Este estudo teve como objetivo identificar mecanismos pelos quais rearranjos cromossômicos aparentemente equilibrados possam estar associados de maneira causal a determinados quadros clínicos. Para isso estudamos seis translocações cromossômicas aparentemente equilibradas, detectadas em pacientes com malformações congênitas, comprometimento neuropsicomotor ou déficit intelectual. Os pontos de quebra desses rearranjos foram mapeados por hibridação in situ fluorescente (FISH). A busca por microdeleções e duplicações genômicas foi realizada por a-CGH. Estudamos duas translocações esporádicas, t(7;17)(p.13;q24) e t(17;20)(q24.3;q11.2), nas quais os pontos de quebra no cromossomo 17 foram localizados, respectivamente, a 917-855 kb e 624-585 kb upstream ao gene SOX9, em segmentos sem genes mapeados. Ambos os portadores apresentavam alterações esqueléticas que indicaram o diagnóstico de displasia campomélica acampomélica. Não foram detectados desequilíbrios cromossômicos submicroscópicos por a-CGH. Essas translocações podem levar à expressão alterada do gene SOX9, ao afetar a região reguladora desse gene. Sequências dos outros cromossomos participantes da translocação, que foram aproximadas ao gene pelo rearranjo, também podem ter afetado sua expressão. O estudo dos rearranjos t(7;17) e t(17;20) forneceu informação para o entendimento da região reguladora do gene. As manifestações clínicas associadas à t(17;20) permitiram redefinir o limite distal do cluster distal de rearranjos do cromossomo 17 associados ao espectro de manifestações clínicas do SOX9. A presença de testículo no portador dessa translocação indicou um elemento conservado candidato a atuar como enhancer do SOX9, para o desenvolvimento do testículo. Duas outras translocações equilibradas estavam associadas a desequilíbrios submicroscópicos em cis aos pontos de quebra. Caracterizamos uma t(10;21)(p13;q22) esporádica associada a atraso do desenvolvimento neuropsicomotor, microcefalia e espasticidade generaliza. Os pontos de quebra dos cromossomos 10 e 21, foram mapeados, respectivamente, em segmentos de 440 kb e 172 kb. Três genes estão mapeados no segmento que contém o ponto de quebra do cromossomo 10 e três outros, no intervalo delimitado para o ponto de quebra no cromossomo 21. O gene CDNF, que pode ter sido interrompido pelo ponto de quebra do cromossomo 10, é altamente expresso no sistema nervoso. A análise por meio de a-CGH detectou quatro deleções no cromossomo 10 todas de novo, indicando a complexidade do rearranjo. Duas deleções estavam próximas ao ponto de quebra: uma deleção de 973 kb em 10p14 e uma outra de 1,15 Mb em 10p13, mapeadas a 3,27 Mb e 210 kb do ponto de quebra da translocação, respectivamente. Outras duas deleções no cromossomo 10 ocorreram no braço longo: uma deleção de 700 kb em 10q26.13 estaria a 110,10 Mb do ponto de quebra da translocação, mas não conseguimos mapeá-la por FISH; uma outra deleção de 1,66 Mb em 10q26.2-q26.3 foi mapeada a 114,68 Mb do ponto de quebra da translocação. Quatorze genes estão localizados nas regiões das microdeleções. Os genes GPR26, OPTN, CUGBP2 são altamente expressos no sistema nervoso e, assim como o CNDF, podem ser considerados candidatos ao efeito fenotípico. O modelo de chromothripsis, em que o rearranjo resulta de uma série de quebras na dupla fita do DNA, seguida de ligação aleatória dos fragmentos resultantes, pode explicar a formação da translocação t(10;21). Aplicando a-CGH no estudo de uma translocação t(X;22)(q22;q13) esporádica, detectamos duplicações de 490 kb e 570 kb, respectivamente, em 22q13 e Xq22. A análise por FISH revelou que as cópias adicionais desses segmentos estavam localizadas nos pontos de quebra dos cromossomos derivativos X (segmento duplicado de 22q13) e 22 (segmento duplicado de Xq22). Não há genes mapeados no segmento duplicado do cromossomo 22. Um dos 14 genes duplicados no cromossomo X é o PLP1 (proteolipid protein 1), cujas mutações de ponto e duplicações causam a doença de Pelizaeus- Merzbacher, caracterizada pela hipomielinização do sistema nervoso central e afetando quase que exclusivamente indivíduos do sexo masculino. O exame neurológico, incluindo ressonância magnética, mostrou que o quadro clínico da paciente é compatível com o da doença de Pelizaeus-Merzbacher. A análise do padrão de inativação do cromossomo X em linfócitos de sangue periférico da paciente, com base na metilação do gene AR e também citologicamente em metáfases, após incorporação de 5-BrdU, revelou que, na maioria das células, o cromossomo X normal está inativo. Esse padrão de inativação torna as células funcionalmente equilibradas