In the present work we perform in situ hybridization with probes to different stretches of rDNA and electron microscopy of nucleoli with different activities, to gain insight into the ultrastructural organization of transcription and processing in the plant nucleolus. The main ultrastructural nucleolar components: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC), are arranged in different ways depending on nucleolar activity. Heterogeneous FCs containing RNP fibrils and nucleolar perichromatin granules are frequently seen in nucleoli in the process of activation. DNA-RNA in situ hybridization with biotinylated probes spanning different sequences of the rDNA unit followed by immunogold detection of biotin, demonstrated the localization of the ribosomal transcripts in DFC, mainly in the zones around the FCs, in GC, and in the periphery of pale FC. The internal region of the heterogeneous FCs is labeled only in cells in the process of activation of transcription after dormancy. The distribution of the U3 probe indicates that the processing of the rRNA takes place in the DFC and inside the heterogeneous FCs, in which transcription occurs. DNA-DNA hybridization demonstrates the presence of rDNA in the compact and extended chromatin located in the interior and at the periphery of FCs and in nucleolar associated chromatin. Our results support the view that the plant nucleolus has a highly dynamic morphological and functional organization composed of a bipartite domain formed by FCs surrounded by DFC, which is associated with rRNA transcription and processing, and the GC representing a store of preribosomal particles.
The largest phylogenetic lineage known to date of the anthrax pathogen Bacillus anthracis is the wide-spread, so-called Trans-Eurasian clade systematically categorized as the A.Br.008/009 group sharing two defining canonical single-nucleotide polymorphisms (canSNP). In this study, we genome-sequenced a collection of 35 B. anthracis strains of this clade, derived from human infections, animal outbreaks or soil, mostly from European countries isolated between 1936 and 2008. The new data were subjected to comparative chromosomal analysis, together with 75 B. anthracis genomes available in public databases, and the relative placements of these isolates were determined within the global phylogeny of the A.Br.008/009 canSNP group. From this analysis, we have detected 3754 chromosomal SNPs, allowing the assignation of the new chromosomal sequences to established sub-clades, to define new sub-clades, such as two new Spanish, one Bulgarian or one German group(s), or to introduce orphan lineages. SNP-based results were compared with that of a multilocus variable number of tandem repeat analysis (MLVA). This analysis indicated that MLVA typing might provide additional information in cases when genomics yields identical genotypes or shows only minor differences. Introducing the delayed mismatch amplification assay (DMAA) PCR-analysis, we developed a cost-effective method to interrogate for a set of ten phylogenetically informative SNPs within genomes of A.Br.008/009 canSNP clade strains of B. anthracis. By this approach, additional 32 strains could be assigned to five of ten defined clades.
A total of 111 clinical and environmental O1, O139 and non-O1/O139 Vibrio cholerae strains isolated between 1978 and 2008 from different geographical areas were typed using a combination of methods: antibiotic susceptibility, biochemical test, serogroup, serotype, biotype, sequences containing variable numbers of tandem repeats (VNTRs) and virulence genes ctxA and tcpA amplification. As a result of the performed typing work, the strains were organized into four clusters: cluster A1 included clinical O1 Ogawa and O139 serogroup strains (ctxA(+) and tcpA(+)); cluster A2 included clinical non-O1/O139 strains (ctxA(-) and tcpA(-)), as well as environmental O1 Inaba and non-O1/O139 strains (ctxA(-) and tcpA(-)/tcpA(+)); cluster B1 contained two clinical O1 strains and environmental non-O1/O139 strains (ctxA(-) and tcpA(+)/tcpA(-)); cluster B2 contained clinical O1 Inaba and Ogawa strains (ctxA(+) and tcpA(+)). The results of this work illustrate the advantage of combining several typing methods to discriminate between clinical and environmental V. cholerae strains.
The geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.
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