Organização da fracção repetitiva nos genomas de Cricetus cricetus, Peromyscus eremicus (Família Cricetidae) e Praomys tullbergi (Família Muridae). Considerações sobre a evolução do cromossoma X
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2007
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O aumento do nosso conhecimento sobre a natureza molecular da heterocromatina constitutiva, permite defini-la como um importante componente genómico, com funções ao nível da estrutura e regulação do genoma (Dimitri et al. 2004), para além de se considerar ainda o seu envolvimento na evolução de cariótipos. É actualmente aceite que a sua presença pode facilitar a ocorrência de rearranjos cromossómicos, considerando-se mesmo as regiões ricas nestas sequências de DNA como “hotspots” de reorganização cromossómica (Yunis e Yasmineh 1971, Peacock et al. 1982, John 1988, Chaves et al. 2004).
No presente trabalho caracterizou-se detalhadamente a heterocromatina constitutiva dos cromossomas de três espécies de roedores, Cricetus cricetus, Peromyscus eremicus (Família Cricetidae), e Praomys tullbergi (Família Muridae), recorrendo-se à metodologia de restrição in situ (com um painel de sete enzimas de restrição) e bandeamento-C sequencial. Este trabalho permitiu detectar uma elevada heterogeneidade molecular nas sequências de heterocromatina constitutiva dos três genomas, provando ser uma ferramenta útil no estudo destas sequências. A comparação geral da quantidade, distribuição e natureza molecular da heterocromatina constitutiva das três espécies em análise, sugere a ocorrência de diferentes percursos evolutivos envolvidos na origem destes cariótipos. Na espécie C. cricetus, que apresenta um cariótipo quase totalmente meta/submetacêntrico, a heterocromatina constitutiva parece estar preferencialmente localizada em regiões (peri)centroméricas, apresentando a maioria dos autossomas dois blocos de heterocromatina constitutiva, o que sugere a ocorrência de, pelo menos, translocações Robertsonianas durante a evolução deste cariótipo. A espécie P. eremicus, com um cariótipo constituído apenas por cromossomas submetacêntricos, apresenta também grandes quantidades de heterocromatina constitutiva essencialmente localizada nas regiões (peri)centroméricas, sendo inclusivamente o braço curto da maioria dos cromossomas completamente heterocromático. A espécie P. tullbergi, com um complemento autossómico acrocêntrico, é das três espécies estudadas aquela cujos cromossomas exibem menos heterocromatina constitutiva centromérica. Em alguns cromossomas, a heterocromatina intersticial é quase tão abundante como a centromérica, contrariamente ao observado para a maioria dos cromossomas das outras duas espécies analisadas. Esta diferente distribuição e o elevado número de subclasses de heterocromatina constitutiva identificado nos cromossomas de P. tullbergi (52 subclasses), sugere que este cariótipo é o mais derivativo dos analisados neste trabalho, tendo sido originado pela ocorrência de um elevado número de complexos rearranjos cromossómicos. A elevada heterogeneidade das sequências de heterocromatina constitutiva identificada nos três genomas analisados, sugere a coexistência de diferentes famílias de DNA satélite, ou variantes destas famílias nestes genomas.
