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Purpose: The Type A standard uncertainty in Monte Carlo (MC) dose calculations is usually determined using the “history by history” method. Its applicability is based on the assumption that the central limit theorem (CLT) can be applied such that the dispersion of repeated calculations can be modeled by a Normal distribution. The justification for this assumption, however, is not obvious. The concept of stochastic quantities used in the field of microdosimetry offers an alternative approach to assess uncertainty. This leads to a new and simple expression. Methods: The value of the MC determined absorbed dose is considered a random variable which is comparable to the stochastic quantity specific energy, z. This quantity plays an important role in microdosimetry and in the definition of the quantity absorbed dose, D. One of the main features of z is that it is itself the product of two other random variables, specifically of the mean dose contribution in a ‘single event’ and of the mean number of such events. The term ‘single event’ signifies the sum of energies imparted by all correlated particles to the matter in a given volume. The similarity between the MC calculated absorbed dose and the specific energy is used to establish the ‘event by event’ method for the determination of the uncertainty. MC dose calculations were performed to test and compare both methods. Results: It is shown that the dispersion of values obtained by MC dose calculations indeed depend on the product of the mean absorbed dose per event, and the number of events. Applying methods to obtain the variance of a product of two random variables, a simple formula for the assessment of uncertainties is obtained which is slightly different from the ‘history by history’ method. Interestingly, both formulas yield indistinguishable results. This finding is attributed to the large number of histories used in MC simulations. Due to the fact that the values of a MC calculated absorbed dose are the product of two approximately Normal distributions it can be demonstrated that the resulting product is also approximately normally distributed. Conclusions: The event by event approach appears to be more suitable than the history by history approach because it takes into account the randomness of the number of events involved in MC dose calculations. Under the condition of large numbers of histories, however, both approaches lead to the same simple expression for the determination of uncertainty in MC dose calculations. It is suggested to replace the formula currently used by the new expression. Finally, it turned out that the concept and ideas that were developed in the field of microdosimetry already 50 years ago can be usefully applied also in MC calculations.

Objectives: Despite the advances in genomics, repetitive DNAs (repeats) are still difficult to sequence, assemble, and identify. This is due to their high abundance and diversity, with many repeat families being unique to the organisms in which they were described. In sugar beet, repeats make up a significant portion of the genome (at least 53%), with many repeats being restricted to the beet genera, Beta and Patellifolia. Over the course of over 30 years and many repeat-based studies, over a thousand reference repeat sequences for beet genomes have been identified and many experimentally characterized (e.g. physically located on the chromosomes). Here, we present the collection of these reference repeat sequences for beets., Data Description: The BeetRepeats_v1.0 resource is a comprehensive compilation of all characterized repeat families, including satellite DNAs, ribosomal DNAs, transposable elements and endogenous viruses. The genomes covered are those of sugar beet and closely related wild beets (genera Beta and Patellifolia) as well as Chenopodium quinoa and Spinacia oleracea (all belonging to the Amaranthaceae). The reference sequences are in fasta format and comprise well-characterized repeats from both repeat categories (dispersed/mobile as well as tandemly arranged). The database is suitable for the RepeatMasker and RepeatExplorer2 pipelines and can be used directly for any repeat annotation and repeat polymorphism detection purposes., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Bioinformatic and molecular characterization of satellite repeats was performed to understand the impact of their diversification on Vaccinium genome evolution. Satellite repeat diversity was evaluated in four cultivated and wild species, including the diploid species Vaccinium myrtillus and Vaccinium uliginosum , as well as the tetraploid species Vaccinium corymbosum and Vaccinium arctostaphylos . We comparatively characterized six satellite repeat families using in total 76 clones with 180 monomers. We observed that the monomer units of VaccSat1, VaccSat2, VaccSat5, and VaccSat6 showed a higher order repeat (HOR) structure, likely originating from the organization of two adjacent subunits with differing similarity, length and size. Moreover, VaccSat1, VaccSat3, VaccSat6, and VaccSat7 were found to have sequence similarity to parts of transposable elements. We detected satellite-typical tandem organization for VaccSat1 and VaccSat2 in long arrays, while VaccSat5 and VaccSat6 distributed in multiple sites over all chromosomes of tetraploid V. corymbosum , presumably in long arrays. In contrast, very short arrays of VaccSat3 and VaccSat7 are dispersedly distributed over all chromosomes in the same species, likely as internal parts of transposable elements. We provide a comprehensive overview on satellite species specificity in Vaccinium , which are potentially useful as molecular markers to address the taxonomic complexity of the genus, and provide information for genome studies of this genus.

