Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive "organelles" involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity 35 S-GTP-␥ -S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate G ␣ subunits and fluoroaluminates, were detected. In one-and two-dimensional immunoblot analyses, an antiserum directed against G i ␣ -type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against G q ␣ subunits. Green light below 1 E m ؊ 2 sec ؊ 1 suppressed cholera toxin-dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the G ␣ subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca 2 ؉ . Dissociation of the putative G ␣ subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of G ␣ subunits in rhodopsin-based signaling of flagellate green algae.

Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive "organelles" involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity 35 S-GTP-␥ -S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate G ␣ subunits and fluoroaluminates, were detected. In one-and two-dimensional immunoblot analyses, an antiserum directed against G i ␣ -type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against G q ␣ subunits. Green light below 1 E m ؊ 2 sec ؊ 1 suppressed cholera toxin-dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the G ␣ subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca 2 ؉ . Dissociation of the putative G ␣ subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of G ␣ subunits in rhodopsin-based signaling of flagellate green algae.

Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms.

Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms.

No abstract