FISH is a useful method to identify individual chromosomes in a karyotype and to discover their structural changes accompanying genome evolution and speciation. DNA probes for FISH should be chromosome specific and/or exhibit specific patterns of distribution along each chromosome. Such probes are not available in many plants including meadow fescue (Festuca pratensis Huds.), an important forage grass species. In the present study, various DNA repeats identified in Illumina shotgun sequences specific to chromosome 4F of F. pratensis were used as probes for FISH to develop the molecular karyotype of meadow fescue and to reveal a long-range molecular organization of its chromosomes. Five tandem repeats produced specific patterns on individual chromosomes. Their use in combination with probes for rRNA genes enabled the establishment of the molecular karyotype of meadow fescue. Most of the mobile genetic elements were dispersed along all the chromosomes except for the DNA transposon CACTA, which was localized preferentially to telomeric and subtelomeric regions, and a putative LTR element, which was localized to (peri)centromeric regions. Cytogenetic mapping of the 5 tandem repeats in other accessions of meadow fescue showed a highly similar distribution and confirmed the versatility and robustness of these probes.

• Klíčová slova
• Fluorescence in situ hybridization, Karyotyping, Meadow fescue, Repetitive DNA, Tandem organized repeats,
• MeSH
• chromozomy rostlin MeSH
• DNA rostlinná MeSH
• Festuca genetika   MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• karyotyp MeSH
• karyotypizace metody   MeSH
• tandemové repetitivní sekvence * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA rostlinná MeSH

BACKGROUND: The genus Pulmonaria (Boraginaceae) represents a taxonomically complex group of species in which morphological similarity contrasts with striking karyological variation. The presence of different numbers of chromosomes in the diploid state suggests multiple hybridization/polyploidization events followed by chromosome rearrangements (dysploidy). Unfortunately, the phylogenetic relationships and evolution of the genome, have not yet been elucidated. Our study focused on the P. officinalis group, the most widespread species complex, which includes two morphologically similar species that differ in chromosome number, i.e. P. obscura (2n = 14) and P. officinalis (2n = 16). Ornamental cultivars, morphologically similar to P. officinalis (garden escapes), whose origin is unclear, were also studied. Here, we present a pilot study on genome size and repeatome dynamics of these closely related species in order to gain new information on their genome and chromosome structure. RESULTS: Flow cytometry confirmed a significant difference in genome size between P. obscura and P. officinalis, corresponding to the number of chromosomes. Genome-wide repeatome analysis performed on genome skimming data showed that retrotransposons were the most abundant repeat type, with a higher proportion of Ty3/Gypsy elements, mainly represented by the Tekay lineage. Comparative analysis revealed no species-specific retrotransposons or striking differences in their copy number between the species. A new set of chromosome-specific cytogenetic markers, represented by satellite DNAs, showed that the chromosome structure in P. officinalis was more variable compared to that of P. obscura. Comparative karyotyping supported the hybrid origin of putative hybrids with 2n = 15 collected from a mixed population of both species and outlined the origin of ornamental garden escapes, presumably derived from the P. officinalis complex. CONCLUSIONS: Large-scale genome size analysis and repeatome characterization of the two morphologically similar species of the P. officinalis group improved our knowledge of the genome dynamics and differences in the karyotype structure. A new set of chromosome-specific cytogenetic landmarks was identified and used to reveal the origin of putative hybrids and ornamental cultivars morphologically similar to P. officinalis.

• Klíčová slova
• Pulmonaria, Comparative karyotyping, Genome size, Repeatome, Satellite DNA,
• MeSH
• chromozomy rostlin * genetika   MeSH
• délka genomu MeSH
• fylogeneze MeSH
• genom rostlinný * MeSH
• karyotyp MeSH
• karyotypizace * MeSH
• plicník genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours--neuroblastoma and Wilms' tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods.

