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Influence of a composition of hybridization mixtures on fluorescence intensity during the in-situ hybridization
Janíček, Tomáš ; Kejík, Lukáš (referee) ; Vojtěch,, Hudzieczek (advisor)
Fluorescenční in situ hybridizace (FISH) je široce používaná metoda pro detekci určité sekvence DNA na chromozomech. Cílem práce je porovnání tří různých chemických sloučenin (formamidu, ethylenkarbonátu a sodných kationtů) používaných ve směsích pro in situ hybridizaci. Složení těchto směsí ovlivňuje renaturaci DNA a je důležité porovnat jejich fyzikální vlastnosti. Práce je rozdělena do dvou hlavních částí. První část se zabývá otázkou termodynamických parametrů používaných pro experimenty FISH, jako je teplota tání, entalpie přechodu DNA ze dvoušroubovice na vlákno nám dává přehled o energii potřebné k tomto přechodu a interakcích mezi bázemi a každou složkou směsi. Kromě toho hodnoty entropie určují poř uvnitř směsi - systém DNA. Druhá část porovnává intenzitu fluorescenčního signálu při optimalizovaných teplotách tání sondy použité pro in situ hybridizaci. Jako sonda byla použita sub-telomerní repetice X43.1, která je umístěna na Y chromozomu rostlinného modelového organismu Silene latifolia. Směs obsahující formamid má nejlepší výkon při delším postupu hybridizace, zatímco ethylenkarbonát poskytuje vyšší intenzitu signálu, a proto je vhodnější pro rychlé FISH protokoly.
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Evolution of karyotype, sex chromosomes and gene clusters in selected taxa of araneomorph spiders
Voráčková, Diana ; Forman, Martin (advisor) ; Sadílek, David (referee)
Spiders (Araneae) are a diverse group with various sex chromosome systems. The most common is X1X20 sex determination, X1X2 denotes two non-homologous X chromosomes and 0 the absence of Y. The presence of Y chromosome is rare among spiders and has been described mainly in Synspermiata, X1X2Y is probably ancestral constitution for this clade. The family Sicariidae was chosen to elucidate the evolution of sex chromosomes in Synspermiata. Karyotypes of 10 species whose 2n ranged from 19 to 23 were investigated in this study. The most common finding was X1X2Y in all studied Loxosceles species. The derived XY system was discovered in Sicarius. Furthermore, the distributional pattern of molecular cytogenetic markers (genes for 18S rRNA, 5S rRNA, histone H3 and U2 snRNA) was analysed using fluorescence in situ hybridization (FISH). These markers were found exclusively on autosomes, on one or two pairs. FISH with whole-genome probes of both sexes was performed to reveal distribution of Y specific sequences. Various patterns of Y differentiation were found in certain species. Y chromosome was small in all Sicariidae. A larger Y with a pronounced accumulation of Y-specific sequences has been detected in previous works - in the X1X2Y system of a synspermiate Pholcus phalongoides (Pholcidae). Pilot...
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Analysis of gonosomal mosaic using FISH method
ŠUTKOVÁ, Jana
In my bachelor thesis I dealt with the analysis of gonosomal mosaics. Mosaicism can be described as a condition in which two or more distinct cell lines originating from a single zygote are found in one organism. In women, the most common mosaic forms are Turner syndrome and triple X syndrome or superfemale. In the male population, Klinefelter syndrome and XYY syndrome (supermale) are most commonly detected. Mosaicism can be investigated by fluorescence in situ hybridization. In the theoretical part of my bachelor's thesis I focused on congenital chromosomal mosaicism, especially on its variants such as Klinefelter syndrome, Turner syndrome, triple X and XYY syndrome. Furthermore, in this part I dealt with fluorescence in situ hybridization. I described fluorescence per se and then its application in clinical practice, where several types of fluorescence in situ hybridization are used. In the practical part I examined the number of gonosomal chromosomes using fluorescence in situ hybridization. I prepared this part in the laboratory of the Faculty of Health and Social Sciences of the University of South Bohemia in České Budějovice. I examined 40 anonymized samples from 20 probands. Capillary blood samples and buccal swabs were taken from each of them. After hybridization of samples with centromeric probe for X and Y chromosomes microscopy was performed. In the last part of this bachelor thesis I dealt with the processing of the results obtained by fluorescence microscopy. Thus, a total of 6 cases of mosaicism were detected in women (the percentage of mosaic cells was above the admissible limit). In males there were a total of 2 cases of increased percentage of mosaic cells.
