Transcriptional Silencing of 35S rDNA in Tragopogon porrifolius Correlates with Cytosine Methylation in Sequence-Specific Manner.

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.

Analyses of the Updated "Animal rDNA Loci Database" with an Emphasis on Its New Features.

We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively.The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci showed, on average, less than 40% of active loci. In conclusion, the updated version of the database provides some new insights into the organization of rRNA genes in chromosomes. We expect that its updated content will be useful for taxonomists, comparative cytogeneticists, and evolutionary biologists. .

Evolutionary trends in animal ribosomal DNA loci: introduction to a new online database.

Ribosomal DNA (rDNA) loci encoding 5S and 45S (18S-5.8S-28S) rRNAs are important components of eukaryotic chromosomes. Here, we set up the animal rDNA database containing cytogenetic information about these loci in 1343 animal species (264 families) collected from 542 publications. The data are based on in situ hybridisation studies (both radioactive and fluorescent) carried out in major groups of vertebrates (fish, reptiles, amphibians, birds, and mammals) and invertebrates (mostly insects and mollusks). The database is accessible online at www.animalrdnadatabase.com . The median number of 45S and 5S sites was close to two per diploid chromosome set for both rDNAs despite large variation (1-74 for 5S and 1-54 for 45S sites). No significant correlation between the number of 5S and 45S rDNA loci was observed, suggesting that their distribution and amplification across the chromosomes follow independent evolutionary trajectories. Each group, irrespective of taxonomic classification, contained rDNA sites at any chromosome location. However, the distal and pericentromeric positions were the most prevalent (> 75% karyotypes) for 45S loci, while the position of 5S loci was more variable. We also examined potential relationships between molecular attributes of rDNA (homogenisation and expression) and cytogenetic parameters such as rDNA positions, chromosome number, and morphology.

Gene conversion events and variable degree of homogenization of rDNA loci in cultivars of Brassica napus.

BACKGROUND AND AIMS: Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. METHODS: We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. KEY RESULTS: Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH ('Darmor', 'Yudal' and 'Asparagus kale') harboured the same number (12 per diploid set) of loci. In B. napus 'Darmor', the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus 'Darmor'. In contrast, B. napus 'Yudal' showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus 'Asparagus kale' showed an intermediate pattern to 'Darmor' and 'Yudal'. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar ('Norin 9') showed co-dominance. CONCLUSIONS: The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion.

Release 4.0 of the Plant rDNA Database: A Database on Plant Ribosomal DNA Loci Number, Their Position, and Organization: An Information Source for Comparative Cytogenetics.

This paper presents the latest update to the Plant rDNA database (Release 4.0), a valuable resource for researchers in the field of plant cytogenetics. The database provides information on the number, position, and arrangement of ribosomal DNA loci in plants, including angiosperms, gymnosperms, bryophytes, and pteridophytes. The new release includes new data for 820 species coming from additional 173 papers. In the updated version of the Plant rDNA database, 4948 entries comprising 2760 organisms can be found. A brief guide on how to navigate the database and obtain the desired information is also provided. The regular updating of the database is important for ensuring the information it contains is accurate, up-to-date, and useful for the research community. The Plant rDNA database continues to be beneficial for phylogenetic and cytogenetic studies in a wide range of taxa including angiosperms, gymnosperms, and early diverging groups, such as bryophytes and lycophytes.

Standardisation of (56)Co and (57)Co using software coincidence counting system.

Activities of the radionuclides (56)Co and (57)Co were determined by the efficiency extrapolation method applied to 4pi(PC)-gamma coincidence counting. Solutions of (56)Co usually contain a significant amount of (57)Co and (58)Co, so the measured activity of (56)Co requires correction. When the conventional coincidence method is used for (56)Co standardisation, the corrections are derived from the dependence of Proportional Counter (PC) detection efficiencies for (57)Co and (56)Co measured using sources with mixture of (56)Co and (57)Co, which is complicated. These difficulties were reduced by means of a software coincidence method, with a HPGe detector in gamma channel, where the detection efficiencies were evaluated directly from the records of coincidence measurements of standardised sources. In the case of (57)Co standardisation, the software coincidence counting system was applied for the evaluation of optimal setting of coincidence parameters. The results obtained by software coincidence counting system were compared with those obtained by the conventional coincidence method.

The half-life of 129I.

The radionuclide 129I is a long-lived fission product that decays to 129Xe by beta-particle emission. It is an important tracer in geological and biological processes and is considered one of the most important radionuclides to be assessed in studies of global circulation. It is also one of the major contributors to radiation dose from nuclear waste in a deep geological repository. Its half-life has been obtained by a combination of activity and mass concentration measurements in the frame of a cooperation of 6 European metrology institutes. The value obtained for the half-life of 129I is 16.14 (12) × 106 a, in good agreement with recommended data but with a significant improvement in the uncertainty.

Standardisation of 54Mn and 65Zn using a software coincidence counting system.

