.rskolin treatment induced the expression of luciferase by 2.3-fold when the reporter plasmid contained the full promoter region (-582 to +iti,. 5futation or deletion of the renin CRE diminished (1.7-fold) but did not not abolish completely CAMP induced transcription demonstrating that the (-582 to -148) region containing the CRE and the (-148 to -3~) r-gion containing a Pit- 1 like site were both necessary t’ol CAMP maximal induction. Mutagenesis of the Pit-l like site confirmed its role in CAMP induced transcription. DNase I footprinting and electromobility shift assays (EMSA) were performed with renin-producing chorionic cell and kidney cortex cell nuclear extracts, to studv the molecular events mediating the CAMP induction. EMSA *showed two specific DNA/protein complexes within the renin CRE region which were displaced bv the somatostatin consensus CRE but not bv the mutated ren’n CRE,-showing that CREB interacts with the CRg. EMSA also showed that factors from kidney and chorionic cells, bind the renin Pit-l like site, distinct from the Pit-1 factor. These results show an additional role of CREB and Pit-l like factors in CAMP regulation of the human renin gene transcription.

REVERSIBLE VOLUME VARIATIONS OCCUR IN THE CELLS

OF THE BENDING ZONE DURING CIRCUMNUTATION OF

THE TWINING SHOOTS

CA& Anne-Francoise, BADOT Pierre-Marie, NEFED’EV Leonid, BONNET Bernard, MILL.ET Bernard

Diparternent des Sciences de In Vie

Laboratoire des Sciences V&t!tales, Univ. de Fmnche-Cornfi,

Place Leclerc, F-25030 BESANCON CEDEX

The Phaseolus orrlgaris L. twining shoots display high

growth and circumnutation movement. This movement affects

the terminal part of shoot which shows a curvature. Reversible

volume variations in the cells of the bending zone were supposed

to be the cause of movement (MILLET B., MELIN D. et BADOT P.M.

(1988). Physid. Planf., 72, 133-138).

Simultaneous measurements of circumnutation and cell

length variations document this hypothesis. Epidermal cells were

marked at each end with a dye droplet. In sitrl cell length variations were measured with a reverse microscope. The apex

position was followed with a video camera perpendicular to the

plant. The cell length showed partially reversible variations. Cells

have a minimal length in the concave side and a maximal length

in the convex side. Data analysis with Fourier method showed

that the period of these variations corresponded to that of the

circumnutation movement.

THE MOUSE PLASMA GLUTATHIONE PEROXIDASE- ENCODING GENE: ORGANIZATION, TISSUE- DISTRIBUTION AND CHROMOSOMAL LOCALIZATION.

SCHWAAB VBronique, BAUD Eric’, GHYSELINCK Norbert*, MATTEI Marie-GeneviBve3, DUFAURE Jean-Pierre and DREVET JogI. Laboratoire de Biologic Cellulaire, CNRS, ERS63, Univ. Blaise Pascal, 24. avenue des Landais. 63177 Aubi&e Cedex and lLabo;atoke d’Oncologie Moi&ulaire, CHRU, Centre Jean Perrin, 63011 Clermont-Ferrand Cedex. 2IGBMC. Part d’innova tion, BP 163, 67404 lllkirch Cedex. 3Centre de Gdn&ique Mgdicale, lnstitut National de la Sante et de la Recherche Mbdicale lJ242, 18385 Marseille Cedex 5.

As a molecular comparative tool to analyze the restriction of expression of an epididymis specific glutathione peroxidase like gene (previously described in GHYSELINCK N.G., DUFAURE I., IAREYRE J.J., RIGAUDIERE N.. MAnEI M.G. and DUFAURE J.P. (1993). Mol. endocfinol., 1: 25%272), a genomic clone encoding the mouse plasma glutathione peroxydase (mGSH-Px.pl). a major enzyme in reducing lipid hydroperoxide and hydrogen peroxyde in plasma, was isolated and 6400 nucleotides (nt) were sequenced. Comparisons with known GSH-Px sequences as well as PCR amplifications of exonnntron boundaries revealed that the single copy mGSH-Px.pl gene is constituted of five exons spanning approximatively 6 kb. Primer extension analysis revealed one major transcription start site at position 101 upstream from the predicted start codon. Thirty-six nt upstream from the cap site, a non consensus “TATA” box was found. No “CAAT” box was observed. Two other additional initiation sites were observed around positions 245 and 249 nt. Northern-Blot experiments as well as RT-PCR assays performed with RNA extracted out of various tissues from normal or castrated animals showed that mGSH-Px.pl gene expression is androgen- independent and tissue-specific. A computer search for known DNA-binding protein recognition sequences was carried out. Finally, the mouse GSH-Px.pl gene was localized on chromosome 9 using in situ hybridization