Gene expression in plastids of higher plants: evolutionary and functional aspects of different RNA polymerases - coordinated assembly of multiprotein-complexes

Plastid gene organisation maintains characteristics typical of its prokaryotic ancestry. The regulation of plastid gene expression however strongly deviates from that one its free-living cyanobacteria relatives. This intriguing complication of plastid gene expression characteristics is the result of an integration process of the cellular subgenomes that introduced eukaryotic traits into the formerly prokaryotic compartment (Herrmann et al. 1997). An interesting example for this process is the transcription system, which consists of both prokaryotic (PEP) and eukaryotic (NEP) RNA polymerases. In order to understand, how far the transcriptional apparatus within plastids was adapted to nuclear needs, three approaches have been undertaken. Firstly, the tobacco homologues of the NEP-enzymes known from Arabidopsis were determined and characterised. Secondly, an extensive transcript analysis for all plastid operons was carried out with wild-type and PEP-lacking material in order to assess the contribution of the two systems to transcription. In order to rapidly screen this plant material, an array-based technique was established. As no arrays for the plastid chromosome had been described, the preparation of the filters, optimisation of hybridisation conditions and probe preparation together with the use of the proper controls was one of the main challenges of this work, in particular as macroarrays are interesting for various other applications. Thirdly, to assess the regulation of PEP expression, an in vivo analysis of the promoter for the rpoB operon was performed. According to data from other groups, this operon is controlled by NEP, which - in addition to other data - led to the suggestion that PEP is switched on by NEP (Hajduckiewicz et al., 1997). To test this, point mutations of the described NEP rpoB promoter (Liere and Maliga; 1999) should be prepared and introduced into the plastid compartment by particle transformation. Insight into the regulation of PEP is crucial in order to understand the interplay of the two RNA polymerase systems. Finally, in addition to studies on plastid transcription, later steps in plastid gene expression were examined in the course of this work as well. As a model to study assembly of a multisubunit complex, the cytochrome b6/f complex was analysed by a gene-disruption approach. Deletion and/or insertion mutants of all plastid encoded subunits of the complex were prepared in order to evaluate the assembly strategy for the holocomplex

Biopharmaceutical classification of desloratadine – not all drugs are classified the easy way

The biopharmaceutical classification of drugs was designed as a basis for bio-waivers – a mechanism with the double ethical benefit of delivering new drug formulations to the market with less human testing and lower cost. However, many drugs defy simple classification because in vitro permeability and stability assessment can be challenging as shown in this study for desloratadine. Literature shows that desloratadine is highly soluble, while data on luminal stability and permeability are circumstantial. Combined with borderline bioavailability and not really known fraction of absorbed dose, desloratadine was found to be a good example for showing the innovative in vitro approaches necessary to unambiguously classify desloratadine according to Biopharmaceutical Classification System (BCS) guideline. Presented study undoubtedly confirmed that desloratadine solubility is high and dissolution is very rapid for immediate release reference tablets. We have demonstrated desloratadine stability under legally required conditions and also in more physiologically relevant media. High in vitro desloratadine permeability was confirmed using Caco-2 and Parallel Artificial Membrane Permeability Assay (PAMPA). Well-established in vitro model with rat intestinal tissue could not be used due to reasons elaborated in this paper

