Abstract: We describe the application of a novel genome walking (GW) strategy for the one-shot identification of members of multigene families. The method was used to study the spinach Lhcb1 family (encoding the light harvesting complex protein Lhcb1), for which three cDNAs were known. Two additional genes and regulatory regions of the five members of the family were identified. For one of the newly detected genes, sequencing of full-length cDNA and analysis of regulatory regions by gel-shift are also reported. To our best knowledge, this is the first report on the use of a GW approach for the study of a multigene family.
Abstract: AIMS: To investigate the surviving capability of Rhodobacter sphaeroides under phototrophic conditions in the presence of high cobalt concentration and its influence on the photosynthetic apparatus biosynthesis. METHODS AND RESULTS: Cells from R. sphaeroides strain R26.1 were grown anaerobically in a medium containing 5.0 mmol l(-1) cobalt ions and in a control medium. Metal toxicity was investigated comparing the soluble proteome of Co(2+)-exposed cells and cells grown in control medium by two-dimensional gel electrophoretic analysis. Significant changes in the expression level were detected for 43 proteins, the majority (35) being up-regulated. The enzyme porphobilinogen deaminase (PBGD) was found down-regulated and its activity was investigated. CONCLUSIONS: The up-regulated enzymes mainly belong to the general category of proteins and DNA degradation enzymes, suggesting that part of the catabolic reaction products can rescue bacterial growth in photosynthetically impaired cells. Furthermore, the down-regulation of PBGD strongly indicates that this key enzyme of the tetrapyrrole and bacteriochlorophyll synthesis is directly involved in the metabolic response. SIGNIFICANCE AND IMPACT OF THE STUDY: Data and experiments show that the cobalt detrimental effect on the photosynthetic growth of R. sphaeroides is associated with an impaired expression and functioning of PBGD.
Abstract: PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.
Abstract: Nad6 orf, encoded in the sunflower mitochondrial genome in single copy contains a non-conserved 3'-extension. The transcription of the nad6 locus generates a highly complex pattern. Three main transcripts of 1240, 960 and 870 nt have been characterized by different approaches. The two smaller ones are apparently the most abundant of the steady-state RNA population and are generated as primary transcription products as well as by processing of the 1240 nt transcript. Their 5'-UTRs are absent or very short. Whereas the 3'-ends of the 960 nt transcripts contain a TGA codon the shorter one terminates at positions excluding the stop codon. The fate of transcripts to promote the synthesis of NAD6 sunflower protein seems, thus, to rely on the occurrence of mechanisms yet to be identified.
Abstract: In recent decades, the most successful strategy for controlling blood pressure has been inhibition of the angiotensin-converting enzyme (ACE). ACE inhibitors of chemical synthesis (captopril, enalapril, ramipril and trandolapril) have been widely used clinically to reduce mortality in patients with heart failure, and in patients with recent myocardial infarction and heart failure or marked left ventricular dysfunction. In addition to preventive and therapeutic drugs, increased attention has been paid to identifying dietary compounds that may contribute to cardiovascular treatment and prevention. ACE inhibitory peptides, derived from a multitude of plant and animal proteins such as milk, soy or fish, represent sources of health-enhancing components. These ACE inhibitory peptides can be enzymatically released from precursor proteins in vitro and in vivo, respectively during food processing and gastrointestinal digestion. They have shown the ability to lower blood pressure by limiting the vasoconstrictory effects of Angiotensin II and potentiating the vasodilatory effects of Bradykinin. By using specific procedures they may be generated in or incorporated into functional foods for the development of 'natural' beneficial health products. Several products containing peptides with ACE inhibitory properties are currently on the market or in development. This review focuses on the use, application and future perspective of bioactive peptides with properties relevant to cardiovascular health.
Abstract: Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill this aim and overcome the time-consuming approach of screening genomic libraries. Usually these protocols rely on specific requirements and strategies, such as the presence of suitable nucleotide restriction sites and ligation of specific single- or double-strand linkers, thus limiting their application to a certain extent. In this paper we present an alternative PCR-based protocol, consisting of four main steps: (i) extension of a sequence-specific primer; (ii) 3'-tailing of extended single-strand DNA; (iii) PCR; and (iv) nested PCR amplifications. This method, which appears to be a valid alternative to the other PCR-based protocols, was used for the identification of sequences flanking the cDNA encoding region of the Lhcb 1.1 gene (one member of the multigene family coding for the light harvesting protein Lhcbl) in the spinach genome.
Abstract: Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.
Abstract: Three proteinase inhibitor genes have been identified in the rapeseed (Brassica napus) genome. They are highly homologous to other genes of the mustard inhibitor (MSI) family of proteinase inhibitors characteristic of Cruciferae. In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti-2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI-2 protein strongly inhibits (Ki=44 nM) a glutamyl proteinase from Streptomyces griseus.