• MeSH
• dítě MeSH
• hybridizace nukleových kyselin MeSH
• indoly MeSH
• krevní buňky cytologie   účinky léků   MeSH
• lidé MeSH
• metafáze účinky léků   MeSH
• neuroblastom genetika   patologie   MeSH
• odběr biologického vzorku metody   MeSH
• proteasy farmakologie   MeSH
• spektrální karyotypizace metody   MeSH
• stárnutí buněk účinky léků   MeSH
• Wilmsův nádor genetika   patologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• indoly MeSH
• proteasy MeSH

The Festuca genus is thought to be the most numerous genus of the Poaceae family. One of the most agronomically important forage grasses, Festuca pratensis Huds. is treated as a model plant to study the molecular mechanisms associated with tolerance to winter stresses, including frost. However, the precise mapping of the genes governing stress tolerance in this species is difficult as its karyotype remains unrecognized. Only two F. pratensis chromosomes with 35S and 5S rDNA sequences can be easily identified, but its remaining chromosomes have not been distinguished to date. Here, two libraries derived from F. pratensis nuclear DNA with various contents of repetitive DNA sequences were used as sources of molecular probes for fluorescent in situ hybridisation (FISH), a BAC library and a library representing sequences most frequently present in the F. pratensis genome. Using FISH, six groups of DNA sequences were revealed in chromosomes on the basis of their signal position, including dispersed-like sequences, chromosome painting-like sequences, centromeric-like sequences, knob-like sequences, a group without hybridization signals, and single locus-like sequences. The last group was exploited to develop cytogenetic maps of diploid and tetraploid F. pratensis, which are presented here for the first time and provide a remarkable progress in karyotype characterization.

• MeSH
• chromozomy rostlin genetika   MeSH
• diploidie MeSH
• Festuca genetika   růst a vývoj   MeSH
• fyziologický stres genetika   MeSH
• genová knihovna MeSH
• hybridizace genetická MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• nízká teplota MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• RNA ribozomální 5S genetika   MeSH
• tetraploidie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální 5S MeSH

BACKGROUND: In Turner syndrome (TS), fluorescent in situ hybridization (FISH) karyotyping offers an alternative to classical karyotyping. OBJECTIVE: We tested the added value of FISH karyotyping from lymphocytes (mesodermal origin), buccal cells (ectodermal origin), and a rear-tongue smear (endodermal origin) to determine the 45,X cell line fraction and its impact on patient phenotype. DESIGN AND PATIENTS: Classical karyotyping and three FISH assays were done in 153 girls and women previously diagnosed with TS in four university hospitals. The 45,X cell line fraction was determined for each method and correlated with the major phenotypic signs. RESULTS: Classical karyotyping identified 45,X/46,XX mosaicism in 77/153 subjects (50%), 45,X monosomy in 52/153 (34%), and other karyotypes in 24/153 (16%). FISH from lymphocytes verified 45,X in 47/52 original cases, whereas 4/52 had 45,X/46,XX and 1/52 45,X/47,XYY mosaicism. The 45,X cell line fraction was higher in FISH from lymphocytes compared to classical karyotyping (median 86.4% vs. 70.0%; p < 0.001), while there was no difference for FISH from buccal or rear-tongue smear cells. The mean 45,X cell line fraction was more abundant in patients with several of the characteristic phenotypic signs compared to patients without them (p < 0.01), but the predictive power was insufficient. CONCLUSION: FISH analysis confirmed the findings of classical karyotyping; only a few 45,X monosomy cases were reclassified as mosaics. The 45,X cell line fraction did not show clinically meaningful prediction of the phenotype. FISH analysis of buccal or rear-tongue epithelial cells may be a non-inferior, less invasive alternative to classical karyotyping.

• Klíčová slova
• Cytogenetics, Fluorescence in situ hybridization, Germ layers, Karyotype, Phenotype, Turner syndrome,
• MeSH
• epitelové buňky MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• lidé MeSH
• lymfocyty metabolismus   MeSH
• monozomie MeSH
• mozaicismus MeSH
• Turnerův syndrom * metabolismus   MeSH
• ústní sliznice MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

• Klíčová slova
• Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
• Publikační typ
• časopisecké články MeSH

A high-yield method for isolation of barley chromosomes in suspension, their analysis and sorting using flow cytometry is described. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea for 18 h, 2.5 micromol/L amiprophos methyl for 2 h, and ice water (overnight). This treatment resulted in metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer by mechanical homogenization, producing, on average, 5 x 10(5) chromosomes from 50 root tips. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis and sorting. While it was possible to discriminate and sort only one chromosome from a barley cultivar with standard karyotype, up to three chromosomes could be sorted in translocation lines with morphologically distinct chromosomes. The purity of chromosome fractions, estimated after PRINS with primers specific for GAA microsatellites, reached 97%. PCR with chromosome-specific primers confirmed the purity and suitability of flow-sorted chromosomes for physical mapping of DNA sequences.