quanto aos segmentos translocados. O PLP1, entretanto, tem uma cópia adicional no cromossomo 22, além das cópias localizadas nos cromossomos X e der(X). Portanto, duas cópias ativas do gene estão presentes nas células da portadora da t(X;22). O mecanismo de formação de rearranjos cromossômicos baseado em bolhas de replicação explicaria a formação de translocações com duplicação em ambos os pontos de quebra, como ocorreu nessa t(X;22). Estudamos também uma aparente t(2;22)(p14;q12) familial que cossegregava com quadro de atraso do desenvolvimento neuropsicomotor e dificuldade de aprendizado associados a dismorfismos craniofaciais e alterações de mãos. A identificação de duplicações e deleções submicroscópicas, por meio de a-CGH e sua validação por FISH revelaram que se tratava, na verdade, de rearranjo, complexo entre três cromossomos 2, 5 e 22: um segmento de 1,2 Mb de 2p14 inseriu-se no braço curto do cromossomo 5, um evento que pode ter causado a deleção de um segmento de 1,4 Mb em 5p15.1; no cromossomo derivativo der(22) um segmento adicional de 5q23.2- 23.3 inseriu-se no ponto de quebra. Todos os afetados da família eram portadores do der(2) e do der(22). No entanto, o der(5) não segregava com o quadro clínico e foi detectado em um individuo fenotipicamente normal da família. Todos os afetados eram portadores da duplicação de 6,6 Mb do braço longo do cromossomo 5 (5q23.2-23.3). Os 17 genes duplicados são candidatos para o quadro clínico, por aumento da dosagem de seus produtos. Outra alteração comum a todos os afetados foi a haploinsuficiência do gene SLC1A4 mapeado em 2p14 e altamente expresso no sistema nervoso. É interessante que a deleção em 2p14, consequente à ausência do der(5), está restrita aos dois afetados que aparentam tem maior déficit cognitivo. Além do SLC1A4 , quatro genes mapeados nesse segmento CEP68, RAB1A, ACTR2 e SPRED2 podem contribuir para a variabilidade clínica dos afetados. A translocação t(2;5;22) pode ter-se originado a partir de duas quebras no braço curto do cromossomo 2, duas no braço curto e duas outras no braço longo do cromossomo 5 e uma quebra no braço longo do cromossomo 22. As quebras teriam ocorrido simultaneamente em um único evento. Após reunião de extremidades quebradas, formaram-se os cromossomos derivativos. Investigamos por a-CGH uma t(2;16)(q35;q24.1) esporádica cujos pontos de quebra foram mapeados anteriormente por FISH; nenhum gene estava mapeado nos segmentos que continham esses pontos de quebra. Não detectamos desequilíbrios cromossômicos submicroscópicos. A paciente portadora da translocação t(2;16) tinha quatro dígitos nas duas mãos e hexadactilia nos pés. A cerca de 1 Mb do ponto de quebra do cromossomo 2 está mapeado o gene IHH, que atua no desenvolvimento dos membros. A translocação pode ter interrompido elemento regulador do IHH ou separado o gene de elemento(s) regulador(es), levando à alteração de sua expressão e ao fenótipo. Este estudo fornece evidência adicional da importância da busca de desequilíbrios cromossômicos submicroscópicos em associação com rearranjos aparentemente equilibrados. Em três das seis translocações estudadas - t(10;21), t(2;22), t(X;22) - foram detectados desequilíbrios cromossômicos submicroscópicos em cis aos pontos de quebra, que podem ser responsáveis pelas manifestações clínicas dos portadores. Este estudo ressalta ainda a importância da técnica de FISH na análise dos desequilíbrios cromossômicos detectados por array, permitindo determinar a relação entre as perdas ou ganhos de segmentos submicroscópicos e os rearranjos equilibrados. A caracterização de rearranjos equilibrados neste estudo também contribuiu para sugerir mecanismos para sua formação / This study aimed at identifying mechanisms that lead to phenotypic abnormalities in carriers of balanced chromosomal rearrangements. We studied six apparently balanced chromosomal translocations detected in patients with congenital malformations, intellectual impairment or neuropsychomotor delay. Breakpoint mapping of apparently balanced chromosomal rearrangements was performed by fluorescence in situ hybridization (FISH), and cryptic genomic imbalances were investigated by array comparative genomic hybridization (a-CGH). We studied two sporadic translocations, t(7;17) (p13;q24) and t(17;20) (q24.3,q11.2). The breakpoints were located on chromosome 17, respectively, 917-855 kb and 624-585 kb upstream the SOX9 gene. There are no genes mapped to these segments. Patients had skeletal abnormalities that led to the diagnosis of acampomelic campomelic dysplasia. No submicroscopic chromosomal imbalances were