Trichinelosis - zoonosis of world proportion

Trihineloza ljudi i životinja je značajan javno zdravstveni problem u svijetu. To se odnosi i na Republiku Hrvatsku u kojoj se trihineloza suzbija po Zakonu. Sprečavanje invazije ljudi se u svijetu pa i u nas temelji na već tradicionalnim metodama trihineloskopije i umjetne probave kao i opće naobrazbe pučanstva glede načina pripreme mesa. Rizik za invaziju svinja uzgajanih u modernim farmama je zanemariv, stoga je već u Sad-u i Europskoj Uniji promovirana ideja o registraciji i evidentiranju farmi s dobrom proizvođačkom praksom u kojima je rizik od trihineloze gotovo zanemariv. Za napomenuti je da bi se takav alternativni način kontrole mogao provesti samo u nekim neendemskim područjima poput SAD-a i nekih država u sklopu Europske Unije. Prethodna zamisao Komisije Europske Unije o proglašavanju područja slobodnih od trihineloze (Trichinella - free area) je opovrgnuta posebice nakon što su u posljednje vrijeme dokazane nemogućnosti potpune kontrole u divljih životinja. Glede toga jedina moguća alternativa pored tradicionalne postmortalne pretrage je implementacija načela programa “Od štale do stola”Trichinelosis among people and animals is a significant public health issue in the world. This also refers to the Republic of Croatia in which trichinelosis is kept under control by law. Prevention of invasion among people, in Croatia as well as in the world, is based on methods of trichineloscopy and artificial digestion, which have become traditional, as well as on comprehensive education of people in relation to meat preparation. The risk of invasion among pigs bred in modern farms is irrelevant, so the idea of registering and recording farms that have a good manufacturing practice, where the risk of trichinelosis is almost negligible, has been promoted in the USA and EU. It is also important to mention that this alternative way of control could be implemented only in some non-endemic areas like the USA and some countries within the EU. The former idea about declaring Trichinella-free areas, supported by the European Union Comission, has been disproved, especially since impossibilities of complete control of wild animals have been proved. In regard to this, the only possible alternative, apart from the traditional post-mortal examinations, is the implementation of principles stated in «From stable to table» programme

A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts

Introduction: Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. Objectives: To demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. Methods: To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. Results: We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Conclusions: Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.German Research Foundation, DFGPeer Reviewe

Uhranjenost djece školske dobi u Međimurskoj županiji u školskoj godini 2007/2008 i uloga Školske medicine u mjerama prevencije pretilosti školske djece

Pretilost je jedan od vodećih javnozdravstvenih problema suvremenog svijeta, poglavito usljed njezinog epidemijskog karaktera, povećanog morbiditeta, mortaliteta i visokih troškova liječenja. Posebice zabrinjava porast pretilosti kod djece i adolescenata koja je izražena kako u svijetu tako i u Hrvatskoj.Po procjenama Svjetske zdravstvene organizacije 2005. god. prekomjernu tjelesnu težinu imalo je oko 1,6 milijardi ljudi starijih od 15 godina,a najmanje 400 milijuna bilo je pretilo(1). U dječjoj i adolescentnoj dobi učestalost debljine se utrostručila u odnosu na stanje prije 25 godina, pri čemu je čak 10% dječje svjetske populacije pretilo ili rizično za razvoj pretilosti. Prema podacima HZJZ iz 2003.(studija Croatian Health Survey) u Hrvatskoj nešto više od polovice populacije žena i 2/3 muškaraca ima tjelesnu težinu veću od preporučene. Prema podacima HZJZ o stanju uhranjenosti djece od 7-14 god. u razdoblju od 2000. do 2005.godine bilo je 11.9% ispitanika s prekomjernom tjelesnom masom i 6,9 % pretile djece. Problem pretilosti djece i mladih je značajan faktor rizika za pojavu većine kroničnih nezaraznih bolesti u odrasloj dobi kao što su kardiovaskularne bolesti, dijabetes tip 2, maligne bolesti, degenerativne bolesti lokomotornog aparata. Međutim pretilost u školske djece nije samo faktor rizika za obolijevanje u odrasloj dobi već i kronična bolest čije se posljedice uočavaju već u dječjoj dobi kao što su razne ortopedske anomalije (varus koljena, valgus deformiteti, epifizioliza glave bedrene kosti), idiopatski povišeni intrakranijalni tlak, opstruktivna apneja u snu, razvoj žučnih kamenaca, nealkoholni steatohepatitis, te konačno povećanje incidencije diabetes mellitusa tip 2 u sve ranijoj dobi(2,3). Također pretilost u dječjoj dobi dovodi do pojave psihosocijalnih problema kao što su gubitak samopouzdanja, depresija, lošija socijalna prilagodba u društvu vršnjaka. Liječenje pretilosti je vrlo važno jer je dokazano da smanjenje težine od samo 5-10% od početne težine smanjuje rizik obolijevanja od niza bolesti povezanih s debljinom(4). UHRANJENOST DJECE ŠKOLSKE DOBI U MEĐIMURSKOJ ŽUPANIJI U ŠKOLSKOJ GODINI 2007/2008 Analizirali smo uhranjenost djece školske dobi u Međimurskoj županiji u tri kompletne generacije učenika prvih, petih i osmih razreda u školskoj godini 2007/08. Obzirom da se tijekom sistematskog pregleda za upis u 1.razred te sistematskih pregleda učenika petih i osmih razreda svake godine vrši praćenje rasta i razvoja učenika na temelju izmjerenih vrijednosti tjelesne težine i visine, izračunat je ITM za svakog učenika. ISPITANICI I METODE Ispitanici su bili svi učenici prvih petih i osmih razreda međimurske županije, njih ukupno 3895

