Tolerance of blackgrass (Alopecurus myosuroide) to sylfonylurea herbicides in the Czech Republic

Seeds of three blackgrass populations fromSouthern Bohemiawere collected in 2007–2008. Biological tests with chlorsulfuron were performed at the 2–3 leaf stage. Some plants survived after treatment with the highest dose 37.5 g/ha. Biological tests showed a resistant phenotype to chlorsulfuron. Leaves of these plants were analysed by dCAPS assay. Two domains of ALS gene: domain A ‒ P197 and domain B ‒ W574 were targeted by PCR with regenerated primers P197 containing BamHI site and W574 containing site BstXI. PCR products of all tested samples were cleaved by BamHI in the codon P197. No mutation of proline in P197 was found out. The codon W574 PCR product of the samples was not cleaved by BstXI.

A new BSMV-based vector with modified β molecule allows simultaneous and stable silencing of two genes

Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:β molecule was engineered with a unique BamHI site between the open reading frame of βc (ORF βc) and poly(A). The mixture of RNA particles α, βBamHI and γMCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to βBamHI and γMCS. Delivery of the target gene fragment in γMCS induced stronger silencing, while delivery in βBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of β enables simultaneous silencing of two genes. Quantifying the β and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than β, while the stability of the insert was higher in β compared with γ. The possible influence of the relative quantity of β and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.

Study of the 49 kDa excretory-secretory protein gene of Trichinella nativa and Trichinella spiralis

To study the function of the 49 kDa excretory-secretory (ES) protein gene (P49) of Trichinella, the genes was amplified by RT-PCR from RNA of Trichinella spiralis and Trichinella nativa and several Chinese Trichinella isolates of domestic animals, and sequenced after being cloned. The amplified products of these parasites produced bands of about 950 bp. The 97.2 % to 100 % nucleotides identity and 94.3 % to 100 % identity of deduced amino acids among P49 gene of these Trichinella strains showed the close relationship of these parasites. The P49 gene of T. nativa was cloned into the BamHI site of the prokaryotic expression vector pET-30a, and the recombinant vector was expressed. The expressed product was 40.8 kDa in size. In Western blot analysis, the expressed product was reactive to sera of mice infected with T. nativa, T. spiralis and their Chinese geographical strains.

A rapid means of identifying wild rice species DNA using dot blots and genome-specific rDNA probes

Intergenic spacer fragments from the rDNA repeat unit were isolated from a single accession of each of 9 species that cover the range of genomes found in the Oryza genus (A-F). Seven of the 9 species contained 1 size class of rDNA repeat unit only, while Oryza sativa and Oryza latifolia contained 3 and 2 size classes, respectively, of which fragments were cloned for the major size class only. Oryza australiensis contained an additional BamHI site in the intergenic spacer. Dot blots were prepared and hybridised with a repeat unit from each species. Under high stringency conditions, all probes were specific to species possessing the same genome or genomes.Key words: rDNA, rice, genome-specific, dot blots.

Duplication of small segments within the major breakpoint cluster region in chronic myelogenous leukemia

The t(9;22) in chronic myelogenous leukemia (CML) may be reciprocal or, in a minority of cases, may result in an extensive deletion of a portion of the major breakpoint cluster region (M-bcr) of the BCR. This report provides evidence of the duplication of small segments within the M-bcr in a small group of patients with CML. Southern blots of Bgl II and Bgl II/BamHI double-digested DNA from the blood or bone marrow of 46 patients with CML were probed with a 5' 1.4-kb Taq I/HindIII M- bcr probe and a 3' 2-kb HindIII/BamHI M-bcr probe. In three patients, rearrangements were noted with both probes in Bgl II-digested DNA, but were not present in Bgl II/BamHI-digested DNA with either probe. Southern analysis of DNA samples double-digested with Bgl II and BspHI from two of these three cases showed no rearrangements with either probe; the M-bcr BspHI site is located 26 bp 3' of the BamHI site in the second intron of the M-bcr. The presence of a rearranged M-bcr with both probes in Bgl II-digested DNA and the lack of rearrangement in Bgl II/BamHI and Bgl II/BspHI double-digested DNA suggest the presence of M- bcr BamHI and BspHI sites on both 9q+ chromosome (9q+) and the Philadelphia chromosome (Ph). This implies a duplication of at least the 26-bp M-bcr BamHI/BspHI fragment in these two samples. Sequence data from one of these two cases confirmed the M-bcr breakpoints to be staggered; the Ph M-bcr breakpoint occurred 258 bp downstream from the 9q+ M-bcr breakpoint. It is concluded that a duplication of small segments within the M-bcr occurs in a small group of patients with CML, which may lead to pseudogermline patterns on Southern blot. Such a duplication may provide insight into the mechanism of some chromosomal translocations in neoplasia.

