Expression and localization of PCSK9 in rat hepatic cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), recently cloned in several laboratories, including ours, causes a third form of autosomal dominant hypercholesterolemia. Its mechanism of action remains unclear. We studied the expression and subcellular localization of PCSK9 in fetal and adult rat tissues associated with cholesterol homeostasis using quantitative reverse transcriptase--PCR, Western blot analysis, subcellular fractionation, and confocal immunofluorescent microscopy. PCSK9 mRNA is most abundant in yolk sac and fetal liver, but the highest expression of the protein was found in differentiated hepatoma FAO-1 cell line, which also shows the highest expression of LDLR. In FAO-1 cells PCSK9 expression is downregulated by cholesterol and 25-hydroxycholesterol and upregulated in the absence of sterols following the same pattern of expression as HMG-CoA reductase, synthase, and LDLR. Subcellular fractionation, combined with Western blotting, showed that PCSK9 is localized in the ER and intermediate vesicular compartment of the cell but not in Golgi cisternae. The mature enzyme is secreted from the liver and hepatoma cells. Double labeling with antibodies to PCSK9 and LDLR or clathrin revealed some colocalization of PCSK9 with clathrin-coated vesicles and LDLR. In conclusion, our results show that PCSK9 is processed in the ER, and the mature convertase is secreted in the plasma.

Transcription of ribosomal DNA intergenic spacer sequences in normal and regenerating rat liver.

In regenerating rat liver both the transcriptional activity of the intergenic spacer rDNA promoter and the steady-state abundance of spacer transcripts are increased about 2-fold, as compared to normal liver. These changes are parallel to the observed 2.5-fold increase in regenerating liver of rRNA gene promotor activity and gene promotor transcripts abundance. These results suggest that both gene and spacer rDNA promotors are subject to common regulatory mechanisms. Our results indicate also that the stability of spacer transcripts in regenerating liver is not significantly altered.

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N(6)-benzyladenine and N1-(2-chloro-4-pyridyl)-N2-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4-6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5'-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10(3) RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.

Isolation of active transcription complexes from animal cell nuclei by nitrocellulose binding.

A method for the rapid isolation of active transcription complexes from animal cell nuclei is described. The method is based on the observation that, after lysis of nuclei with the detergents Sarkosyl and Triton X-100, transcription complexes are selectively bound to nitrocellulose. The nitrocellulose filters retain 80-90% o the RNA labelled briefly in vitro and about 10% of the nuclear DNA. The bulk of the retained DNA is in the size range of 20 kb. Transcription complexes involving both RNA polymerase I and II are retained by nitrocellulose. The nitrocellulose-bound transcription complexes preserve almost all of their RNA polymerase activity. The size distribution of the RNA product shows that bound transcription complexes retain also most of their growing RNA chains. The possibility to use selective retention by nitrocellulose in the analysis of transcriptionally active genes is discussed.

Effect of cycloheximide on the in vivo and in vitro synthesis of ribosomal RNA in rat liver.

The action of low (5 mg/kg body wt;) and high (20 mg/kg body wt.) doses of cycloheximide, both causing a rapid and almost complete inhibition of protein synthesis in rat liver is investigated. Short-term (15 min) [14C]orotate incorporation into nucleolar rRNA in vivo is inhibited only by the high dose acting for periods longer than 1 h. The effect may be correlated with a strongly reduced labelling of the cellular pool of free uridine nucleotides. These results indicate that in vivo transcription of rRNA genes may not be under stringent control. The activity of template-bound RNA polymerase A in nuclei isolated from animals treated with both doses of cycloheximide is reduced within 1 h to about 50% of controls reaching nearly plateau levels at longer times of action of the drug. The differential effect of cycloheximide inhibition of protein synthesis on in vivo and in vitro rRNA synthesis suggests the existence of elongation control protein(s) characterized by a rapid turnover and a loose association with the nucleus.

Transcription of DNA-histone complexes by yeast RNA polymerase B.

Transcription of denatured DNA complexed with histones (total, H1 or H2A/H2B/H3/H4) by yeast RNA polymerase B is investigated. Binding of histones to DNA restricts its template activity by decreasing the formation of active, heparin-resistant, RNA polymerase initiation complexes. The elongation of pre-initiated RNA on denatured DNA, complexed with histones, is possible, although resulting in somewhat shorter RNA chains. It is suggested that RNA polymerase B can elongate on a DNA strand covered with histones.