The patchwork pattern of rodent genomes: species-specific organization of orthologous DNA sequences
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2016
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Comparar, acto tão comum no nosso quotidiano, sempre assumiu uma dimensão
primordial na área das ciências da vida, como a biologia e a medicina. O estudo da
evolução dos genomas e espécies baseia-se em vários tipos de comparação, desde as
morfológicas às paleontológicas, a comparações moleculares das sequências de DNA e
RNA, de proteínas ou dos cromossomas.
A tese está dividida em seis capítulos, sendo o primeiro o capítulo da introdução.
O segundo capítulo desta tese apresenta a análise das histórias evolutivas dos genomas de
dois roedores atuais, Cricetus cricetus (Cricetidae, Cricetinae) e Peromyscus eremicus
(Cricetidae, Neotominae). Para tal, os dois genomas foram comparados com um genoma
referência, um genoma ancestral comum, utilizando três abordagens moleculares
conjugadas: “Comparative Chromosome Painting”; sequenciação dos genomas de Mus
musculus e de Rattus norvegicus (dados disponíveis na base de dados Ensembl); e análise
das regiões de heterocromatina constitutiva nas espécies Cricetus cricetus e Peromyscus
eremicus. Verificou-se que: (a) a espécie Peromyscus eremicus apresenta um genoma
muito conservado relativamente ao cariótipo ancestral comum, enquanto que o genoma
de Cricetus cricetus divergiu significativamente do genoma de referência; (b) os
rearranjos cromossómicos evolutivos mais comuns nestas espécies foram fusões, fissões e
inversões, associados a alterações no padrão da heterocromatina; e (c) as regiões de
quebra evolutivas colocalizam-se preferencialmente com regiões de heterocromatina
constitutiva, sugerindo a importância destas regiões na ocorrência dos rearranjos
evolutivos.
No terceiro capítulo foram estudados os genomas dos roedores Phodopus
roborovskii e Phodopus sungorus (Cricetidae, Cricetinae) quanto à localização da
heterocromatina constitutiva e de sequências teloméricas intersticiais. Os resultados
permitiram relacionar estas regiões/sequências com a evolução dos genomas destas
espécies.
Há ainda muitas lacunas no conhecimento da constituição das regiões de
heterocromatina constitutiva, já que são as regiões genómicas mais difíceis de sequenciar
e de analisar com as técnicas disponíveis. Sabe-se que são essencialmente constituídas por sequências repetitivas, sobretudo por sequências de DNA satélite, mas que estas não são as únicas sequências presentes nessas regiões. No quarto capítulo isso ficou
demonstrado no genoma de seis espécies de roedores: Tatera gambiana (Muridae,
Gerbillinae), Acomys sp. (Muridae, Deomyinae), Cricetomys sp. (Nesomyidae,
Cricetomyinae), Microtus arvalis (Cricetidae, Arvicolinae), Phodopus roborovskii e
Phodopus sungorus (Cricetidae, Cricetinae). Neste trabalho verificou-se que as
sequências repetitivas LINE-1 (L1) se localizam quer em regiões eucromáticas, quer em
regiões heterocromáticas. Este estudo também concluiu que a localização das sequências
L1 é específica da espécie.
As sequências repetitivas mais comuns nas regiões de heterocromatina
constitutiva – DNA satélite – são sequências ainda pouco estudadas, e até negligenciadas,
persistindo muitas incertezas quanto à sua organização, função e evolução. No trabalho
que deu origem ao quinto capítulo, foi isolada pela primeira vez uma família de DNA
satélite no genoma de Peromyscus eremicus – PMSat –, tendo-se verificado que se
encontra nas regiões pericentroméricas e nos braços curtos heterocromáticos desta espécie.
Esta sequência foi analisada em espécies aparentadas, tendo-se verificado a sua presença
nessas espécies de roedores. Estes resultados indicam que a família de DNA satélite
PMSat já se encontrava num ancestral comum. Este conhecimento possibilitou uma
“datação” da sequência PMSat e sugestões quanto à forma como terá decorrido a sua
evolução.
Em resumo, esta tese focou-se no estudo das regiões de heterocromatina
constitutiva, a dois níveis: perceber qual o envolvimento das regiões de heterocromatina
constitutiva na evolução dos genomas e conhecer a sua composição molecular, ou seja, quais as sequências que as constituem. O sexto capítulo consiste na discussão geral dos resultados obtidos e no esboço de algumas sugestões para estudos futuros.