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Evolutionary dynamics of satellite DNA in plant genomes
ÁVILA ROBLEDILLO, Laura
Satellite DNA (satDNA) belongs to the highly repetitive fraction of eukaryotic genomes. It is best characterized by the formation of long arrays of almost identical sequences that are tandemly repeated. These repeats are widely distributed in plant species where they can make up a substantial proportion of their genomes. Despite the long history of satDNA research, the classic methodology did not allow for its comprehensive characterization. Consequently, the fragmentary information gathered during the last 60 years does not answer the many questions surrounding the evolution of these elements. The development of new techniques in sequencing, together with the availability of new bioinformatics tools for analyzing different genome fractions, has presented an opportunity to advance studies of tandem repeats. This thesis describes the landscape characterization of satDNA in the genome of Fabeae species by exploring the diversity of satDNA within a genome, the association of these elements with functional centromeres, as well as their genome-wide organization. We employed new computational pipelines specifically designed for the analysis of tandem repeats from next generation sequencing data, and combined their results with molecular and cytogenetic methods to achieve comprehensive characterization of the satellite repeats.
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Karyotype evolution of African clades of theraphosid mygalomorphs
Košátko, Prokop ; Král, Jiří (advisor) ; Nguyen, Petr (referee)
Karyotypes of mygalomorph spiders are not satisfactorily known. This thesis is focused on the basic cytogenetic analysis of selected species of African clades of theraphosid mygalomorphs. It includes four subfamilies: Eumenophorinae, Harpactirinae, Ischnocolinae and Stromatopelminae. Diploid numbers, chromosome morphology, sex chromosome systems and chromosome behaviour in male germline in the selected species of African theraphosid subfamilies were studied. The findings support published results, that refer of high karyotype diversity in Theraphosidae. Diploid chromosome number reduction is probably a basic trend of theraphosid karyotype evolution. The majority of analysed species exhibited one, two or three sex chromosomes. In some species neo-sex chromosome systems were found. In some species one or two sex chromosome pairs (SCP), composed of chromosomes which lack morphological differentiation were detected. Nucleolus organizer regions were detected by fluorescent in situ hybridization in several species. Constitutive heterochromatin detection was performed by C-banding in two species. Keywords: constitutive heterochromatin, diploid number, karyotype, fluorescence in situ hybridization, Mygalomorphae, nucleolus organizer region, SCP, sex chromosome, spider, Theraphosidae
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Molecular-cytogenetic diagnostics of marker chromosomes
Tesner, Pavel ; Kočárek, Eduard (advisor) ; Zemanová, Zuzana (referee) ; Šubrt, Ivan (referee)
Supernumerary marker chromosomes (sSMCs) are a relatively rare cytogenetic phenomenon. Their laboratory examination is often difficult, and the clinical interpretation is even more challenging. The main reason is that most sSMC carriers have no clinical manifestations. The chromosome origin and exact range of the aberration are very important, as well as the fact that sSMCs are often found in mosaics that can strongly influence both the phenotype and the interpretation of result. Prenatal sSMC finding is one of the most challenging situations in both clinical and laboratory genetics. This work deals with the investigation process of sSMC carriers using molecular cytogenetic techniques, especially fluorescence in situ hybridization (FISH). We investigated a total of 67 families collected both prospectively and retrospectively, and we found 70 unique sSMCs in a total of 74 individuals. Six cases were familial and in three cases two sSMCs were found in one individual. According to the initial karyotype finding, the cases were divided into two groups, sSMCs supernumerary to a normal karyotype (group A) and sSMCT s supernumerary to the Turner karyotype (group B). The chromosomal origin was successfully determined in 88,6 % sSMCs. In group A the most common findings were sSMCs derived from chromosome 15,...
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Karyotype analysis of selected representatives of two pedipalpid orders, Amblypygi and Uropygi
Sember, Alexandr ; Král, Jiří (advisor) ; Ráb, Petr (referee)
Karyotype analysis of selected species from arachnid orders Amblypygi and Uropygi Whip spiders (Amblypygi) and whip scorpions (Uropygi) represent relict arachnid orders which has been found already at Upper Carboniferous strata. Although cytogenetic data from amblypygids and uropygids might be important to reconstruct karyotype evolution of arachnids, cytogenetics of these orders is almost unknown. Presented study is aimed in analysis of karyotype and meiosis in 16 species of Amblypygi and 4 species of Uropygi. Both groups are characterized by considerable range of diploid chromosome numbers (2n = 24 - 86 in Amblypygi and 36 - 66 in Uropygi). Analysed species does not exhibit morfologically differentiated sex chromosomes. Differentiation of sex chromosomes on molecular level was revealed in amblypygid Paraphrynus mexicanus by comparative genome hybridization. Obtained data indicate XY/XX sex chromosome system in this species. Comparison of karyotype data indicates reduction of chromosome numbers during evolution of both orders. In Amblypygi, this reduction was accompanied by increase of number of biarmed chromosomes. This trend is not apparent in Uropygi. Karyotypes of most analysed amblypygids and uropygids are also characterized by low amount of heterochromatin. Most studied species exhibit two pairs...