Saffron crocus (Crocus sativus) is the source of the most expensive spice of the world, produced from manually harvested stigmas, thus serving as a cash crop for rural communities. However, despite its economic importance, its genome and chromosomes are poorly studied. C. sativus is a sterile triploid species harboring eight chromosome triplets, and propagated only as a clonal lineage by corms. Saffron's evolutionary origin, parental species and allo- or autotriploidy has been a matter of discussion for almost a century. We performed a survey sequencing of the saffron genome and selected cytogenetic landmark sequences consisting of major tandem repeats, which we used as probes in comparative multicolor fluorescent in situ hybridization (FISH). We tagged 92 chromosomal positions and resolved the chromosomal composition of saffron triplets. By comparative FISH of six Crocus species from 11 accessions, we demonstrate that C. sativus is an autotriploid hybrid derived from heterogeneous Crocus cartwrightianus cytotypes. The FISH reference karyotype of saffron is crucial for integrating genome sequencing data with chromosomes and for investigating the relationship among Crocus species. We provide an evolutionary model of the saffron emergence; the knowledge of the parental origin offers a route towards the resynthesis of C. sativus from C. cartwrightianus to broaden saffron's gene pool., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Sequencing of plant genomes often identified the hAT superfamily as the largest group of DNA transposons. Nevertheless, detailed information on the diversity, abundance and chromosomal localization of plant hAT families are rare. By in silico analyses of the reference genome assembly and bacterial artificial chromosome (BAC) sequences, respectively, we performed the classification and molecular characterization of hAT transposon families in Musa acuminata. Musa hAT transposons are organized in three families designated MuhAT I, MuhAT II and MuhAT III. In total, 70 complete autonomous elements of the MuhAT I and MuhAT II families were detected, while no autonomous MuhAT III transposons were found. Based on the terminal inverted repeat (TIR)-specific sequence information of the autonomous transposons, 1722 MuhAT I- and MuhAT II-specific miniature inverted-repeat transposable elements (MuhMITEs) were identified. Autonomous MuhAT I and MuhAT II elements are only moderately abundant in the sections of the genus Musa, while the corresponding MITEs exhibit an amplification in Musa genomes. By fluorescent in situ hybridization (FISH), autonomous MuhAT transposons as well as MuhMITEs were localized in subtelomeric, most likely gene-rich regions of M. acuminata chromosomes. A comparison of homoeologous regions of M. acuminata and Musa balbisiana BACs revealed the species-specific mobility of MuhMITEs. In particular, the activity of MuhMITEs II showing transduplications of genomic sequences might indicate the presence of active MuhAT transposons, thus suggesting a potential role of MuhMITEs as modulators of genome evolution of Musa.

Background and Aims: The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification., Methods: A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling., Key Results: Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species., Conclusions: The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Background and Aims: Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera., Methods: In order to isolate highly repetitive sequences, a c(0)t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH)., Key Results: Characterization of the c(0)t-1 library resulted in the detection of repetitive sequences including the (GA)(n) dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S-5.8S-25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species., Conclusions: The repeated sequences isolated from D. moschatum c(0)t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.