A análise in silico das sequências LINE-1 (“Long Interspersed Elements-1”) permitiu estabelecer relações filogenéticas entre espécies, já que são caracteres não homoplásicos (Verneau et al. 1998, Waters et al. 2007). O alinhamento das sequências LINE-1 de C. cricetus, P. eremicus, P. tullbergi (isoladas pela primeira vez neste trabalho), M. musculus, R. norvegicus e de 10 espécies pertencentes à família Cricetidae, disponíveis na base de dados “NCBI Nucleotide”, demonstrou que todas as sequências correspondem a uma determinada região da ORFII da sequência LINE-1 das espécies índex M. musculus e R. norvegicus. Sugere-se assim que esta região da sequência está conservada nas várias espécies de roedores, observando-se valores de similaridade entre sequências acima dos 75%. O alinhamento de todas as sequências permitiu construir um dendograma que relaciona filogeneticamente todas as espécies analisadas, comprovando-se uma maior proximidade filogenética entre os roedores pertencentes à mesma família
O mapeamento físico das sequências LINE-1 de C. cricetus, P. eremicus e P. tullbergi, nos respectivos cromossomas, demonstrou que estas sequências se encontram “interspersed” ao longo de todos os cromossomas, não se verificando uma localização preferencial destas sequências no cromossoma X. Estes resultados não suportam a hipótese de Lyon (1998), que sugere o envolvimento dos sequências LINE-1 na inactivação do cromossoma X, ao observar uma localização preferencial destas sequências no cromossoma X do Homem e rato. Duas hipóteses podem ser formuladas para justificar os resultados obtidos. Pode ter ocorrido perda de sequências LINE-1 no cromossoma X das três espécies no decorrer da evolução, sendo que este acontecimento foi anteriormente sugerido para outras espécies de roedores (Casavant et al. 2000), embora neste último caso, a perda destas sequências seja relativa a todo o complemento cromossómico. Outro cenário possível é a existência de diferentes mecanismos de inactivação do cromossoma X (pelo menos nestes roedores) sem que haja participação das sequências LINE-1, e neste caso, o genoma não precisaria de conservar estas sequências maioritariamente localizadas no cromossoma X, observando-se alternativamente uma distribuição indiferenciada por todo o complemento cromossómico
A realização de “Comparative Chromosome Painting”, com uma sonda representativa do cromossoma X de R. norvegicus (RNOX) em preparações cromossómicas das três espécies, revelou três segmentos sinténicos num dos braços do cromossoma X de C. cricetus, oito segmentos sinténicos no cromossoma X de P. eremicus e seis segmentos sinténicos no braço longo do cromossoma X de P. tullbergi. Esta análise revelou ainda a ocorrência de uma inversão pericêntrica durante a evolução do cromossoma X de P. eremicus. A caracterização da heterocromatina constitutiva realizada durante este trabalho, revelou a existência de bandas-C a flanquear alguns segmentos sinténicos discriminados pelo “paint” RNOX no cromossoma X das três espécies analisadas. Estes resultados parecem assim sugerir a participação das sequências de heterocromatina na ocorrência dos rearranjos cromossómicos, que originaram estes cromossomas, evidenciada pela presença de sequências repetitivas nas regiões de “breakpoints” evolutivos. Ainda no âmbito deste trabalho, realizou-se uma análise in silico entre a distribuição cromossómica de sequências repetitivas em tandem e a localização de regiões de “breakpoints” evolutivos no cromossoma X da espécie índex M. musculus. Nesta análise recorreu-se às sintenias entre cromossomas do M. musculus, R. norvegicus e H. sapiens, para a identificação de regiões de “breakpoints” evolutivos no cromossoma X de M. musculus, e a ferramentas bioinformáticas que permitiram a avaliação da densidade de sequências repetitivas em tandem neste cromossoma. Resultados preliminares desta análise demonstram que as regiões que apresentam maior densidade de sequências repetitivas em tandem, correspondem de facto, a regiões de “breakpoints” evolutivos, o que vem comprovar o importante papel destas sequências na reorganização da arquitectura dos genomas ao longo da evolução.
Quando se compararam os cromossomas X de C. cricetus, P. eremicus e P. tullbergi, observaram-se diferenças ao nível da sua morfologia, e na quantidade, localização e natureza molecular da sua heterocromatina, sugerindo que se originaram através da ocorrência de rearranjos cromossómicos como inversões, transposições centroméricas, adições/eliminações de heterocromatina e translocações Robertsonianas, como é o caso do cromossoma X de C. cricetus, que apresenta dois blocos de heterocromatina constitutiva na região (peri)centromérica. O maior número de subclasses de heterocromatina constitutiva identificado no cromossoma X de P. tullbergi, comparativamente às diferentes espécies em análise, sugere que o cromossoma X desta espécie é o mais rearranjado. No entanto, a análise dos segmentos sinténicos no cromossoma X das três espécies de roedores, com a sonda de “painting” RNOX, sugere o cromossoma X de P. eremicus como sendo o mais rearranjado. Esta aparente discordância deve-se provavelmente às diferentes análises efectuadas, nomeadamente, identificação de subclasses de heterocromatina e de segmentos sinténicos entre cromossomas de diferentes espécies, sendo indispensável uma investigação mais detalhada, de modo a clarificar efectivamente os mecanismos subjacentes à evolução destes cromossomas
Futuros trabalhos que visem o estudo e caracterização de sequências de heterocromatina constitutiva, nomeadamente sequências de DNA satélite, isoladas por exemplo, por microdissecção de regiões centroméricas de cromossomas, e a construção de mapas comparativos para diferentes espécies de roedores, irão certamente contribuir para um melhor entendimento dos processos evolutivos que moldaram estes genomas e das relações filogenéticas entre espécies.
