97 research outputs found
Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS
No full textAbstract Background The β-glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated. Results We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. Conclusions In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.</p
Un nuovo metodo per l'analisi quantitativa dell'attività GUS nelle piante utilizzando MUG come substrato
No full textPhylogeny, evolution and systematics of Moehringia (Caryophyllaceae) as inferred from molecular and morphological data: a case of homology re-assessment
No full textInhibition of glucuronidase activity (GUS) in plant extracts in relation to the use of GUS as reporter gene.
No full textThe glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented
A novel method for fluorometric continuous measurement of β-glucuronidase (GUS) activity using 4-methyl-umbelliferyl-β-D-glucuronide (MUG) as substrate
No full textBeta-Glucuronidases (GUS) are histochemically and fluorometrically detectable enzymes that cleave the glycosidic bond of glucuronides in living organisms. Previously thought to be absent in plants, endogenous GUS activity has recently been demonstrated ubiquitous, and its function is being widely investigated in model plants. Further, the GUS gene from Escherichia coli is the most widely used reporter gene in plant transformation experiments. Fluorometric assay of GUS activity is universally performed using fluorogenic substrate 4 methylumbelliferyl-beta-D-glucuronide (MUG) by discontinuous measurement, based on the general notion that basification of the solution following enzymatic cleavage is necessary to detect the fluorophore 4-methyl-umbelliferone (MU). In this report, we analyze MU and MUG spectroscopic characteristics at different pHs, and show that MU is a fluorescent compound also at pHs below its pKa, although with a different excitation spectrum compared to the ionized form. On the basis of this evidence, we show that MUG is a suitable substrate to perform continuous measurement of GUS activity at the pH optima of plant
endogenous and E. coli GUS enzymes. An efficient and straightforward method which greatly improves the procedure to assess GUS activity is described, and insights are given on the principles leading to optimal setting of instrumental parameters
Seed morphology in Moehringia L. and its taxonomic significance in comparative studies within the Caryophyllaceae.
No full textSeeds of 30 species of Moehringia and 12
representatives of the Caryophyllaceae were examined
with the Scanning Electron Microscope. High
diversity was found in seed coat micromorphology,
and a number of novel morphological features have
been observed. Comparison with other representatives
of the Caryophyllaceae has provided new
insight into the potential taxonomic value of
discrete morphological characters. Though many
characters appear informative within Moehringia,
they are affected by a high level of homoplasy when
considered in a wider phylogenetic context. Smooth
seed is the common condition in Moehringia, except
for Eastern Balkan and Iberian species that maintain
a secondary ornamentation of the testa cells
resembling the plesiomorphic Caryophyllaceaetype.
Five types of strophioles were recognized, in
accordance with previous literature. Among these,
the strophiole of the Iberian species appears very
distinctive and a proper strophiole could not be
verified in M. glochidisperma and M. fontqueri
Genetic drift linked to heterogeneous landscape and ecological specialization drives diversification in the Alpine endemic columbine Aquilegia thalictrifolia
No full textThe European Alpine system is an extensive mountain range, whose heterogeneous landscape together with Quaternary climatic oscillations significantly affected organismal diversity and distribution in Europe. The model genus Aquilegia representsa textbook example of a rapid and recent radiation through the Northern hemisphere, with the majority of the European taxa occuring in the Alpine system. However, the processes governing genetic differentiation of the genus in this complex geographicarea are still widely unexplored. In this work, we used 9 microsatellite loci to study the genetic structure and diversity of 11 populations of Aquilegia thalictrifolia Schott & Kotschy, an alpine taxon characterized by a marked ecological specificity.We found that, despite the endemic and fragmented distribution, A. thalictrifolia has overall high levels of heterozygosity, which is consistent to the substantial inbreeding depression that characterizes the genus. Strong spatial genetic structuring ofpopulations suggests a historical prevalence of genetic drift over gene flow, with natural barriers and ecological niche hindering migration. An analytical comparison of fixation and population differentiation indexes allowed us to infer hypothesesof the postglacial history and more recent demographic events that have influenced the genetics of the species. Overall, ourresults indicate allopatry as a major force of differentiation in the European scenario, likely to underlie the development oftaxonomic boundaries in a broader geographic context. This adds to previous notions on the primary evolutionary forcesshaping the Aquilegia radiation in Europ
Crescita in vitro ed in vivo dei tubetti pollinici: possibile ruolo delle glucuronidasi nel modulare la plasticità della parete e dell'inibitore delle glucuronidasi presente nello stilo nel regolare la crescita dei tubetti polinici.
No full textEsocitosi ed endocitosi intervengono nella regolazione dei processi di crescita e morfogenesi nelle cellule eucariotiche, in particolare in quelle altamente polarizzate come il tubetto pollinico la cui crescita è localizzata nella regione apicale e avviene attraverso un meccanismo conosciuto come "tip growth" (52). Nella crescita apicale dei pt, oltre al trasporto regolato di vescicole endo - ed eso- citotiche, è necessaria un'azione concertata di enzimi che modifichino la parete (45-48). Recentemente è stata dimostrata la presenza di ß-glucuronidasi (GUS) nelle piante ed è stato proposto un loro ruolo nella crescita per distensione e, in particolare, nella formazione dei peli radicali (49). L’allungamento dei peli radicali è caratterizzato da una crescita polarizzata analoga, in meccanismi e regolazione, a quella presente nei pt (52, 53). E’ quindi particolarmente interessante investigare la presenza di GUS nel polline e il suo eventuale ruolo nella germinazione e crescita dei tubetti pollinici. Contemporanea analisi proteomica condotta in collaborazione con l’unità di Siena potrà identificare eventuali isoforme proteiche attive nel tubetto in crescita.