Abstract: Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.
Abstract: Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor-containing diet. The amino-acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66% of the mature protein amino acid sequence. This trypsin, called HzTrypsin-S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76% identical) to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin-S and HzTrypsin-C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin-S could not be inhibited by > 1000-fold molar excess of any of these. The same inhibitors inhibited HzTrypsin-C with apparent equilibrium dissociation constants ranging from 1 nm to 30 nm. Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.
Abstract: The updated version of PLMItRNA reports information and multialignments on 609 genes and 34 tRNA molecules active in the mitochondria of Viridiplantae (27 Embryophyta and 10 Chlorophyta), and photosynthetic algae (one Cryptophyta, four Rhodophyta and two Stramenopiles). Colour-code based tables reporting the different genetic origin of identified genes allow hyper-textual link to single entries. Promoter sequences identified for tRNA genes in the mitochondrial genomes of Angiospermae are also reported. The PLMItRNA database is accessible at http://bighost.area.ba.cnr.it/PLMItRNA/.
Abstract: The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 x 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 microg ml-1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.
Abstract: PLANT-PIs is a database developed to facilitate retrieval of information on plant protease inhibitors (PIs) and related genes. For each PI, links to sequence databases are reported together with a summary of the functional properties of the molecule (and its mutants) as deduced from literature. PLANT-PIs contains information for 351 plant PIs, plus several isoinhibitors. The database is accessible at http://bighost.area.ba.cnr.it/PLANT-PIs.
Abstract: The divergent transcription of two tRNA genes encoded in sunflower mitochondrial DNA, proposed as genes of different genetic origin, has been studied in detail. The transcription initiation site (TIS) for both transcript precursors has been identified by hybridization with in vitro (32)P-capped total RNAs and primer extension. The location of two TISs and the analysis of distribution of sequence elements (motifs) usually present in higher plant mitochondrial promoters led to the identification of two short regions (about 30-40 bp) which can be proposed as the promoters for the transcription of two genes. This conclusion is supported by the observation that within the short intergenic region included between the 5' termini of two genes (1924 bp) the distribution of those specific motifs is unique around the TISs, although not identical for the two promoters. Based on specific experimental results the trnE promoter shows a higher efficiency in comparison with that of the trnH promoter. This result is in good agreement with its structure which strictly conforms to those described for mitochondrial genes of dicot plants. Instead the other promoter shows some divergences which could be responsible for its lower efficiency. The context in which trnH lies in the sunflower mitochondrial genome and other features described in the paper may suggest that, despite the high similarity with the chloroplast counterpart, the trnH gene could have a native origin.
Abstract: The PLMItRNA database for mitochondrial tRNA molecules and genes in VIRIDIPLANTAE: (green plants) [Volpetti,V., Gallerani,R., DeBenedetto,C., Liuni,S., Licciulli,F. and Ceci,L.R. (2000) Nucleic Acids Res., 28, 159-162] has been enlarged to include algae. The database now contains 436 genes and 16 tRNA entries relative to 25 higher plants, eight green algae, four red algae (RHODOPHYTAE:) and two STRAMENOPILES: The PLMItRNA database is accessible via the WWW at http://bio-www.ba.cnr.it:8000/PLMItRNA.
Abstract: The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants. It can be applied to study the adaptations of digestive proteases in pest insects. Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities. Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants. Active and inactive mutants of MTI-2 are constructed and displayed on phage. These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification.
Abstract: Transcription analysis of a mustard (Sinapis alba L.) serine proteinase inhibitor gene revealed identical 5' termini of mRNAs synthesized during seed maturation and chemical or wounding induction. Polyadenylation of mRNAs on multiple or single sites differentiated gene expression, increasing the availability of stable mRNAs during seed maturation compared with chemical and wounding induction. Expression of the beta-glucuronidase (GUS)-encoding region of the UidA reporter gene, detected under the control of deleted segments of the region flanking on the 5' side the mit-2 gene, identified a stretch of about 520 bp essential for gene expression. The presence in this region of two ABRE motifs is relevant for plant response to gene induction. Expression of GUS was detectable under different induction stimuli in several organs such as seedlings and leaves and was active to varying extents in the vascular tissues and meristem.
Abstract: The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.
Abstract: The current version of PLMItRNA has been realized to constitute a database for tRNA molecules and genes identified in the mitochondria of all green plants ( Viridiplantae ). It is the enlargement of a previous database originally restricted to seed plants [Ceci,L.R., Volpicella,M., Liuni,S., Volpetti,V., Licciulli,F. and Gallerani,R. (1999) Nucleic Acids Res., 27, 156-157]. PLMItRNA reports information and multialignments on 254 genes and 16 tRNA molecules detected in 25 higher plants (one bryophyta and 24 vascular plants) and seven green algae. PLMItRNA is accessible via the WWW at http://bio-WWW.ba.cnr.it:8000/srs6/
Abstract: The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40-160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine beta-trypsin compared to the precursor and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.