Duplication of small segments within the major breakpoint cluster region in chronic myelogenous leukemia

The t(9;22) in chronic myelogenous leukemia (CML) may be reciprocal or, in a minority of cases, may result in an extensive deletion of a portion of the major breakpoint cluster region (M-bcr) of the BCR. This report provides evidence of the duplication of small segments within the M-bcr in a small group of patients with CML. Southern blots of Bgl II and Bgl II/BamHI double-digested DNA from the blood or bone marrow of 46 patients with CML were probed with a 5' 1.4-kb Taq I/HindIII M- bcr probe and a 3' 2-kb HindIII/BamHI M-bcr probe. In three patients, rearrangements were noted with both probes in Bgl II-digested DNA, but were not present in Bgl II/BamHI-digested DNA with either probe. Southern analysis of DNA samples double-digested with Bgl II and BspHI from two of these three cases showed no rearrangements with either probe; the M-bcr BspHI site is located 26 bp 3' of the BamHI site in the second intron of the M-bcr. The presence of a rearranged M-bcr with both probes in Bgl II-digested DNA and the lack of rearrangement in Bgl II/BamHI and Bgl II/BspHI double-digested DNA suggest the presence of M- bcr BamHI and BspHI sites on both 9q+ chromosome (9q+) and the Philadelphia chromosome (Ph). This implies a duplication of at least the 26-bp M-bcr BamHI/BspHI fragment in these two samples. Sequence data from one of these two cases confirmed the M-bcr breakpoints to be staggered; the Ph M-bcr breakpoint occurred 258 bp downstream from the 9q+ M-bcr breakpoint. It is concluded that a duplication of small segments within the M-bcr occurs in a small group of patients with CML, which may lead to pseudogermline patterns on Southern blot. Such a duplication may provide insight into the mechanism of some chromosomal translocations in neoplasia.

Ribosomal DNA repeat unit polymorphism in six Aegilops species

Total genomic DNA of 27 accessions representing six Aegilops species was restricted with BamHI, EcoRI, and BamHI plus EcoRI, and the restriction fragments were probed with the ribosomal clones pMF2 containing the ribosomal RNA coding regions. The rDNA repeat lengths varied between 9.0 and 10.8 kb. Intraspecific variation among the 10 accessions of Ae. squarrosa var. strangulata ranged from 9.0 to 9.6 kb, suggesting a diversity of genotypes within as well as between species. These variations were not related to their geographical distributions. Each of 24 accessions had two BamHI sites in the coding region (type A), while three accessions of Ae. squarrosa var. strangulata had four BamHI sites (type B, two sites in the intergenic region). Results for these three accessions of Ae. squarrosa var. strangulata suggest genotypic diversity in this species. In BamHI restriction of each accession, a third DNA fragment, ranging between 9.0 and 10.8 kb in type A and 6.0 kb in type B, resulted from lack of digestion at the 26S BamHI site. In double digestion, all rDNA repeat units were restricted by EcoRI, yielding 3.9-, 0.9-, and 4.8-kb fragments, the last of which arose from the lack of digestion at the 26S BamHI site, estimated to occur in 5–20% of the repeat units, depending on the accession.Key words: Triticum tauschii, RFLP, diversity.