detected by a-CGH. These translocations can alter gene expression by directly disrupting regulatory elements or by a position effect. The translocation t(7;17) and (17;20) provided additional information regarding the regulatory region of SOX9. The clinical manifestations associated with the translocation t(17;20) allowed the redefining of the limits of the distal breakpoint cluster of rearrangements on chromosome 17, which are associated with SOX9-related disorders. A conserved element was identified as a candidate SOX9 enhancer for testis development. Two additional sporadic translocations were associated with submicroscopic imbalances in cis to the breakpoints: t(10;21) and t(X;22). The translocation t(10;21)(p13;q22) was present in a girl with delayed motor development, microcephaly and generalized spasticity. The breakpoints on chromosomes 10 and 21 were mapped to 440 kb and 172 kb segments, respectively. Among the genes mapped to these breakpoint regions, only CDNF on chromossome 10, is highly expressed in the nervous system. Four de novo deletions on chromosome 10 were identified by a-CGH, revealing the complexity of the rearrangement. Two deletions were located at the vicinity of the translocation breakpoint: a 973 kb deletion on 10p14 and a 1.15 Mb deletion on 10p13 located, respectively, 3.27 Mb and 210 kb distal to the translocation breakpoint. Two other deletions were detected on the long arm of chromosome 10: a 700 kb deletion on 10q26.13, located 110.10 Mb distal to the translocation breakpoint, which we could not mapped by FISH; and a 1.66 Mb deletion on 10q26.2-q26.3, located 114.68 Mb distal to the translocation breakpoint. Fourteen genes are mapped to the microdeletion regions. Among these genes, GPR26, OPTN, CUGBP2 are highly expressed in the nervous system and, together with CNDF, are candidates for having clinical effects. The chromothripsis model, in which rearrangements result from a series of simultaneous double-stranded breaks followed by random joining of chromosomal fragments, might explain the formation of this t(10,21) translocation. Applying a-CGH to the apparently balanced translocation t(X;22)(q22;q13) carried by a girl, we detected duplicated segments on 22q13 and Xq22, encompassing 490 kb and 570 kb, respectively. FISH analysis revealed that the additional copies were located to the breakpoints of the derivative X chromosome (22q13 duplicated segment) and of the derivative 22 chromosome (Xq22 duplicated segment). No genes are mapped to the duplicated segment of chromosome 22. One of the 14 duplicated genes on the X chromosome is PLP1 (proteolipid protein 1). PLP1 point mutations and duplications cause Pelizaeus-Merzbacher disease, characterized by hypomyelination of the central nervous system, and affecting almost exclusively males. Neurological examination of the patient, including MRI showed that her clinical manifestations were compatible with Pelizaeus-Merzbacher disease. The pattern of X chromosome inactivation was determined in peripheral blood lymphocytes, based on the AR gene methylation, and cytologically, in metaphases spreads, after 5-BrdU incorporation, and showed that the normal X chromosome was the inactive one in the majority of cells. This pattern of X inactivation makes cells functionally balanced for the translocated segments. A copy of the PLP1 gene, however, is present on chromosome 22, in addition to the copies located on the chromosomes X and der(X). Thus, two active copies of the gene are present in the cells, irrespective of the X-inactivation pattern. A mechanism based on replication bubbles can explain the formation of translocations with duplication at the breakpoints, such as this t(X;22). An apparently balanced familial translocation t(2;22)(p13;q12.2) was detected in association with learning disability and craniofacial and hand dysmorphisms. The combination of a-CGH and FISH revealed that the rearrangement, identified by Gbanding as a two-break balanced translocation, was a more complex three-chromosome rearrangement: a segment from chromosome 2 was inserted into chromosome 5 short arm, an event that probably caused a 5p15.1 deletion; on chromosome 22 a segment from 5q23.2-23.3 was inserted into the breakpoint. Chromosomes der(2) and der(22) were present in all affected individuals. However, the der(5) did not segregate with the clinical phenotype, and was detected in a phenotypically normal individual. The 6.6 Mb duplication of the long arm of chromosome 5 was the imbalance common to all affected individuals. The 17 genes in this region are candidates for the clinical phenotypes through dosage effect. In addition, common to all affected individuals is the haploinsufficiency of SLC1A4, a gene highly expressed in the nervous system, which is encompassed by the deletion on chromosome 2. Interestingly, learning disabilities were more pronounced in those patients who also carried chromosome 2 deletion. CEP68, RAB1A, ACTR2 and SPRED2, mapped to this deleted segment, might contribute to the variability of the clinical phenotype in the family. The translocation t(2;5;22) might have originated from a series of simultaneously occurring brakes, two on the short arm of chromosome 2, four breaks on the short arm and two on the long arm of chromosome 5, and one break on the long arm of chromosome 22. We also investigated by a-CGH a sporadic translocation t(2;16)(q35;q24.1) whose carrier had hand and feet defects. Submicroscopic imbalances were not detected. Previously performed FISH delimited the breakpoints segments on chromosomes 2 and 16, which encompassed no genes. The IHH gene, which is involved in limb development, is located approximately 1 Mb upstream chromosome 2 breakpoint. Therefore, the translocation might have disrupted a regulatory element of IHH or, alternatively, separated the gene from a regulatory region, thus altering IHH expression. This study provides further evidence for the occurrence of submicroscopic chromosomal imbalances in association with apparently balanced rearrangements. In three out of six translocations - t(10,21), t(2;5;22), t(X;22) - cryptic duplications/deletions in cis to the breakpoints were detected, which might account for the clinical manifestations of the patients. This study also highlights the importance of FISH in the analysis of genomic imbalances detected by array in determining how losses and gains of submicroscopic segments relate to the rearranged chromosomes. The characterization of the balanced translocations in this study also contributed to suggest mechanisms for their formation

La déficience intellectuelle (DI) est une affection fréquente à causes multiples et souvent inconnues. Durant les 30 dernières années, l’examen utilisé pour l’exploration des anomalies chromosomiques chez des patients libanais présentant une DI était le caryotype standard. Le but de ce projet de thèse était d'appliquer pour la 1ère fois au Liban les technologies d'hybridation sur puces à ADN dans la recherche de nouveaux microremaniements (CNV) impliquant des gènes susceptibles d'engendrer une DI.Ainsi, les ADNs de 99 contrôles et de 185 patients libanais présentant une DI inexpliquée ont été hybridés sur des puces à ADN. Nous avons, par la suite, classé les CNVs identifiés en groupes selon leur transmission, leur contenu en gènes et leur localisation.Nous avons identifié 29 CNVs pathogènes associés à des syndromes ou gènes morbides responsables du phénotype recherché et 90 variants de signification inconnue dont 25 ont été investigués. On a retrouvé 18 de ces derniers comme probablement bénins, 5 comme probablement pathogènes, et 2 à investiguer puisqu'ils sont rapportés comme pathogènes dans la littérature mais hérités d’un parent sain dans l’étude. Nous avons élaboré 4 cas des CNVs pathogènes, 3 des CNVs probablement pathogènes qui sont des microdélétions à l'état homozygote chez des sujets issus de mariages consanguins; et les 2 CNVs à investiguer.Finalement, nous avons discuté les avantages de cette technique permettant l'identification chez 8% des patients ayant une DI inexpliquée, des microremaniements non identifiables par caryotype standard. Cependant nous avons aussi souligné la complexité, les limites et certaines incertitudes d’interprétation des résultats. / Chromosomal imbalances are the most frequent cause of intellectual disability (ID). In Lebanon, during the past 30 years, screening of these imbalances was done using standard karyotyping. However, the resolution of this test was insufficient to detect submicroscopic chromosomal imbalances (CNV). The aim of this thesis was to apply, for the first time in Lebanon, advanced techniques like the chromosomal microarray analysis (CMA), capable of detecting CNVs. Therefore, we screened the DNAs of 185 Lebanese subjects having unexplained ID and those of 99 healthy controls.CNVs identified were classified into groups upon their inheritance status, gene/microRNA content, and their localization.We identified 29 pathogenic CNVs associated to known syndromes or to morbid genes responsible for the patient's phenotype. We also found 90 variants of unknown significance of which 25 were investigated. 18 of the latter were likely benign, 5 were considered as probably pathogenic, and 2 needed future investigations to be classified, as they were considered as pathogenic in the literature but were inherited from a normal parent in this study.We discussed interesting cases by developing 4 pathogenic CNVs, 3 probably pathogenic that were homozygous microdeletions found in patients issued from consanguineous parents, and the 2 CNVs that required further investigations.Finally, we discussed the advantage of this CMA technique that lead to the identification of microimbalances unseen by a standard karyotype in 8% (14/174) of the patients with unexplained ID. Moreover, we mentioned the complexity, limitation and difficulty of interpretation of some results.

No description available.

Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure. First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin. Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.

In this thesis work, I have used a comparative genomics approach to study a fungal model organism, Neurospora tetrasperma. My specific focus has been on genomic introgression, intron evolution, chromosomal structural rearrangements and codon usage. All of the studies are based on large-scale dataset generated by next-generation sequencing technology (NGS), combined with other techniques, such as Optical Mapping. In the introgression study, we detected large-scale introgression tracts in three N. tetrasperma lineages, and the introgression showed allele-specific and chromosomal-specific pattern. In the study of introns, we found indications of mRNA mediated intron loss and non-homologous end joining (NHEJ) mediated intron gains in N. tetrasperma. We found that selection is involved in shaping intron gains and losses, and associated with intron position, intron phase and GC content. In the study of chromosomal structural rearrangements, we found a lineage specific chromosomal inversion pattern in N. tetrasperma, which indicates that inversions are unlikely to associate with the origin of the suppressed recombination and the mating system transition in N. tetrasperma. The result suggests inversions are the consequences, rather than the causes, of suppressed recombination on the mating-type chromosome of N. tetrasperma. In the final study, analyses of codon usage indicated that the region of suppressed recombination in N. tetrasperma is subjected to genomic degeneration, and selection efficiency has been much reduced in this region.

Toksino-antitoksino (TA) sistemos – tai poros viename operone esančių bakterijų ir archėjų genų, kurių vienas koduoja toksišką baltymą, o antras – jį neutralizuojantį baltymą-antitoksiną. Tol, kol ląstelėje gaminamas pakankamas abiejų baltymų kiekis, antitoksinas jungiasi su toksinu ir jį išaktyvina. Tačiau, esant nepalankioms aplinkos sąlygoms, labilesnis antitoksinas suardomas aktyvintų proteazių, o likęs laisvas stabilesnis toksinas slopina gyvybiškai svarbius ląstelinius procesus – baltymų arba DNR biosintezę, dėl ko stabdomas ląstelių augimas arba jos žūva. Escherichia coli chromosomoje aprašyta daugiau nei dešimt TA sistemų, kurių viena yra dinJ-yafQ, apie kurią žinoma labai nedaug. Anksčiau laboratorijoje atliktuose darbuose nustatyta, kad dinJ-yafQ koduoja transliaciją slopinantį toksiną YafQ, o DinJ ir YafQ baltymai sudaro stiprų baltymų kompleksą, slopinantį YafQ toksišką poveikį. Kol kas nieko nėra žinoma apie YafQ molekulės sritis, svarbias sąveikai su antitoksinu DinJ. Šiame darbe sekai atrankios mutagenezės metodu buvo tirtos YafQ baltymo sritys, svarbios sąveikai su „savuoju“ toksinu DinJ. TA sistemoms būdinga savo operono transkripcijos autoreguliacija. DNR sulėtinimo gelyje eksperimentais parodėme atrankią DNR ir antitoksino DinJ bei DinJ-YafQ baltymų komplekso sąveiką. Laisvas antitoksinas DinJ silpniau sąveikauja su DNR nei būdamas komplekse su YafQ, o sąveikai su DNR svarbi DinJ baltymo N galinė dalis. Iš dviejų dinJ-yafQ operono promotoriaus srityje... [toliau žr. visą tekstą] / Prokaryotic toxin antitoxin systems consist of two adjacent genes, where one encodes a stable toxin harmful to essential cellular processes (translation or DNA synthesis), and the other a labile antitoxin, capable of blocking the toxin's activity by binding into stable protein complex. TA systems are proposed to be involved in bacterial adaptation to stress conditions by modulating the level of essential biological processes. There are at least ten characterized chromosome-encoded TA loci in Escherichia coli. The dinJ-yafQ operon codes for YafQ toxin which is neutralized by its cognate antitoxin, DinJ. YafQ is known to inhibit translation in vivo and belongs to the RelE toxin family of toxin ribonucleases. By using site-specific mutagenesis of YafQ, we have investigated the protein regions important for its interaction with DinJ antitoxin. Transcriptional autoregulation has been reported for members of all known TA gene families and appears to be general characteristic of regulation of TA loci. In this work electrophoretic mobility shift assay was used to investigate the interaction between the antitoxin DinJ and DinJ-YafQ complex and dinJ-yafQ operon promoter DNA. Antitoxin DinJ in the complex with YafQ had an enhanced DNA-binding affinity compared to free DinJ. N-terminal domain of antitoxin is crucial for interaction with DNA. Bioinformatic analysis of dinJ-yafQ operon promoter region revealed several palindromic DNA islands and their importance for interaction with DinJ... [to full text]

The genetic structure of 49 natural populations of four species (P. attwateri, P. boylii, P. pectoralis, and P. polius) of the Peromyscus boylii species group was analyzed through application of chromosomal and electrophoretic techniques. Chromosomal variation within and among populations of the boylii species group was analyzed from 178 specimens. Electrophoretic techniques were utilized for the demonstration of variation in enzymes and other proteins encoded by structural loci and applied to the study of the evolution of the boylii species group by estimation of levels of genetic heterozygosity within populations, estimation of degree of genetic similarity between conspecific populations and between species, and determination of patterns of geographic variation in allelic frequencies and levels of heterozygosity. Six distinct chromosomal patterns were observed among the populations of the four species of the boylii species group. All specimens had a diploid number of 48 and the major difference in chromosomal morphology was in the number of pairs of large to medium biarmed autosomes. Little or no chromosomal variation was observed in three species (attwateri, pectoralis and polius), but considerable chromosomal variation occurred among populations of P. boylii. Generally, the chromosomal variation in P. bylii was between allopatric populations, with each chromosomal pattern limited to a recognized subspecies. Polymorphism was observed in two populations. The polymorphism observed in P. polius was the result of pericentric inversion involving the smallest pair of metacentric autosomes. The polymorphism observed in P. bolii cileus was interpreted at the result of gene flow between P. boylii rowleyi and P. boylii spicilegus. In addition to chromosomal evidence, analysis of electrophoretic data demonstrated and suggested effective gene flow between the chromosomal forms of P. boylii. Electrophoretically demonstrable variation was analyzed in 11 proteins encoded by 17 autosomal loci. Of the 17 structural loci, 11 were polymorphic in one or more populations . No more than five loci were observed to be polymorphic within a single population, with a mean number of polymorphic loci of 2.26. The levels of genetic variability, as measured by the proportion of loci in a heterozygous state in the average individual of a population, for populations of the boylii species group were within the range reported for other rodents. Populations of P. attwateri exhibited levels of heterozygosity considerably lower than those reported for other species of Peromyscus. The low level of genetic variability in P. attwateri was probably the result of founder effect of the original population which was isolated on the Edwards Plateau during the late Pleistocene and subsequent genetic drift which allowed fixation of genes within this isolated population. Geographic variation in levels of heterozygosity observed among populations of P. attwateri and P. boylii indicates a north to south cline of increasing heterozygosity with the most variable populations occurring in the southern portion of the range of each species. A wide range of the levels of heterozygosity and genetic similarity was observed among populations of P. pectoralis and is the result of genetic contribution from three Pleistocene refugia. Analysis of paired combinations of populations of P. boyli indicated that the various chromosomal forms of P. boylii. form a conspecific unit. Populations of P. attwateri were genetically distinct from other members of this group. Examination of specific loci for allelic homology suggested that P. attwateri arose from genetic divergence of a population of P. boylii isolated on the Edwards Plateau during the late Pleistocene.