Mladi i sredstva ovisnosti – rezultati istraživanja o stavovima, navikama i korištenju sredstava ovisnosti kod djece i mladih Međimurske županije

UVOD Tijekom odrastanja djeca i mladi susreću se s brojnim rizicima i izazovima među kojima su i sredstva ovisnosti. Svjedoci smo stalnog trenda porasta učestalosti korištenja alkohola i nekih drugih sredstava među mladima kako u cijelom svijetu, tako i u našoj zemlji. Pušenje, pijenje alkohola i korištenje ostalih sredstava ovisnosti u mladih adolescentne dobi česta su tema razgovora odraslih, tema napisa u novinama i drugih medijskih priloga . Različite su reakcije i komentari odraslih na ovu temu i kreću se od izuzetne prestrašenosti i izbjegavanja razgovora o tome, do onih koji smatraju da je problem preuveličan i da je želja za eksperimentiranjem i probanjem nečeg novog normalan i sastavni dio odrastanja. Svjedoci smo i transformacije uloge obitelji i škole i sve veće otuđenosti mladog čovjeka koji je sve više vremena u svom odrastanju prepušten sebi i utjecaju svojih vršnjaka . Identificiranje najčešćih rizičnih i zaštitnih činitelja prvi je korak u planiranju rješavanja problema, a moguće ga je započeti ako prikupimo sve potrebne podatke i saznanja o problemu kojeg želimo rješavati . Cilj našeg istraživanja bio je utvrditi učestalost, oblik i načine korištenja sredstava ovisnosti, stavove i navike koji utječu na uzimanje tih sredstava te utvrditi glavne socijalne i psihološke čimbenike koji su u osnovi uzimanja psihoaktivnih sredstava. Također smo nastojali utvrditi kako o tome razmišljaju mladi u našoj županiji, koriste li ta sredstva u jednakoj mjeri kao i drugdje u Hrvatskoj te kako objašnjavaju razloge takvog ponašanja. Opći ciljevi : • sprečavanje i smanjenje zluouporabe alkohola, cigareta i drugih sredstava ovisnosti • podizanje opće svijesti i informiranje javnosti o štetnim posljedicama konzumiranja sredstava ovisnosti • smanjenje obolijevanja, smrtnosti i opterećenja bolestima povezanih s ovisničkim ponašanjem • smanjenje broja nesreća, naročito u prometu • smanjenje pojave društvenih poremećaja, raspada obitelji i nasilja u obitelji • zaštita zdravlja mladih kroz promicanje zdravih stilova života i promicanje duševnog zdravlja Specifični ciljevi : • ispitati učestalost i vrstu korištenja sredstava ovisnosti u populaciji učenika 7. i 8. razreda osnovne škole i 2. razreda srednje škole • utvrditi glavne socijalne i psihološke čimbenike koji su u osnovi uzimanja sredstava ovisnosti • ispitati stavove i navike učenika koji utječu na uzimanje psihoaktivnih sredstava Istraživanje je potaknuo Tim za zdravlje Međimurske županije u sklopu programa «Rukovođenje i upravljanje za zdravlje u lokalnoj zajednici», a vezano uz javnozdravstveni prioritet «Prekomjerno pijenje i pušenje u mladih», odabran tijekom izrade Županijske slike zdravlja

Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms

Most studies of eukaryotic gene regulation have been done looking at mature mRNA levels. Nevertheless, the steady-state mRNA level is the result of two opposing factors: transcription rate (TR) and mRNA degradation. Both can be important points to regulate gene expression. Here we show a new method that combines the use of nylon macroarrays and in vivo radioactive labeling of nascent RNA to quantify TRs, mRNA levels, and mRNA stabilities for all the S. cerevisiae genes. We found that during the shift from glucose to galactose, most genes undergo drastic changes in TR and mRNA stability. However, changes in mRNA levels are less pronounced. Some genes, such as those encoding mitochondrial proteins, are coordinately regulated in mRNA stability behaving as decay regulons. These results indicate that, although TR is the main determinant of mRNA abundance in yeast, modulation of mRNA stability is a key factor for gene regulation

Cluster Analysis and Comparison of Various Chloroplast Transcriptomes and Genes in Arabidopsis thaliana

Chloroplast RNA metabolism is integrated into wider gene regulatory networks. To explore how, we performed a chloroplast genome-wide expression analysis on numerous nuclear Arabidopsis mutants affected in diverse chloroplast functions and wild-type plants subjected to various stresses and conditions. On the basis of clustering analysis, plastid genes could be divided into two oppositely regulated clusters, largely congruent with known targets of nucleus- and plastid-encoded RNA polymerases, respectively. Further eight sub-clusters contained co-transcribed and functionally tightly associated genes. The chloroplast transcriptomes could also be classified into two major groups comprising mutants preferentially affected in general plastid gene expression and other chloroplast functions, respectively. Deviations from characteristic expression profiles of transcriptomes served to identify novel mutants impaired in accumulation and/or processing of specific plastid RNAs. Expression profiles were useful to distinguish albino mutants affected in plastid gene expression from those with defects in other plastid functions. Remarkably, biotic and abiotic stressors did not define transcriptionally determined clusters indicating that post-transcriptional regulation of plastid gene expression becomes more important under changing environmental conditions. Overall, the identification of sets of co-regulated genes provides insights into the integration of plastid gene expression into common pathways that ensures a coordinated response

Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns. MatK is essential for plant cell survival in tobacco, and thus null mutants have not yet been isolated. We therefore attempted to over-express MatK from a neutral site in the chloroplast, placing it under the control of a theophylline-inducible riboswitch. This ectopic insertion of MatK lead to a variegated cotyledons phenotype. The addition of the inducer theophylline exacerbated the phenotype in a concentration-dependent manner. The extent of variegation was further modulated by light, sucrose and spectinomycin, suggesting that the function of MatK is intertwined with photosynthesis and plastid translation. Inhibiting translation in the transplastomic lines has a profound effect on the accumulation of several chloroplast mRNAs, including the accumulation of an RNA antisense to rpl33, a gene coding for an essential chloroplast ribosomal protein. Our study further supports the idea that MatK expression needs to be tightly regulated to prevent detrimental effects and establishes another link between leaf variegation and chloroplast translation

Genome-wide studies of mRNA synthesis and degradation in eukaryotes

In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected, with large differences between genes at each stage of the process, from initiation to mRNA degradation, but with striking similarities for functionally related genes, indicating that all steps are coordinately regulated. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing