Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.

The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures: expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.

Major Cellular and Physiological Impacts of Ocean Acidification on a Reef Building Coral

As atmospheric levels of CO2 increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO2 conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation assessment

Background: Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes. Methods: The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays. Results: A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15). Conclusion: This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.

Construction of a global map of human gene expression : the process, tools and analysis

This thesis studies human gene expression space using high throughput gene expression data from DNA microarrays. In molecular biology, high throughput techniques allow numerical measurements of expression of tens of thousands of genes simultaneously. In a single study, this data is traditionally obtained from a limited number of sample types with a small number of replicates. For organism-wide analysis, this data has been largely unavailable and the global structure of human transcriptome has remained unknown. This thesis introduces a human transcriptome map of different biological entities and analysis of its general structure. The map is constructed from gene expression data from the two largest public microarray data repositories, GEO and ArrayExpress. The creation of this map contributed to the development of ArrayExpress by identifying and retrofitting the previously unusable and missing data and by improving the access to its data. It also contributed to creation of several new tools for microarray data manipulation and establishment of data exchange between GEO and ArrayExpress. The data integration for the global map required creation of a new large ontology of human cell types, disease states, organism parts and cell lines. The ontology was used in a new text mining and decision tree based method for automatic conversion of human readable free text microarray data annotations into categorised format. The data comparability and minimisation of the systematic measurement errors that are characteristic to each lab- oratory in this large cross-laboratories integrated dataset, was ensured by computation of a range of microarray data quality metrics and exclusion of incomparable data. The structure of a global map of human gene expression was then explored by principal component analysis and hierarchical clustering using heuristics and help from another purpose built sample ontology. A preface and motivation to the construction and analysis of a global map of human gene expression is given by analysis of two microarray datasets of human malignant melanoma. The analysis of these sets incorporate indirect comparison of statistical methods for finding differentially expressed genes and point to the need to study gene expression on a global level.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

Effect of long-wave UV radiation on mouse melanoma: an in vitro and in vivo study

The skin cancer incidence has increased substantially over the past decades and the role of ultraviolet (UV) radiation in the etiology of skin cancer is well established. Ultraviolet B radiation (280-320 nm) is commonly considered as the more harmful part of the UV-spectrum due to its DNA-damaging potential and well-known carcinogenic effects. Ultraviolet A radiation (320-400 nm) is still regarded as a relatively low health hazard. However, UVA radiation is the predominant component in sunlight, constituting more than 90% of the environmentally relevant solar ultraviolet radiation. In the light of the recent scientific evidence, UVA has been shown to have genotoxic and immunologic effects, and it has been proposed that UVA plays a significant role in the development of skin cancer. Due to the popularity of skin tanning lamps, which emit high intensity UVA radiation and because of the prolonged sun tanning periods with the help of effective UVB blockers, the potential deleterious effects of UVA has emerged as a source of concern for public health. The possibility that UV radiation may affect melanoma metastasis has not been addressed before. UVA radiation can modulate various cellular processes, some of which might affect the metastatic potential of melanoma cells. The aim of the present study was to investigate the possible role of UVA irradiation on the metastatic capacity of mouse melanoma both in vitro and in vivo. The in vitro part of the study dealt with the enhancement of the intercellular interactions occurring either between tumor cells or between tumor cells and endothelial cells after UVA irradiation. The use of the mouse melanoma/endothelium in vitro model showed that a single-dose of UVA to melanoma cells causes an increase in melanoma cell adhesiveness to non-irradiated endothelium after 24-h irradiation. Multiple-dose irradiation of melanoma cells already increased adhesion at a 1-h time-point, which suggests the possible cumulative effect of multiple doses of UVA irradiation. This enhancement of adhesiveness might lead to an increase in binding tumor cells to the endothelial lining of vasculature in various internal organs if occurring also in vivo. A further novel observation is that UVA induced both decline in the expression of E-cadherin adhesion molecule and increase in the expression of the N-cadherin adhesion molecule. In addition, a significant decline in homotypic melanoma-melanoma adhesion (clustering) was observed, which might result in the reduction of E-cadherin expression. The aim of the in vivo animal study was to confirm the physiological significance of previously obtained in vitro results and to determine whether UVA radiation might increase melanoma metastasis in vivo. The use of C57BL/6 mice and syngeneic melanoma cell lines B16-F1 and B16-F10 showed that mice, which were i.v. injected with B16-F1 melanoma cells and thereafter exposed to UVA developed significantly more lung metastases when compared with the non-UVA-exposed group. To study the mechanism behind this phenomenon, the direct effect of UVA-induced lung colonization capacity was examined by the in vitro exposure of B16-F1 cells. Alternatively, the UVA-induced immunosuppression, which might be involved in increased melanoma metastasis, was measured by standard contact hypersensitivity assay (CHS). It appears that the UVA-induced increase of metastasis in vivo might be caused by a combination of UVA-induced systemic immunosuppression, and to the lesser extent, it might be caused by the increased adhesiveness of UVA irradiated melanoma cells. Finally, the UVA effect on gene expression in mouse melanoma was determined by a cDNA array, which revealed UVA-induced changes in the 9 differentially expressed genes that are involved in angiogenesis, cell cycle, stress-response, and cell motility. These results suggest that observed genes might be involved in cellular response to UVA and a physiologically relevant UVA dose have previously unknown cellular implications. The novel results presented in this thesis offer evidence that UVA exposure might increase the metastatic potential of the melanoma cells present in blood circulation. Considering the wellknown UVA-induced deleterious effects on cellular level, this study further supports the notion that UVA radiation might have more potential impact on health than previously suggested. The possibility of the pro-metastatic effects of UVA exposure might not be of very high significance for daily exposures. However, UVA effects might gain physiological significance following extensive sunbathing or solaria tanning periods. Whether similar UVA-induced pro-metastatic effects occur in people sunbathing or using solaria remains to be determined. In the light of the results presented in this thesis, the avoidance of solaria use could be well justified.

