3-Hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34) mediates the first regulatory step (HMG-CoA reduction to mevalonate) in isoprenoid biosynthesis. The tomato genome contains at least four differentially regulated hmg isogenes encoding HMGR. Functions of tomato hmg2 in defense responses were studied by promoter analyses of hmg2:GUS gene fusions, overexpression of hmg2 cDNA, and antisense inhibition of hmg1 and hmg2 in transgenic plants. Activity of the hmg2 promoter is developmentally regulated showing expression in seedling cotyledons and hypocotyls, in trichomes, and in reproductive tissues including pollen, stigmas, ovules, petals and mature seeds. hmg2:GUS activity is rapidly induced by wounding or in response to pathogenic viruses or bacteria. hmg2:GUS expression is localized to tissue surrounding lesions generated through interactions with either TMV or the bacterial pathogen, Erwinia carotovora subsp. carotovora (Ecc). Tomato hmg2 cDNA was cloned by PCR, expressed in E. coli to confirm its HMGR activity, inserted behind the double enhanced CaMV 35S promoter, and engineered into tobacco. Southern and northern analyses confirmed transformation and message expression. Enzyme activity was enhanced compared to nontransformed plants. Selected transgenic plants were significantly reduced for Ecc tissue maceration. The size of necrotic lesions induced by TMV was also significantly reduced compared to the nontransformed or vector controls. Thus, genetic manipulation of the rate-limiting step in a major defense pathway provides a novel strategy for enhancing disease resistance. We also generated transgenic tobacco and tomato containing antisense constructs for tomato hmg1 and hmg2 to study their effect on disease resistance. Full-length hmg2 and 5' regions of hmg1 or hmg2 were inserted in the antisense orientation behind a 35S promoter. Tomato expressing the full-length hmg2 antisense showed lower HMGR enzyme activity and were more susceptible to soft rot by Ecc than control plants. In contrast, expression of either antisense hmg/ or antisense hmg2 in the heterologous tobacco system resulted in plants with enhanced resistance to Ecc and reduced TMV lesion sizes. These results may indicate that antisense inhibition is non-specifically exerted on isogenes other than the defense-specific HMGR gene. / Ph. D.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

No description available.

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

No description available.

Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of the plant secondary metabolites, isoflavonoids. These phenolic compounds, which are well-known for their multiple biological effects, are produced mostly in leguminous plants (family Fabaceae). However, at least 225 of them have also been described in 59 other families, without any knowledge of orthologues to hitherto known IFS genes from legumes (with the single exception of sugar beet - Beta vulgaris, from the family Chenopodiaceae). In view of these facts, this masters thesis has focused on two main objectives: (1) to identify isoflavone synthase genes in selected leguminous and non-leguminous plants exploiting the PCR strategy with degenerate and non-degenerate primers, and (2) to find a system for the verification of the correct function of these genes. Our methodology for the identification of IFS orthologues was successfully demonstrated in the case of two examined legumes - Phaseolus vulgaris L. and Pachyrhizus tuberosus (Lam.) Spreng, in the genomic DNA of which the complete IFS sequences have been newly identified. To design a procedure for ascertaining the correct function of these genes and others once they have been completely described, a pilot study with IFS from Pisum sativum L. (CYP93C18; GenBank number...