Comparison is an activity that we frequently perform in our daily lives. It is also an activity that has always had a prime role in life sciences such as biology and medicine. The study of the evolution of species is based on several kinds of comparison: morphological comparison, paleontological comparison and molecular comparison of DNA sequences, RNA sequences, proteins and chromosomes across genomes and ultimately species. The thesis is divided in six chapters, where the first is an introductory chapter. The second chapter of this thesis presents our analysis of the evolutionary histories of the genomes of two current rodent species, Cricetus cricetus (Cricetidae, Cricetinae) and Peromyscus eremicus (Cricetidae, Neotominae). We compared both genomes with a reference genome, a common ancestral genome, and used three molecular approaches in combination: “Comparative Chromosome Painting”; sequencing data of the genomes of Mus musculus and Rattus norvegicus (both available in the Ensembl database); and constitutive heterochromatin analysis performed in Cricetus cricetus and Peromyscus eremicus. It was found that: (a) the Peromyscus eremicus species has a very conserved genome when compared with the common ancestor karyotype, while the Cricetus cricetus genome differs significantly from the reference genome; (b) the most common evolutionary chromosome rearrangements in these species were fusions, fissions and inversions, associated with changes in the pattern of heterochromatin; and (c) the evolutionary breakpoint regions are preferentially collocated with regions of constitutive heterochromatin, suggesting the importance of these regions in the occurrence of evolutionary rearrangements. The third chapter of this thesis describes our study of the genomes of the rodents Phodopus roborovskii and Phodopus sungorus (Cricetidae, Cricetinae) with regard to the location of constitutive heterochromatin and interstitial telomeric sequences. The results allowed us to relate these regions and sequences with the evolution of the genomes of these species. There are still many gaps in our understanding of the establishment of the regions of constitutive heterochromatin, as these regions are some of the most difficult to sequence and analyze with the available techniques. It is known that they consist essentially of repetitive sequences, most of which are satellite DNA sequences, but other types of sequences are present in these regions. The fourth chapter reports the study of transposable elements in the genome of six species of rodents: Tatera gambiana (Muridae, Gerbillinae), Acomys sp. (Muridae, Deomyinae), Cricetomys sp. (Nesomyidae, Cricetomyinae), Microtus arvalis (Cricetidae, Arvicolinae), Phodopus roborovskii and Phodopus sungorus (Cricetidae, Cricetinae). This study showed that LINE-1 (L1) repetitive sequences are located in the euchromatic and heterochromatic regions, and that the location of these sequences is species-specific. In spite of satellite DNA being the most common type of repetitive sequence found in constitutive heterochromatin, its study has been neglected, and many uncertainties remain regarding its organization, function and evolution. Chapter five reports the first isolation ever of the PMSat family of satellite DNA in the genome of the cactus mouse Peromyscus eremicus. We verified that this satellite DNA family is mainly located in the pericentromeric regions and in the heterochromatic p-arms of the cactus mouse genome. This sequence was analyzed and detected in the genome of related rodent species, which indicates that PMSat was already present in a common ancestor. This discovery allowed us to date the PMSat sequence and indicates how its evolution proceeded. In summary, this thesis focused on the study of the regions of constitutive heterochromatin, on two levels: how are they involved in the evolution of genomes and what is their constitution, i.e. which sequences do they harbour. Chapter six consists of a general discussion of the collected results and an outline of a few suggestions for further study.
Comparison is an activity that we frequently perform in our daily lives. It is also an activity that has always had a prime role in life sciences such as biology and medicine. The study of the evolution of species is based on several kinds of comparison: morphological comparison, paleontological comparison and molecular comparison of DNA sequences, RNA sequences, proteins and chromosomes across genomes and ultimately species. The thesis is divided in six chapters, where the first is an introductory chapter. The second chapter of this thesis presents our analysis of the evolutionary histories of the genomes of two current rodent species, Cricetus cricetus (Cricetidae, Cricetinae) and Peromyscus eremicus (Cricetidae, Neotominae). We compared both genomes with a reference genome, a common ancestral genome, and used three molecular approaches in combination: “Comparative Chromosome Painting”; sequencing data of the genomes of Mus musculus and Rattus norvegicus (both available in the Ensembl database); and constitutive heterochromatin analysis performed in Cricetus cricetus and Peromyscus eremicus. It was found that: (a) the Peromyscus eremicus species has a very conserved genome when compared with the common ancestor karyotype, while the Cricetus cricetus genome differs significantly from the reference genome; (b) the most common evolutionary chromosome rearrangements in these species were fusions, fissions and inversions, associated with changes in the pattern of heterochromatin; and (c) the evolutionary breakpoint regions are preferentially collocated with regions of constitutive heterochromatin, suggesting the importance of these regions in the occurrence of evolutionary rearrangements. The third chapter of this thesis describes our study of the genomes of the rodents Phodopus roborovskii and Phodopus sungorus (Cricetidae, Cricetinae) with regard to the location of constitutive heterochromatin and interstitial telomeric sequences. The results allowed us to relate these regions and sequences with the evolution of the genomes of these species. There are still many gaps in our understanding of the establishment of the regions of constitutive heterochromatin, as these regions are some of the most difficult to sequence and analyze with the available techniques. It is known that they consist essentially of repetitive sequences, most of which are satellite DNA sequences, but other types of sequences are present in these regions. The fourth chapter reports the study of transposable elements in the genome of six species of rodents: Tatera gambiana (Muridae, Gerbillinae), Acomys sp. (Muridae, Deomyinae), Cricetomys sp. (Nesomyidae, Cricetomyinae), Microtus arvalis (Cricetidae, Arvicolinae), Phodopus roborovskii and Phodopus sungorus (Cricetidae, Cricetinae). This study showed that LINE-1 (L1) repetitive sequences are located in the euchromatic and heterochromatic regions, and that the location of these sequences is species-specific. In spite of satellite DNA being the most common type of repetitive sequence found in constitutive heterochromatin, its study has been neglected, and many uncertainties remain regarding its organization, function and evolution. Chapter five reports the first isolation ever of the PMSat family of satellite DNA in the genome of the cactus mouse Peromyscus eremicus. We verified that this satellite DNA family is mainly located in the pericentromeric regions and in the heterochromatic p-arms of the cactus mouse genome. This sequence was analyzed and detected in the genome of related rodent species, which indicates that PMSat was already present in a common ancestor. This discovery allowed us to date the PMSat sequence and indicates how its evolution proceeded. In summary, this thesis focused on the study of the regions of constitutive heterochromatin, on two levels: how are they involved in the evolution of genomes and what is their constitution, i.e. which sequences do they harbour. Chapter six consists of a general discussion of the collected results and an outline of a few suggestions for further study.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Evolução molecular , Genoma , Heterocromatina , DNA satélite , Genética evolutiva , Ratazana , Modelo animal , Rearranjos cromossómicos