Background and Aims: The aim of this work was the identification and molecular characterization of novel sugar beet (Beta vulgaris) repetitive sequences to unravel the impact of repetitive DNA on size and evolution of Beta genomes via amplification and diversification., Methods: Genomic DNA and a pool of B. vulgaris repetitive sequences were separately used as probes for a screening of high-density filters from a B. vulgaris plasmid library. Novel repetitive motifs were identified by sequencing and further used as probes for Southern analyses in the genus Beta. Chromosomal localization of the repeats was analysed by fluorescent in situ hybridization on chromosomes of B. vulgaris and two other species of the section Beta., Key Results: Two dispersed repetitive families pDvul1 and pDvul2 and the tandemly arranged repeat family pRv1 were isolated from a sugar beet plasmid library. The dispersed repetitive families pDvul1 and pDvul2 were identified in all four sections of the genus Beta. The members of the pDvul1 and pDvul2 family are scattered over all B. vulgaris chromosomes, although amplified to a different extent. The pRv1 satellite repeat is exclusively present in species of the section Beta. The centromeric satellite pBV1 by structural variations of the monomer and interspersion of pRv1 units forms complex satellite structures, which are amplified in different degrees on the centromeres of 12 chromosomes of the three species of the Beta section., Conclusions: The complexity of the pBV1 satellite family observed in the section Beta of the genus Beta and, in particular, the strong amplification of the pBV1/pRv1 satellite in the domesticated B. vulgaris indicates the dynamics of centromeric satellite evolution during species radiation within the genus. The dispersed repeat families pDvul1 and pDvul2 might represent derivatives of transposable elements.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species.

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.

Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.

The cell cycle is controlled by cyclin-dependent protein kinases (CDKs). The activity of these enzymes is directed by inhibitors of CDKs. The 21-kD protein product (P21) of the WAF1/CIP1 gene, which can be transactivated by the protein product of the tumor suppressor gene p53, acts as an inhibitor of cyclin-dependent kinases. To assess whether both P21 and p53 may play a role in the control of cellular proliferation in atherosclerotic lesions, the topographical association between p53, P21, and the proliferation marker MIB1/Ki-67, was analyzed by immunohistochemistry in human carotid atheromatous plaques of 26 patients. p53 immunoreactivity (IR) was present in 26 of 26 cases in the nuclei of virtually all cell types (macrophages [MPs], smooth muscle cells [SMCs], endothelial cells [ECs]) in areas with chronic inflammation in 71.08 +/- 8.28% of the nuclei. p53 staining in the control tissue from human coronary arteries was present in 0.3 +/- 0.45% of the cells (P < .002): P21-IR was present in 24 of 26 specimens in 64.38 +/- 10.13% of the cells (controls: 3.8 +/- 1.85%, P < .002) and localized to nuclei of MPs (CD68 positive) and SMCs (alpha-actin positive), as well as ECs of microvessels present in 21 specimens (21 of 21) and luminal ECs present in 18 specimens (16 of 18). As shown by double labeling, P21-IR colocalized with p53-IR in most MPs (24 of 24), intimal SMCs (22 of 24), ECs of microvessels (19 of 21), and luminal ECs (10 of 16). Interestingly, few p53-positive cells did not show simultaneous P21-IR, and, conversely, not all P21-positive cells demonstrated p53-IR. MIB1/Ki-67-positive cells were identified in 21 of 26 tissue specimens in 3.53 +/- 1.79% of the nuclei (controls: 0%, P < .002) and localized principally to MPs bordering the atheromatous lipid core (21 of 26) and to a few scattered SMCs (16 of 26), ECs of microvessels (13 of 21), and luminal ECs (2 of 18). Most importantly, none of the cells coexpressing P21 and p53 were positive for MIB1/Ki-67-IR, indicating the absence of proliferating activity. In summary, this study demonstrates that P21-IR is present in the atherosclerotic plaque and colocalizes with p53 in most MPs, SMCs, and ECs. The lack of proliferation markers in cells coexpressing p53 and P21 suggests that transcriptional activation of the WAF1/CIP1 gene by p53 may be involved in the control of cellular proliferation in advanced human atherosclerotic plaques.

To understand the molecular mechanisms involved in the transition from vegetative to reproductive growth, the photoperiodic-controlled induction of flowering in the long-day plant Sinapis alba was used to screen for regulatory genes which are expressed upon induction in the apical meristem. By using the conserved MADS box-encoding region of the organ identity gene AGAMOUS, the genes SaMADS A and SaMADS B were identified which are expressed in transition stages of mustard. RNA blot analysis has confirmed that the transcript levels of both genes are drastically increased shortly after the induction of flowering and that both genes are expressed earlier than the known MADS box genes. In situ hybridization studies have shown that the expression of the genes is restricted to the apical meristem of induced plants during early phases of reproductive development. The expression of SaMADS A is first detectable in the central zone of the meristem, a region where the earliest changes of an evoked meristem could be detected by classical physiological methods, suggesting that SaMADS A may have an important function during the transition to flowering.