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Infectious transcripts play a key role in the research on plant viruses at the molecular level. A number of cDNA clones covering the whole genome of the Polish isolate of potato leafroll virus were constructed. Four overlapping clones were selected and assembled using restriction sites. The full copy was positioned between T7 RNA polymerase promoter and unique ScaI site. The full-length capped transcripts of the sequence of the viral genome synthesised in vitro were able to replicate in protoplasts and to produce the viral coat protein
Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immuno- screening of sporocyst 2gt11 library and by random sequencing of clones from 2Zap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion pro­tein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore po­tentially usefull for vaccine development.
W describe an effective systematic approach to genetic mapping of cDNA clones, including those obtained from EST sequencing. The EST of interest is first partially sequenced from the 3'-end. PCR primers which bracket the 3'-UTR segment of the cDNA are designed. The corresponding gene segment is amplified from the parents of the mapping population, using primers equipped with 3'- and 5'-extensions to facilitate direct sequencing of PCR products. Comparison of the sequences obtained from the mapping parents frequently reveals single nucleotide polymorphisms or insertion / deletion polymorphisms, which can then be genotyped in a mapping population. The genotyping of SNPs is performed by pyrosequencing, a sequencing-by-synthesis method that has been used successfully in SNP diagnostics. SNP analysis of up to 96 samples, a number required to produce meaningful genetic segregation data, can be rapidly accomplished in parallel. The parental genotype of three loci, stearoyl-ACP desaturase, nucleoside-diphosphate kinase and sucrose synthetase-1 were determined by conventional sequencing, and the polymorphism so identified were scored by the pyrosequencing of 94 individuals of a maize recombinant-inbred population. These loci were successfully placed onto chromosomes 3, 7 and 9 respectively. This method is generally applicable to most plant species, which show sufficient sequence diversity in the 3'-UTR region of genes.
Selected cDNA clones of Arabidopsis thaliana, isolated previously by suppression subtractive hybridisation, were used to differentiate between abiotic stress factors. Changes in expression patterns of 79 genes were examined by array analysis in Arabidopsis thaliana after fumigation with ozone and after short- or long-term mercuric-ion exposure. Substantial changes in the abundance of 42 transcripts were recorded in response to the treatments, and 6 transcript clusters were observed. The abundance of 37 mRNAs was increased more then 1.5-fold, whereas that of 5 mRNAs was reduced. The abundances of 5, 6 and 9 mRNAs were specifically in creased by short-term mercury application, ozone fumigation, and long-term mercuric-ion exposure, respectively. The transcription of the other 5 transcripts was induced by both ozone and short-term mercuric-ion treatment. The abundance of 10 different mRNAs was in creased by the different mercuric-ion applications. Two transcripts were induced by ozone fumigation, as well as long-term, mercury treatment. Finally, 5 transcripts were repressed by ozone exposure, and 3 out of them by short-term mercuric-ion treatment. These results show that the array technique can be used to analyse the expression pattern in Arabidopsis thaliana under ozone and mercuric-ion stress. Searches against the Arabidopsis database furthermore provide a classification of most genes. In addition possible cis-acting regulatory elements were identified by an in silico approach using the MIPS Arabidopsis thaliana database.
Recent studies showed, that ozone-induced gene expression occurs via at least two different signalling mechanisms that are ethylene-dependent (fi-1,3-glucanases) and ethylene-independent independent (stilbene synthase). To identify transacting factors involved in ozone-induced gene expression we analyzed a 150 bp PCR fragment of an ozone-responsive promoter segment of the grape vine resveratrol synthase gene (Vst1) in combination of a cDNA library prepared from ozone-treated tobacco plants, using the yeast one-hybrid screening system. Two cDNA clones that encode WRKY binding proteins were isolated by this screening technique. The open reading frame of NtWRKY10 and NtWRKYll showed an identity of 93.5 % and the deduced amino acid sequence an identity of 89.3 %. According to the WRKY domain classification in Arabidopsis, both proteins belong to subgroup II. Comparison with known tobacco WRKY proteins indicate that WRKY10 and WRKY11 belong to a new class of tobacco WRKY transcription factors. Electrophoretic mobility shift assays (EMSA) of yeast extracts, containing the WRKY fusion protein showed a weak binding to the radioaclively labelled 150 bp ozone-responsive Vst1 fragment. These results are consistent with an involvement of WRKY proteins in ozone-induced phytoalexin gene expression.
In this study, we found flower cDNA clones which may be connected with the development of flower sex in cucumber. Two pairs of nearly-isogenic lines: gynoecious GY3 (FFMMGG) versus hermaphrodite HGY3 (FFmmGG) and monoecious B10 (ffMMGG) versus gynoecious 2gg (ffMMgg) were used for clone isolation. To obtain differentially-expressed clones, we applied the differential screening method. 454 clones from GY3 and 478 from B10 cDNA libraries were isolated. The results of RFLP analysis with 56 cDNA clones showed no clones which cosegregated with sex in cucumber. The 28 cDNA B10 and 33 cDNA GY3 clones isolated using the differential screening method were sequenced. Some of them seem to may play a role in cell differentiation or flower development. Among the 61 identified clones, 14 show high homology to plant proteins, although of unknown function. 11 show high homology to known proteins, and the possible function of some of them is discussed. For 3 clones, no significant similarity was found. The 31 clones displayed high homology to plant cDNA in EST database. The patterns of expression of five differential cDNA clones, 35GY3, 216GY3, 47GY3, 100B10 and 157B10, were analyzed in cucumber flower buds using in situ RT-PCR. The most interesting clone is 35GY3, because of its possible role in the inhibition of the development of male specific elements in the female cucumber flower.
The cDNA clones coding for two pathogenesis-related protein homologues of PR10 class, LlPR10.lA and LlPR10.1B, were identified in yellow lupin expres­sion library of uninfected roots. The contribution of PR10 proteins to the overall mechanism of plant defence still remains unknown. In order to elucidate the structure and function of lupin PR10.1A protein, a substantial quantity of the protein was produced in an E. coli expression system using plasmids of pET-se- ries: pET-3a and pET-15b, carrying the T7 promoter. Both plasmids with sub- cloned LlprlO.la gene were overexpressed in E. coli, strain BL21(DE3)pLysS. The recombinant L/PR10.1A protein, overproduced in bacterial cells trans­formed with the pET-3a/LlprlO.la plasmid, was purified to homogeneity from the insoluble "inclusion bodies" by ammonium sulphate fractionation and two sequential chromatographic steps: ion-exchange chromatography on DE 52 cellulose followed by size exclusion chromatography on Superdex 75 FPLC column. The (His)e LlPR10.lA protein overproducted in E. coli cells harbour­ing the pET-15b/LlprlO.la plasmid was purified by chromatography on Ni2+- charged His.Bind Resin. Western blot analysis with rabbit serum containing anti-£/PR10.1AN antibody revealed identical immunochemical properties of the two recombinant polypeptides and native LIPR10.1A protein. The recombinant protein produced in pET-3a plasmid was renatured from its insoluble form, concentrated up to 22 mg/ml and submitted to crystallisation. However, the LIPR10.1A protein expressed in pET-15b plasmid precipitated from the solution when at a higher concentration (10 mg/ml). This preparation was used at a lower concentration as an antigen for the preparation of polyclonal antibodies for immunochemical studies.
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