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Evolutionary dynamics of rDNA clusters in arthropods with holocentric chromosomes
Tymich, Alexandr ; Šťáhlavský, František (advisor) ; Dalíková, Martina (referee)
Nowadays, rDNA clusters displayed by fluorescence in situ hybridization (FISH) are one of the standard cytogenetic markers used to identify chromosome homology, allowing the determination of mechanisms and possible rate of rearrangements during karyotypic evolution of the studied groups. This bachelor thesis presents a review of the variability of rDNA clusters in arthropods with holocentric chromosomes. Most of the groups studied so far (Odonata, Psocoptera, Hemiptera, Lepidoptera, Buthidae and the genus Rhipicephalus) show a conservative number and position of rDNA clusters. However, this fact may be due to the limited amount of data available. Nowadays, best studied is order Lepidoptera, in which a relatively large variability in the number and position of rDNA clusters has been found. This is probably due to chromosome fusion/cleavage, chromosome rearrangements, or transposition of rDNA clusters. Key words: rDNA clusters, holocentric chromosomes, fluorescence in situ hybridization, arthropods, evolutionary dynamics, NOR
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Karyotype evolution of African clades of theraphosid mygalomorphs
Košátko, Prokop ; Král, Jiří (advisor) ; Nguyen, Petr (referee)
Karyotypes of mygalomorph spiders are not satisfactorily known. This thesis is focused on the basic cytogenetic analysis of selected species of African clades of theraphosid mygalomorphs. It includes four subfamilies: Eumenophorinae, Harpactirinae, Ischnocolinae and Stromatopelminae. Diploid numbers, chromosome morphology, sex chromosome systems and chromosome behaviour in male germline in the selected species of African theraphosid subfamilies were studied. The findings support published results, that refer of high karyotype diversity in Theraphosidae. Diploid chromosome number reduction is probably a basic trend of theraphosid karyotype evolution. The majority of analysed species exhibited one, two or three sex chromosomes. In some species neo-sex chromosome systems were found. In some species one or two sex chromosome pairs (SCP), composed of chromosomes which lack morphological differentiation were detected. Nucleolus organizer regions were detected by fluorescent in situ hybridization in several species. Constitutive heterochromatin detection was performed by C-banding in two species. Keywords: constitutive heterochromatin, diploid number, karyotype, fluorescence in situ hybridization, Mygalomorphae, nucleolus organizer region, SCP, sex chromosome, spider, Theraphosidae
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Influence of a composition of hybridization mixtures on fluorescence intensity during the in-situ hybridization
Janíček, Tomáš ; Kejík, Lukáš (referee) ; Vojtěch,, Hudzieczek (advisor)
Fluorescenční in situ hybridizace (FISH) je široce používaná metoda pro detekci určité sekvence DNA na chromozomech. Cílem práce je porovnání tří různých chemických sloučenin (formamidu, ethylenkarbonátu a sodných kationtů) používaných ve směsích pro in situ hybridizaci. Složení těchto směsí ovlivňuje renaturaci DNA a je důležité porovnat jejich fyzikální vlastnosti. Práce je rozdělena do dvou hlavních částí. První část se zabývá otázkou termodynamických parametrů používaných pro experimenty FISH, jako je teplota tání, entalpie přechodu DNA ze dvoušroubovice na vlákno nám dává přehled o energii potřebné k tomto přechodu a interakcích mezi bázemi a každou složkou směsi. Kromě toho hodnoty entropie určují poř uvnitř směsi - systém DNA. Druhá část porovnává intenzitu fluorescenčního signálu při optimalizovaných teplotách tání sondy použité pro in situ hybridizaci. Jako sonda byla použita sub-telomerní repetice X43.1, která je umístěna na Y chromozomu rostlinného modelového organismu Silene latifolia. Směs obsahující formamid má nejlepší výkon při delším postupu hybridizace, zatímco ethylenkarbonát poskytuje vyšší intenzitu signálu, a proto je vhodnější pro rychlé FISH protokoly.
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