Sulla base di sequenze note dei geni GUS in Arabiopsis (51) e Scutellaria (50) studi di biologa-molecolare verranno condotti sull’mRNA estratto dal polline e dai tubetti pollinici, allo scopo di individuare il/i geni GUS espressi nel polline. Questo potrà consentire in futuro esperimenti di silenziamento genico volti allo studio del ruolo del GUS nella maturazione del polline e nella germinazione e crescita dei tubetti pollinci.
Nel nostro laboratorio, è stata inoltre dimostrata la presenza di un inibitore dell'attività glucuronidasica negli stili di Nicotiana (43). Indicazioni preliminari sembrano indicare una sua presenza nello spazio apoplastico e, quindi, un suo possibile ruolo nella regolazione dell’attività GUS associata ai tubetti pollinici e forse della loro crescita. Si intende quindi purificare tale inbitore, caratterizzarlo e studiarne la distribuzione nello stilo in relazione alla crescita dei tubetti pollinici compatibili e incompatibili.
Considerato che dati sperimentali indicano che l'inibitore dell'attività GUS viene internalizzato all'interno dei pt durante la crescita nel tessuto di trasmissione stilare, la caratterizzazione-purificazione dell’inibitore e l’utilizzo di polline trasformato con il costrutto “promotore LAT52-GUS” (espresso specificatamente nel polline) apriranno nuove possibilità per studiare in vitro e semi-vivo il fenomeno di endocitosi, particolarmente rilevante per le interazioni pt-stilo.
Nell’ambito di questo progetto si intendono quindi perseguire i seguenti obiettivi:
- Studio sulla presenza della ß-glucuronidasi (GUS) nei tubetti pollinici (pt): sua caratterizzazione, purificazione e investigazione del ruolo svolto nella germinazione e crescita dei tubetti pollinici.
- Purificazione e caratterizzazione dell’inibitore stilare del GUS.
- Analisi della distribuzione dell’inibitore in diverse parti dello stilo, in relazione anche al tipo di impollinazione, e suo utilizzo per investigare il processo di internalizzazione nei tubetti polinici
Molecular phylogeny of the Caryophyllaceae (Caryophyllales) inferred from chloroplast matK and nuclear rDNA ITS sequences
No full textCaryophyllaceae is a principally holarctic family including around 2200 species often classified into the three subfamilies Alsinoideae, Caryophylloideae, and Paronychioideae. Complex and possibly homoplasious morphological characters within the family make taxa difficult to delimit and diagnose. To explore part of the morphological evolution within the family, we investigated the phylogeny of the Caryophyllaceae by means of analyzing plastid and nuclear sequence data with parsimony and Bayesian methods. We describe a mode of tracing a stable phylogenetic signal in ITS sequences, and a significant common signal is shared with the plastid data. Parsimony and Bayesian analyses yield some differences in tree resolution. None of the subfamilies appear monophyletic, but the monophyly of the Caryophylloideae is not contradicted. Alsinoideae are paraphyletic, with Arenaria subg. Eremogone and Minuartia subg. Spergella more closely related to the Caryophylloideae. There is strong support for the inclusion of Spergula-Spergularia in an Alsinoideae-Caryophylloideae clade. Putative synapomorphies for these groupings are twice as many stamens as number of sepals and a caryophyllad-type of embryogeny. Paronychioideae form a basal grade, where tribe Corrigioleae are sister to the rest of the family. Free styles and capsules with simple teeth are possibly plesiomorphic for the family
Il Fior novello, musica a cinque voci di Giovanni Maroni, 1596
No full textLa raccolta di madrigali Il Fior novello di Giovanni Maroni costituisce un interessante elemento di collegamento tra diverse figure della letteratura italiana e alcuni protagonisti delle vicende politiche di fine Cinquecento. Il Fior novello è stato infatti pubblicato nel 1596 a Venezia da Ricciardo Amadino ed è dedicato al giovane marchese Ambrogio Spinola, il governatore di Milano all’epoca in cui si svolsero i fatti narrati nei Promessi Sposi di Alessandro Manzoni. L’opera, dimenticata per secoli, attirò l’interesse di Gabriele d’Annunzio durante la sua visita alla città estense avvenuta nel novembre 1898. Numerosi sono gli interrogativi irrisolti aperti da questo libro: le vicende biografiche dell’autore, la paternità dei testi messi in musica e il loro oggetto poetico. Il presente volume propone, insieme allo studio introduttivo, una trascrizione moderna, secondo criteri ecdotici rigorosi e aggiornati, attenta però a soluzioni editoriali che ne consentano l’agevole lettura a chi voglia attualizzarne oggi il contenuto in un’esecuzione dal vivo. Lo stesso formato di stampa è stato scelto a questo scopo.The collection of madrigals Il Fior novello by Giovanni Maroni is an interesting link between different figures in Italian literature and some players of the political events of the late Sixteenth century. Il Fior novello was in fact published in 1596 in Venice by Ricciardo Amadino and is dedicated to the young Marchese Ambrogio Spinola, the governor of Milan at the time when the events narrated in the Promessi Sposi by Alessandro Manzoni took place. Maroni’s work was forgotten, and after three centuries attracted the interest of Gabriele d'Annunzio during his visit to the Ferrara which took place in November 1898. There are numerous unresolved questions opened by this book: the biography of the author, the authorship of the texts and their poetic object. The present volume proposes, together with the introductory study, a modern transcription, according to rigorous and up-to-date ecdotic criteria, but attentive to editorial solutions that allow easy reading to those who want to actualize their content today in a live performance
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