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Biomedical subjects

Diego Breviario

Publications and source records attributed to Diego Breviario.

7 recordsLinked to original sources
PubMed2005

Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene.

Plant calcium-dependent protein kinases (CDPKs) are involved in calcium-mediated signal transduction pathways. Their expression is finely tuned in different tissues and in response to specific signals, but the mechanism of such a regulation is still largely unknown. OsCDPK2 gene expression is modulated in vivo during rice (Oryza sativa L.) flower development and is downregulated by white light in leaves. In order to identify OsCDPK2 regulatory sequences, we amplified and cloned both the 5' and 3'-flanking regions of the gene. Sequence analysis revealed that the leader sequence is interrupted by an intron, whose regulatory role was investigated. Different ss-gucuronidase (GUS) expression vectors, carrying combinations of the putative OsCDPK2 regulatory regions, were generated and GUS expression was analyzed both in transient assays and in transgenic rice plants. The whole 5'-flanking sequence was able to drive GUS expression in rice calli and leaves transiently transformed with the biolistic technique. Analysis of the GUS expression pattern in transgenic plants revealed strong activity in root tips, leaf veins and mesophyll cells, in flower reproductive organs and in mature pollen grains. Expression was also shown to be subject to an intron-mediated enhancement (IME) mechanism, since the deletion of the leader intron sequence from chimeric OsCDPK2::GUS plasmids almost completely abolished GUS activity. Furthermore, in transiently transformed leaves, GUS expression driven by the OsCDPK2 promoter-leader region was constitutively observed regardless of light or dark exposure. Light-regulated expression was restored by inserting the OsCDPK2 3' untranslated region (3'UTR) downstream of the chimeric OsCDPK2::GUS transcription unit, suggesting that light down-regulation is mediated by a mechanism driven by the 3'UTR.

3' Untranslated Regions↗
PubMed2004

Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species.

TBP (tubulin-based polymorphism) is a new molecular marker based tool that relies on the presence of intron-specific DNA polymorphisms of the plant beta-tubulin gene family. The multifunctional and essential role of the tubulin proteins is reflected in the conservation of regions within their primary amino acid sequence. The ubiquitous nature of this gene family can be exploited using primers that amplify the first intron of different beta-tubulin isotypes, revealing specific fingerprints. The method is rapid, simple, and reliable and does not require preliminary sequence information of the plant genome of interest. The ability of TBP to discriminate between accessions and species in oilseed rape, coffee, and lotus is shown. In all cases, TBP was able to detect specific genetic polymorphisms in the context of a simplified and readily appreciable pattern of DNA amplification. The application of TBP for assessing genetic diversity and genome origins in disseminated plant landraces rather than in highly inbred cultivated species is also discussed.

Brassica napus↗
PubMed2003

Introns are key regulatory elements of rice tubulin expression.

The genomic clones containing elements that regulate transcription of the three known rice ( Oryza sativa L.) alpha-tubulin isotypes ( Ostua1, Ostua2 and Ostua3) have been isolated. We have used these genomic regions to identify the regulatory elements that contribute to the expression of a marker gene ( gusA) in transient assays performed on rice calli derived from mature embryos. In all cases, we found that the first intron was required to achieve high levels of expression. This is consistent with data already reported for the alpha-tubulin isotype1 and indicates that a common regulatory mechanism is active on all the members of the rice alpha-tubulin gene family. The enhancing effect of the first intron was then tested by constructing illegitimate combinations of alpha-tubulin promoter and intron sequences ( Ostua1pro- Ostua2intro; Ostua1pro- Ostua3intro; Ostua2pro- Ostua3intro; Ostua3pro- Ostua2intro) and then by assaying beta-glucuronidase (GUS) activity in transformed rice calli. All illegitimate combinations expressed GUS at high level, suggesting that rice alpha-tubulin promoters and introns can be exchanged among the different isotypes. This did not occur when the intron of the rice beta-tubulin isotype16, known to enhance transcription of its own gene, was used in place of the alpha-tubulin intron. We have also analysed the effect of abscisic acid (ABA) on GUS expression in rice calli transformed with chimeric tubalpha2pro-intro:: gusA and tubalpha3pro-intro:: gusA constructs. ABA was able to reduce GUS expression only in the presence of the tubalpha2pro-intro sequence. We discuss these data in terms of mechanisms that in rice, as opposed to other plants, may control tubulin isotype-specific expression and the involvement of ABA in the regulation of alpha-tubulin expression.