Abstract: The PLMItRNA database contains information and multialignments of tRNA genes and molecules detected in higher plant mitochondria. It has been developed from a previous compilation of higher plant mitochondrial tRNA genes [Sagliano,A., Volpicella,M., Gallerani,R. and Ceci,L.R. (1998) Nucleic Acids Res., 26, 154-155] and implemented with data and sequences of tRNA molecules retrieved from the literature. The current version of the database reports information on 171 genes and 16 tRNA molecules from 24 plants. PLMItRNA is accessible via WWW at http://bio-www.ba.cnr.it:8000/srs/
Abstract: This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.
Abstract: A new version of the compilation of higher plant mitochondrial tRNA genes (http://www.ebi.ac.uk/service ) has been obtained by means of the FastA program for similarity searching in nucleotide sequence Databases. This approach improves the previous collection, which was based on literature data analysis. The current compilation contains 158 sequences with an increase of 43 units. In this paper, some interesting features of the new entries are briefly presented.
Abstract: Sunflower mitochondrial DNA contains a single copy of the cob gene. The gene begins with the unusual GTG initiation codon and lies in a transcription unit having a different organization with respect to that common to the other six known sequences from plant species which include both monocotyledons and dicotyledons.
Abstract: The physical map for seventeen tRNA genes on the mitochondrial genome of the dicotyledonous plant Helianthus annuus has been established. Eleven are genuine mitochondrial genes, while the other six show a high degree of similarity with the chloroplast counterparts. The genes, with the exception of the genuine trnS(GCT) and of the chloroplast-like trnV and trnP, are expressed. The comparison of the organization of some tRNA genes in the H. annuus mitochondrial genome with that of similar genes detectable in other plants reveals that their association is common to several dicotyledons.
Abstract: This compilation reports the tRNA genes detected on higher plant mitochondrial genomes subdivided into the widely accepted categories of 'genuine' and 'chloroplast-like' genes. Moreover, it includes a list of pseudo or truncated genes divided in the same way.
Abstract: We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms.
Abstract: The organization of the genes nad3 and rps12 has been investigated in the mitochondrial genome of two dicotyledonous plants - Helianthus and Magnolia - and one monocotyledonous plant (Allium). These plants all contain a complete rps12 gene downstream of the nad3 gene. This arrangement is thus highly conserved within angiosperms. The two genes are co-transcribed and the transcript is modified at several positions by RNA editing of the C to U-type, thus confirming that both genes encode functional proteins. Some 26, 35 and 27 editing events have been identified in the PCR-derived nad3-rps12 cDNA population from sunflower, Magnolia and onion, respectively. Editing of the nad3-rps12 transcript is thus more extensive in Magnolia than in the other angiosperms so far investigated and radically changes the genomically encoded polypeptide sequence. A novel species-specific codon modification was observed in Magnolia. Several homologous sites show differences in editing pattern among plant species. A C-to-U alteration is also found in the non-coding region separating the nad3 and rps12 genes in sunflower. The PCR-derived cDNA populations from the nad3-rps12 loci analysed were found to be differently edited. In addition the plant species show marked variations in the completeness of RNA editing, with only the Magnolia nad3 mRNA being edited fully.
Abstract: Three sunflower mitochondrial HindIII restriction fragments containing the tRNA genes trnI, trnE and trnfM have been sequenced. The genes are present in single copy on the whole genome and are transcribed. Hybridization experiments and sequence analysis of the HindIII fragments allowed the precise mapping and orientation of each gene on the sunflower mitochondrial genome.
Abstract: The gene coding for the mustard trypsin inhibitor-2 has been isolated from a genomic library and characterized. Comparison of genomic and cDNA sequences indicates that the gene is interrupted by an intron of 193 bp. The eukaryotic peculiar regulatory sequences have been detected in the 5' flanking region of the gene. In addition, a decanucleotide has been detected that is highly similar to the proposed G-box and to the ABRE motifs required for the gene expression induced by methyl jasmonate and abscissic acid. Northern blot analysis demonstrates that the gene is expressed in immature seeds as well as in wounded leaves.
Abstract: The genes coding for tRNA-Cys (trnC), tRNA-Asn (trnN) and tRNA-Tyr (trnY) have been sequenced in a region of about 3.0 kb of the sunflower mitochondrial DNA. The trnC and trnY are genuine mitochondrial genes, while the trnN gene has a chloroplast origin. Despite their heterologous origin the three genes are transcribed. Their arrangement is the first detected in a highly conserved form in a specific group of advanced dicots.
Abstract: A tRNA(Val) (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA(Val) gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA(Val) is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA(Val) species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.
