Specific sequence elements in the 5' untranslated regions of rbcL and atpB gene mRNas stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii.

Using a series of point mutations in chimeric reporter gene constructs consisting of the 5' regions of the Chlamydomonas chloroplast rbcL or atpB genes fused 5' to the coding sequence of the bacterial uidA (GUS) gene, RNA-stabilizing sequence elements were identified in vivo in the 5' untranslated regions (5' UTRs) of transcripts of the chloroplast genes rbcL and atpB in Chlamydomonas reinhardtii. In chimeric rbcL 5' UTR:GUS transcripts, replacement of single nucleotides in the 10-nt sequence 5'-AUUUCCGGAC-3', extending from positions +38 to +47 relative to the transcripts' 5' terminus, shortened transcript longevity and led to a reduction in transcript abundance of more than 95%. A similar mutational analysis of atpB 5' UTR:GUS transcripts showed that the 12-nt atpB 5' UTR sequence 5'-AUAAGCGUUAGU-3', extending from position +31 to position +42, is important for transcript stability and transcript accumulation in the chloroplast of Chlamydomonas. We discuss how the 5' UTR sequence elements, which are predicted to be part of RNA secondary structures, might function in RNA stabilization.

[Clinical practice guidelines of the Spanish Society of Cardiology for pulmonary thromboembolism and hypertension]. / Guías de práctica clínica de la Sociedad Española de Cardiología en tromboembolismo e hipertensión pulmonar.

Primary pulmonary hypertension is a progressive disease. Most affected patients are young and middle-aged women. Etiology is unknown, although a familial and genetic factor is present in up to 6% of cases. Endothelial dysfunction and abnormalities in calcium channels of smooth muscle fibers are the present pathogenetics theories. Diagnostic tests try to exclude secondary causes of pulmonary hypertension and to evaluate its severity. Acute vasodilatory test is vital in the selection of treatment. Oral anticoagulation is indicated in all patients. Lung transplant is performed when medical treatment is unsuccessful. Atrial septostomy is an alternative and palliative treatment for selected cases. Chronic thromboembolic pulmonary hypertension is a special form of secondary pulmonary hypertension, clinically undistinguishable from primary primary hypertension, is of mandatory diagnosis because it can be cured with thromboembolectomy. Pulmonary embolism is common in hospitalised patients. The mortality rate for pulmonary embolism continues to be high: up to 30% in untreated patients. The accurate detection of pulmonary embolism remains difficult, as pulmonary embolism can accompany as well as mimic other cardiopulmonary illnesses. Non-invasive diagnostic tests have poor specificity and sensitivity. The D-dimer level and the spiral CT angiography have also been employed as new alternatives and important tools for precise diagnosis of suspected pulmonary embolism. The standard therapy of pulmonary embolism is intravenous heparin for 5 to 10 days in conjunction with oral anticoagulants posteriorly for 3 to 6 months. The incidence of deep venous thrombosis, pulmonary embolism and death due to pulmonary embolism, can be reduced significantly and shown clear benefits only by adoption of a prophylactic strategy with low-molecular-weight-heparins or dextrans in patients at risk.

The redox state regulates RNA degradation in the chloroplast of Chlamydomonas reinhardtii.

A Chlamydomonas reinhardtii chloroplast transformant, designated MU7, carrying a chimeric (rbcL promoter: beta-glucuronidase [GUS]: psaB 3' end) gene whose transcripts have been found previously to be unstable in light (half-life of 20 min in light as opposed to a half-life of 5 h in the dark), was used to study the role of electron transport and of the redox state in the degradation of chloroplast transcripts in the light. Blocking photosynthetic electron transport with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented the light-dependent breakdown of the pool of GUS transcripts in MU7 cells. Diamide, an oxidizing agent, caused a measurable delay in the degradation of GUS transcripts in the light. The addition of dithiothreitol (DTT), a dithiol reductant, to MU7 cells in which GUS transcript levels were stabilized by DCMU induced degradation of GUS transcripts. Similarly, DTT induced a decrease in the levels of GUS transcripts when added to MU7 cells in the dark period of the light/dark cycle, a period in which GUS transcript levels normally increase. The levels of transcripts of endogenous chloroplast genes were affected by DCMU and DTT in the same direction as levels of GUS transcripts. The results suggest a regulatory role of the redox state in the degradation of chloroplast transcripts in C. reinhardtii.

Endogenous fluctuations of DNA topology in the chloroplast of Chlamydomonas reinhardtii.

DNA supercoiling in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii was found to change with a diurnal rhythm in cells growing in alternating 12-h dark-12-h light periods. Highest and lowest DNA superhelicities occurred at the beginning and towards the end of the 12-h light periods, respectively. The fluctuations in DNA supercoiling occurred concurrently and in the same direction in two separate parts of the chloroplast genome, one containing the genes psaB, rbcL, and atpA and the other containing the atpB gene. Fluctuations were not confined to transcribed DNA regions, indicating simultaneous changes in DNA conformation all over the chloroplast genome. Because the diurnal fluctuations persisted in cells kept in continuous light, DNA supercoiling is judged to be under endogenous control. The endogenous fluctuations in chloroplast DNA topology correlated tightly with the endogenous fluctuations of overall chloroplast gene transcription and with those of the pool sizes of most chloroplast transcripts analyzed. This result suggests that DNA superhelical changes have a role in the regulation of chloroplast gene expression in Chlamydomonas.

Activity of the Chlamydomonas chloroplast rbcL gene promoter is enhanced by a remote sequence element.

The chloroplast gene rbcL encodes the large subunit of ribulose bisphosphate carboxylase. In Chlamydomonas reinhardtii, this gene is transcribed more actively than any other protein-encoding chloroplast gene studied to date. To delineate the rbcL gene promoter, chimeric reporter genes containing fragments of the 5' region of the rbcL gene fused to the coding sequence of the bacterial uidA gene, encoding beta-glucuronidase, were stably introduced into the chloroplast genome of Chlamydomonas by microprojectile bombardment. The relative transcription rates of endogenous and introduced genes were determined in transgenic cell lines in vivo. The basic rbcL promoter is located within the region of the gene extending from positions -18 to +63, taking position +1 as the site of initiation of transcription. A chimeric reporter gene containing only the basic promoter is transcribed only 1-15% as actively as the endogenous rbcL gene, depending on the conditions under which cells are grown and tested. However, a chimeric gene containing rbcL sequences extending to position +170 or beyond is transcribed at about the same rate as the endogenous gene. Deletion of the sequence between positions +170 and +126, well within the protein-encoding region, reduces the rate of transcription to that of reporter genes with the basic promoter alone.

Site specificity of pea histone acetyltransferase B in vitro.

Histone acetyltransferase B from pea embryonic axes has been purified approximately 300-fold by a combination of chromatographic procedures, including affinity chromatography on histone-agarose. The enzyme preparation has been used for the in vitro transfer of acetyl groups from [1-14C]acetyl-CoA to non-acetylated pea histone H4. Up to three acetyl groups can be introduced into the histone. The resulting mono-, di-, and triacetylated H4 isoforms were separated and sequenced to determine the acetylated sites. Only sites 5, 12, and 16 were used by histone acetyltransferase B, but no clear preference among them was observed. The absence of modification of other potentially acetylatable sites is another indication that acetylation of the different lysine residues in the N-terminal H4 tail serves as a specific signal in different nuclear processes.

Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation.

Changes in the relative sizes of pools of transcripts of organelle genes during plastid development are common in flowering plants, but technical difficulties have prevented direct determinations of the effects of changes in rates of transcription and degradation on such fluctuations. It has been possible to follow both rates in Chlamydomonas reinhardtii. In synchronous or asynchronous cultures of cells grown in 12 h light/12 h dark periods, sizes of pools of transcripts of the chloroplast genes atpA, atpB, tufA, and psaB fluctuate. Differences in chloroplast transcript abundances in light/dark cycles were found to be cell cycle-independent but controlled by either an endogenous rhythm (atpA, atpB, and tufA) or by light (psaB). In vivo labeling experiments showed that transcriptional regulation and light/dark-regulated degradation both contribute, in gene-specific manners, to the level of transcripts of individual C. reinhardtii chloroplast genes in cells grown in alternating light/dark cycles.

5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts.

We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization. Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle. However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark. Transcripts disappeared rapidly upon illumination of the cells. The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes. The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene. Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells. The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene. The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes.

Characterization of pea histone deacetylases.

The present paper is the first report on histone deacetylases from plants. Three enzyme fractions with histone deacetylase activity (HD0, HD1 and HD2) have been partially purified from pea (Pisum sativum) embryonic axes. They deacetylate biologically acetylated chicken histones and, to a lesser extent, chemically acetylated histones, this being a criterion of their true histone deacetylase nature. The three enzymes are able to accept nucleosomes as substrates. HD1 is not inhibited by n-butyrate up to 50 mM, whereas HD0 and HD2 are only slightly inhibited, thereby establishing a clear difference to animal histone deacetylases. The three activities are inhibited by acetate, Cu(2+) and Zn(2+) ions and mercurials, but are only scarcely affected by polyamines, in strong contrast with yeast histone deacetylase. Several criteria have been used to obtain cumulative evidence that HD0, HD1 and HD2 actually are three distinct enzymes. In vitro experiments with free histones show that HD0 deacetylates all four core histones, whereas HD1 and HD2 show a clear preference for H2A and H2B, the arginine-rich histones being deacetylated more slowly.

Partial purification and properties of two histone acetyltransferases from the yeast, Saccharomyces cerevisiae.

Two histone acetyltransferases, A and B, have been extracted and partially purified from yeast cells. The purification scheme included ammonium sulfate precipitation, and chromatography on DEAE-Sepharose and Sephadex G-200. The basic properties of both enzymes closely correspond to those of acetyltransferase A and B found in higher eucaryotes. Yeast enzyme A elutes from DEAE-Sepharose prior to acetyltransferase B, and it is activated by low concentrations of DNA and strongly inhibited by p-chloromercuribenzoate (PCMB). Enzyme B is inhibited by DNA over the entire range of concentrations tested and it is less sensitive to PCMB than enzyme A. When assayed with yeast whole histones, enzyme B shows a marked specificity toward histone H4, although H3 and H2B are also accepted as substrates. Enzyme A preferentially catalyzes the acetylation of yeast H2B and H3, with the other two core histones being acetylated to a much lesser extent.