Microarrays in the Diagnosis of Human Herpesvirus infections

Currently, there are nine known human herpesviruses and these viruses appear to have been a very common companion of humans throughout the millenia. Of human herpesviruses, herpes simplex viruses 1 and 2 (HSV-1, HSV-2), causative agents of herpes labialis and genital herpes, and varicella-zoster virus (VZV), causative agent of chicken pox, are also common causes of central nervous system (CNS) infections. In addition, human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), all members of the herpesvirus family, can also be associated with encephalitis and meningitis. Accurate diagnostics and fast treatment are essential for patient recovery in CNS infections and therefore sensitive and effective diagnostic methods are needed. The aim of this thesis was to develop new potential detection methods for diagnosing of human herpesvirus infections, especially in immunocompetent patients, using the microarray technique. Therefore, methods based on microarrays were developed for simultaneous detection of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6A, HHV-6B, and HHV-7 nucleic acids, and for HSV-1, HSV-2, VZV, and CMV antibodies from various clinical samples. The microarray methods developed showed potential for efficiently and accurately detecting human herpesvirus DNAs, especially in CNS infections, and for simultaneous detection of DNAs or antibodies for multiple different human herpesviruses from clinical samples. In fact, the microarray method revealed several previously unrecognized co-infections. The microarray methods developed were sensitive and provided rapid detection of human herpesvirus DNA, and therefore the method could be applied to routine diagnostics. The microarrays might also be considered as an economical tool for diagnosing human herpesvirus infections.

Characterisation, cloning and production of industrially interesting enzymes: gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.

Nucleotide sequence of the 3' terminal region of belladonna mottle virus-Iowa (renamed Physalis mottle virus) RNA and an analysis of the relationships of tymoviral coat proteins

The 3prime terminal 1255nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3prime terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy, Several genes have been identified and cloned from the carrot system, These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using a Cuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes.

U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

The genetic associations of acute anterior uveitis and their overlap with the genetics of ankylosing spondylitis

Acute anterior uveitis (AAU) involves inflammation of the iris and ciliary body of the eye. It occurs both in isolation and as a complication of ankylosing spondylitis (AS). It is strongly associated with HLA-B*27, but previous studies have suggested that further genetic factors may confer additional risk. We sought to investigate this using the Illumina Exomechip microarray, to compare 1504 cases with AS and AAU, 1805 with AS but no AAU and 21 133 healthy controls. We also used a heterogeneity test to test the differences in effect size between AS with AAU and AS without AAU. In the analysis comparing AS+AAU+ cases versus controls, HLA-B*27 and HLA-A*02:01 were significantly associated with the presence of AAU (P<10−300 and P=6 × 10−8, respectively). Secondary independent association with PSORS1C3 (P=4.7 × 10−5) and TAP2 (P=1.1 × 10−5) were observed in the major histocompatibility complex. There was a new suggestive association with a low-frequency variant at zinc-finger protein 154 in the AS without AAU versus control analysis (zinc-finger protein 154 (ZNF154), P=2.2 × 10−6). Heterogeneity testing showed that rs30187 in ERAP1 has a larger effect on AAU compared with that in AS alone. These findings also suggest that variants in ERAP1 have a differential impact on the risk of AAU when compared with AS, and hence the genetic risk for AAU differs from AS.