Abscisic Acid↗
PubMed2003

Alternative splicing of NHE-1 mediates Na-Li countertransport and associates with activity rate.

Sodium-lithium countertransport (SLC) is an ouabain-insensitive exchange of Na for Li found in the erythrocyte membrane of several mammalian species. Although increased SLC activity is presently the most consistent intermediate phenotype of essential hypertension and diabetic nephropathy in humans, the gene responsible for this membrane transport has not been identified. Because of functional similarities, SLC was suggested to represent an in vitro mode of operation of the Na-H exchanger (NHE). This hypothesis, however, has been long hampered by the total insensitivity of SLC to amiloride, which is an intrinsic inhibitor of the first isoform of NHE, the only NHE isoform detected in human erythrocytes. We describe here the identification in human reticulocytes and erythrocytes of an alternative splicing of NHE lacking the amiloride binding site. Transfection experiments with this spliced variant restore amiloride-insensitive, phloretin-sensitive SLC activity. Expression of both regular and spliced transcripts of NHE is increased in subjects with high SLC activity. Altogether, these findings, by extending to NHE the characteristics of inheritance and predictivity previously attributed to SLC, eventually restore the candidacy of NHE isoform 1 as a gene involved in the pathogenesis of essential hypertension and diabetic nephropathy.

Alternative Splicing↗
PubMed2002

A dual effect on protein synthesis and degradation modulates the tubulin level in rice cells treated with oryzalin.

The effect of the anti-microtubular drug oryzalin on growth and morphology of cultured rice (Oryza sativa L., cv. Roncarolo) cells was evaluated with specific reference to mechanisms that control intracellular tubulin levels. The addition of oryzalin caused a great reduction in the level of both alpha- and beta-tubulin polypeptides, as detected by Western blot analysis. However, no appreciable decrease was observed in the population of total or isotype-specific alpha- and beta-tubulin mRNAs. Only within the first 24 h of the oryzalin treatment, when the level of both alpha- and beta-tubulin polypeptides was still undiminished, was a consistent reduction in the amount of total beta-tubulin mRNA observed. Pulse-chase experiments performed on rice cells grown in the presence of 1 microM oryzalin revealed the presence of two distinct mechanisms that negatively control alpha- and beta-tubulin polypeptide levels. (i) There was an immediate effect on protein synthesis, which resulted in a reduction in the level of newly synthesized tubulin. (ii) There was a delayed response characterized by a substantial degradation of both alpha- and beta-tubulin monomers; this degradation occurred after 24 h of herbicide treatment. The possible involvement of Ca2+ in the degradation of the unincorporated tubulin monomers is also documented and discussed.

Abscisic Acid↗
PubMed2002

A long leader intron of the Ostub16 rice beta-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro.

A 2 kb DNA fragment, upstream of the rice beta-tubulin isotype 16 (Ostub16) coding sequence, was isolated using inverse PCR and screening of a tubulin-enriched lambda library. An intron (863 bp) present in the 5' untranslated region (5' UTR) is spliced out to produce the most abundant mRNA species which corresponds to the previously cloned Ostub16 cDNA. Transient expression assays performed on rice embryogenic calluses with chimeric Ostub16::GUS constructs demonstrated that the entire 2 kb upstream sequence has a strong promoter activity, and that the 863 bp intron is required for high-level GUS expression. In addition, the intron sequence is capable per se of sustaining a weak but consistent GUS expression. Two rare Ostub16 transcripts, with a start site mapping within this intron sequence, were detected in rice coleoptile cells. The transcription start site mapped at position -290 with respect to the ATG codon, and the shorter molecule originated from splicing of the same precursor mRNA. Therefore transcriptional expression of rice beta-tubulin isotype 16 results in the synthesis of two premRNA molecules (I and II) encoding for three different mRNA species. We discuss these findings in terms of function and molecular evolution of the mechanisms that control plant beta-tubulin gene expression.

5' Flanking Region↗