• MeSH
• chromozomy genetika   MeSH
• DNA primery MeSH
• elektroforéza v agarovém gelu MeSH
• fyzikální mapování chromozomů MeSH
• hybridizace in situ fluorescenční MeSH
• hydroxymočovina farmakologie   MeSH
• insekticidy farmakologie   MeSH
• ječmen (rod) genetika   MeSH
• karyotypizace MeSH
• kořeny rostlin genetika   MeSH
• metafáze MeSH
• mikrosatelitní repetice genetika   MeSH
• mitóza genetika   MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny farmakologie   MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amiprophos MeSH Prohlížeč
• DNA primery MeSH
• hydroxymočovina MeSH
• insekticidy MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH

Electrophoretic karyotyping was used to compare DNA probes of yeasts isolated from blood of preterm neonates (n = 66) in a neonatal intensive care unit (NICU) and from the hands of healthy hospital personnel (n = 10). The yeasts were identified as Candida albicans using standard laboratory methods. DNA was extracted from yeasts and isolation of identical DNA strains from the pairs nurse-neonate suggested that one nurse transmitted one yeast strain by her hands to three neonates. Four neonates harbored two identical strains originating from two nurses, i.e. each nurse transmitted the same strain to two neonates. In the additional 7 cases transmission of 1 yeast strain by 1 nurse to 1 neonate was observed. Our data suggest that nonperinatal nosocomial transmission of C. albicans occurs in neonates, possibly via cross-contamination being transferred on hands of health care workers. The importance of careful hand washing of staff (health care workers) and other infection-control procedures (to prevent the nosocomial transmission of pathogens in the NICU environment) is emphasized.

• MeSH
• Candida albicans genetika   izolace a purifikace   MeSH
• fungemie mikrobiologie   MeSH
• infekce spojené se zdravotní péčí mikrobiologie   přenos   MeSH
• jednotky intenzivní péče o novorozence * MeSH
• kandidóza epidemiologie   mikrobiologie   přenos   MeSH
• karyotypizace MeSH
• lidé MeSH
• novorozenec nedonošený krev   MeSH
• novorozenec MeSH
• personál nemocniční * MeSH
• přenos infekce ze zdravotnického pracovníka na pacienta MeSH
• prospektivní studie MeSH
• ruka MeSH
• zdravotní sestry MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Geografické názvy
• Slovenská republika epidemiologie   MeSH

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4',6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.

• Publikační typ
• časopisecké články MeSH

OBJECTIVE: Shift of indicated invasive examination in prenatal diagnostics towards the earlier phases of pregnancy with preservation of quality of cytogenetic detection. DESIGN: Cytogenetic and molecular-cytogenetic analysis of the chorionic villi after long term culture. SETTING: Department of Medical Genetics and Foetal Medicine, Faculty of Medicine, Palacky University Olomouc, Faculty Hospital in Olomouc. METHODS: Cultivation of fibroblasts developing from chorionic villi after enzymatic or mechanical disintegration and their karyotyping. Using fluorescent in situ hybridisation to identify the most common chromosomal aneuploidies and to determine gonosomes in indicated cases. RESULTS: Testing and optimisation of long term culture method and its routine use. Method was utilised so far in 12 patients and successfulness was 83%. Additional fluorescent in situ hybridisation was performed in 6 cases. CONCLUSION: Using long term culture method of chorionic villi as reliable and routine tool in prenatal diagnostics.

• MeSH
• chromozomální aberace diagnóza   MeSH
• chromozomální poruchy MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace * MeSH
• lidé MeSH
• odběr choriových klků * MeSH
• první trimestr těhotenství MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH