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Barley stripe mosaic virus (BSMV) was found in seeds of 24 varieties originated from barley cultivar collections per 40 tested. The virus more frequently occurred in seeds of winter cultivars where it was detected in seeds of 17 varietes per 19 tested while in seeds of spring cultivars it was found only in 7 cultivars per 21 tested. Similar results were obtained in the case of commercial varieties. The infection of seeds of some varieties exceeded 10%. In all cases occurrence of the mild and no symptoms was confirmed. Preliminary studies showed that BSMV caused the decrease of yield by 9–15% and the decrease of mass of 1 000 seeds ranging from 0.5 to 3%. The risk of isolate mutation of a mild BSMV to the isolate causing severe symptoms was observed in spring barley cultivar Annabell.
Based on 156 sequences of Potato spindle tuber viroid (PSTVd) taken from GenBank, consensus sequence thereof containing 359 nucleotides was determied by means of CLUSTAL W program. For this sequence the secondary structures were derived by using Mfold program. In linear structure of the lowest two apical loops and 27 internal loops and bulges were found. To define 3D structures of those fragments and double-stranded structures connecting them, an RNA FRABASE ver. 1.0 search engine was used. In this way, their 3D equivalents in resolved crystallographic structures and those derived from measurements of NMR spectra were found. For smaller fragments, full compatibility was observed. Bigger ones required usage of our script to isostericity base replacement for fragments of not full homology. The above procedure did not change torsion angles χ around glycosyl bonds. Then these fragments were connected via superimposition of common canonical base pairs. An integrated structure that obtained was then energetically minimized in torsion angles space and Cartesian coordinates space. Then 7 different 2D structures (2 linear and 5 branched) were taken into account and additional, alternative threedimensional ones were generated for them. The length of whole 3D structure of viroid RNA for corresponding linear 2D structure amounted to 500 Å that was in agreement with the data obtained from electron microscopy. RNA of linear structures, due to their extent is able to easily complex with proteins. Asides from previously found in central conservative domain of PSTVd and described in the literature E loop we found another, similar one in pathogenic domain. Both structures have characteristic of sarcin/ricin RNA motif S-turn of one strain.
The simple method of nucleic acids extraction, based on guanidine thiocyanate extraction buffer, was sufficient for obtaining a good templates for RT-PCR. The RT-PCR reactions were performer from the start to the end (both the reverse transcription and the HLVd-cDNA amplification) in the same reaction mixture and in the very small volume of reaction (10 μI). Both pairs of primers designed by authors were good for reverse transcription and later for amplification of the HLVd-cDNA. The presence of gelatin as a stabilizer of DNA polymerase was indispensable for successful performance of RT-PCR.
The simple method of nucleic acids extraction, based on guanidine thiocyanate extraction buffer, was sufficient for obtaining a good templates for RT-PCR. The RT-PCR reactions were performer from the start to the end (both the reverse transcription and the HLVd-cDNA amplification) in the same reaction mixture and in the very small volume of reaction (10 μI). Both pairs of primers designed by authors were good for reverse transcription and later for amplification of the HLVd-cDNA. The presence of gelatin as a stabilizer of DNA polymerase was indispensable for successful performance of RT-PCR.
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