Foetal anomalies found on ultrasound increase the probability of occurrence of chromosomal abnormalities. They cause about one quarter of all abortions and stillbirths and many of inborn defects in newborns. Karyotype analysis is number one method in prenatal diagnosis whereas array CGH is often used as a verification and supplemental method. The aim of this work was to prove that array CGH gives additional chromosomal findings to karyotypes and could substitute conventional karyotyping as a primary examination method in foetuses with ultrasound findings. We examined 45 prenatal samples using both methods. These samples were referred for invasive examination because of abnormal ultrasound findings. Karyotype analyses found two abnormalities in two (4,4 %) patients and array CGH identified aberrations in five (11,1 %) foetuses whereas both anomalies detected by karyotypes were discovered by array CGH too. This means that array CGH identified about 6,7 % more aberrations than karyotype. Our results correspond with scientific articles which refer that array CGH should replace karyotype not only in postnatal examinations but even in prenatal diagnosis. Keywords: chromosomal aberrations, array CGH, karyotype, prenatal diagnosis, ultrasound

Brain gliomas represent a heterogeneous group of tumors of various histological subtypes which differ according to their response to treatment and prognosis. Tumors created from astrocytes and oligodendrocytes occur most often. Histological classification of gliomas is often subjective, as well as their treatment today is still problematic. The aim of this diploma thesis was to carry out a detailed molecular cytogenetic analysis of the genome of tumor cells in patients with histologically confirmed brain gliomas of different subtypes and stages of malignancy, look for recurrent aberration-specific subtypes and assess their potential role in the development and progression of cancer. To observation specific frequency known aberrations in different subtypes of brain tumors, we used the method of interphase FISH (I-FISH) with a panel of specific locus and / or centromeric DNA probes. The whole genome analysis and detection of cryptic unbalanced changes in the genome of tumor cells, we used the method of SNP array. Combining methods I- FISH and SNP array was detected not only the known chromosomal changes that are typical of the different subtypes of tumors, but also new or uncommon recurrent aberrations. In patients with low-grade gliomas are the most commonly observed acquired UPD (aUPD) on the short...

Student Number : 0419128X - MSc Dissertation - School of Animal, Plant and Environmental Sciences - Faculty of Science / Reproductive isolation was investigated in two chromosomally distinct populations of Rhabdomys on the Gauteng highveld. The two populations, Midrand (2n = 48) and Irene (2n = 46), occur 15 km apart, with no known contact or hybrid zone between them. Behavioural experiments, comprising male-female dyadic encounters and female preference tests for same-and different-population male odour, were used to test for premating barriers. Aggression levels were highest in different-population than same-population dyads, and females spent more time with odours of males from their own population than of those of the other population. Breeding and postnatal development studies were conducted to establish postmating barriers. Compared to different-population pairs, reproductive success was markedly reduced in different-population pairings, and the few hybrids that were produced did not breed. My studies indicate that behavioural isolation is well-developed between the Midrand and Irene striped mice, and suggest that the mate recognition system has diverged in allopatry, which would reduce gene flow between the two populations. Such divergence supports the findings of mtDNA studies by other workers who proposed that the two chromosomal forms used in my study represent two subspecies of R. dilectus. Previous studies showed that distant striped mice populations (>900km) displayed behavioural divergence and intermediately located populations (~80km) were behavioural compatible but had hybrid failure; the Irene population was used in both studies. In comparison, the behavioural incompatibility between the closely-located Midrand and Irene populations provides support for the reinforcement of previous postmating isolation seen in the intermediately located populations, particularly since no contact or hybrid zone exists between the two forms. However, I cannot rule out other explanations, such as dissimilar ecological conditions, influencing interfertility.