The activities of 54Mn and 65Zn have been determined by 4pi(PC)-gamma coincidence counting, with efficiency variation performed by the conventional method of altering the self-absorption in the sources as well as by the computer discrimination method. The standardisation of 65Zn presents some complications requiring optimisation of the gamma-ray energy window settings to achieve a linear efficiency-extrapolation curve. Determination of these optimal settings by the conventional coincidence method is a tedious task. These difficulties have been reduced by the utilisation of a software coincidence counting system that records time and amplitude information of individual pulses from coincidence measurements, where the coincidence parameters are set after the data collection process has completed, facilitating multiple data evaluations on a single data set. The optimal gamma-ray energy window settings for the 65Zn standardisation were derived from the results of the 54Mn standardisation, as well as from studies of the 65Zn data itself. The setting of the PC channel thresholds for K and both (K+L) electrons is also discussed. The results are compared with those attained using conventional coincidence counting.

Activity standardization, photon emission probabilities and half-life measurements of (177)Lu.

Activity standardization of (177)Lu and measurement of two nuclear parameters were done. Activity standardization of (177)Lu was done utilizing the 4πß-γ coincidence method with a combined standard uncertainty of 0.28%. Emission probability of 112.95keV and 208.37keV was measured by calibrated spectrometer with HPGe detector. The efficiency was computed with MCNP code and validated using experimental points. Half-life was derived from prolonged measurement of peak area by three different spectrometer systems and also from measurement with ionization chamber.

Application of pulse mixing method in software coincidence counting.

In recent years the software coincidence counting system, designed for absolute activity measurement, has been developed in the Czech Metrology Institute. In this system a true coincidence count rate is calculated from the records of time and amplitude data of individual pulses and may be determined by two different methods. The first one uses a coincidence resolving time, in a manner similar to a classical coincidence measurement. The second method applies the pulse mixing method formulae, so that it does not use the resolving time and the correction for accidental coincidences. Both methods have been tested on the same data from a 4pibeta (PC)-gamma coincidence measurement of 60Co sources. The difference between the results obtained from both calculation methods applied on the data from the same measurement did not exceed 0.015%. The details of both methods and the results of their comparison are presented.

Software coincidence counting.

A system designed for absolute activity measurement is described using a digital method. The system is based on data recording from a coincidence measurement and subsequent software processing of the data records. The data acquisition device collects amplitudes of individual pulses from analogue-to-digital converters and supplies them with time information. Software processing of data records from this system offers many benefits in comparison to conventional coincidence counting, for example it enables to perform time and pulse height analysis and setting of coincidence parameters by using a wide variety of evaluation methods to one data record. The digital system was tested with a 4pi beta-gamma coincidence detectors arrangement consisting of a proportional counter and two NaI(Tl) scintillation detectors. The results obtained with a 60Co source are presented.

Standardisation of 64Cu using a software coincidence counting system.

The activity of the radionuclide (64)Cu was determined by the efficiency extrapolation method applied to 4π(PC)-γ coincidence counting. The standardisation was performed by software coincidence counting-a digital method for primary activity measurement that simplifies the setting of optimal coincidence parameters. The γ-ray-energy window, characterised by identical gamma detection efficiency related to the sum of EC and to the sum of beta decay branches, was found. This setting ensured a linear and zero slope extrapolation curve.

Activity standardization of ¹³4Cs and ¹³7Cs.

The paper presents the results from a primary standardization of (137)Cs using two independent methods - efficiency tracing using PC-NaI coincidence and the TDCR method. The nuclides (60)Co and (134)Cs were used as the tracers. Primary standardization of the (134)Cs is also discussed. The efficiency extrapolation was carried out by measuring samples of varying mass and using the wet extrapolation method. The results obtained are in good agreement; the differences did not exceed 0.5%. The advantages, pitfalls and also possibilities for improvement of the procedures are discussed.

Activity standardisation of 45Ca and 204Tl using the new TDCR system at CMI.

A new triple-to-double coincidence ratio (TDCR) system has been established at CMI as an additional technique for primary activity standardisation. Details of the detectors and the electronics are given. Construction of the optical chamber was validated using measurement of a (3)H standard-the efficiency achieved with Ultima Gold was 58%. Several methods of efficiency variation (coloured bands painted on vial, positioning and photomultipliers defocussing) are compared. Activity concentrations of (204)Tl and (45)Ca were determined with the TDCR method and with the efficiency tracer technique. Advantages of the TDCR method compared to the efficiency tracing method and agreement between results are shown.

Standardisation of 124SB and 152EU using software coincidence counting system.

Activities of the radionuclides (124)Sb and (152)Eu were determined by the efficiency extrapolation method applied to 4pi(PC)-gamma coincidence counting. The (124)Sb sources were prepared from a solution with the chemical form of 50 microg g(-1) SbCl(3) in 2 M HCl. To inhibit the volatility of antimony chlorides, the sources were slowly dried in a H(2)S atmosphere with relative humidity of 76% for about 48 h. This procedure increased the beta detection efficiency up to 0.98, which simplified the standardisation. In the (152)Eu standardisation, the optimal gamma-ray energy window setting to achieve a linear dependency and the correct slope of the extrapolation curve were derived by means of software coincidence counting system using offline evaluation of data with different coincidence parameter settings. The results obtained by the software coincidence counting system were compared with those obtained by the conventional coincidence method.