The increase of our knowledge concerning the molecular nature of constitutive heterochromatin is now allowing to define these sequences as important genomic components, with functions in the structure and regulation of the genome (Dimitri et al. 2004), besides its involvement in karyotype’s evolution. Nowadays it is accepted that the presence of constitutive heterochromatin can facilitate the occurrence of chromosome rearrangements, being the rich regions in these DNA sequences considered as hotspots for chromosome reshuffling (Yunis and Yasmineh 1971, Peacock et al. 1982, John 1988, Keys et al. 2004) In the present work it was described in detail the constitutive heterochromatin of three rodent species chromosomes, Cricetus cricetus, Peromyscus eremicus (Cricetidae Family), and Praomys tullbergi (Muridae Family), using in situ endonuclease restriction (with a panel of seven restriction enzymes) and sequential C-banding. This work allowed to detect a high molecular nature of the constitutive heterochromatin in the three genomes, demonstrating to be an useful tool in the study of these sequences. The general comparison of the amount, distribution and molecular nature of the constitutive heterochromatin in the three species analyzed, suggests the occurrence of different evolutionary pathways in the origin of these karyotypes. In the species C. cricetus, which presents a nearly meta/submetacentric karyotype, the constitutive heterochromatin seems to be preferentially located in (peri)centromeric regions, presenting the majority of the autosomes two constitutive heterochromatin blocks, what suggests the occurrence, of at least, Robertsonian translocations during the course of these karyotypes evolution. The species P. eremicus, with a submetacentric karyotype, also presents great amounts of constitutive heterochromatin, essentially located in (peri)centromeric regions, being inclusively the short arms of the majority of the chromosomes completely heterochromatic. The species P. tullbergi, with an acrocentric autosome complement, is the one whose chromosomes exhibit less centromeric constitutive heterochromatin. In some chromosomes, interstitial heterochromatin is almost as abundant as the centromeric heterochromatin, in opposition to the observed for most chromosomes of the other two analyzed species. This different distribution patterns and the large number of constitutive heterochromatin subclasses identified in the P. tullbergi chromosomes (52 subclasses), suggest that this species has a more derivative karyotype than the other two genomes analyzed, originated by a great number of complex chromosomal rearrangements. The high heterogeneity of constitutive heterochromatin sequences identified, suggests the coexistence of different DNA satellite families, or variants of these families in these three analyzed genomes The in silico analysis of LINE-1 sequences (Long Interspersed Elements-1) allowed to establish phylogenetic relationships among species, since they are homoplasy-free characters (Verneau et al. 1998, Waters et al. 2007). The comparison among LINE-1 sequences from C. cricetus, P. eremicus, P. tullbergi (isolated for the first time in this work), M. musculus, R. norvegicus, and of 10 more species from the Cricetidae Family (available in the NCBI Nucleotide database), demonstrated that all the sequences are similar to a certain region of ORFII LINE-1 sequence of the índex species M. musculus and R. norvegicus. In this way, it is suggested that this region of the sequence is conserved in the rodent species analyzed, presenting similarity values above 75% among the sequences. The alignment of all the sequences allowed obtaining a dendogram that relates phylogenetically the analyzed species, specifically it was observed a larger phylogenetic proximity among rodents belonging to the same family. The physical mapping of C. cricetus, P. eremicus and P. tullbergi LINE-1 sequences, in the respective chromosomes, demonstrated that these sequences are interspersed along all the chromosomes, not presenting any preferential location in the X chromosome. These results do not support the Lyon hypothesis (1998), which suggests the involvement of LINE-1 sequences in the X chromosome inactivation, after observing a preferential location of these sequences in man and mouse X chromosomes. Two hypotheses can thus be drawn to justify the obtained results. It might have happened loss of LINE-1 sequences in the X chromosome of the three species during the evolution course, being this event previously suggested for other rodent species by Casavant et al. (2000), although in this case, the loss of these sequences was considered for the whole chromosome complement. Another possible scenario is the existence of different mechanisms of X chromosome inactivation (at least in these rodents), without the participation of LINE-1 sequences, and in this case, the genome wouldn’t need to conserve these sequences highly located in the X chromosome, being alternatively observed an undifferentiated distribution by the whole chromosome complement. Comparative Chromosome Painting using a R. norvegicus X chromosome (RNOX) specific probe in chromosome preparations of the three species, revealed three syntenic segments in one arm of C. cricetus X chromosome, eight syntenic segments in P. eremicus X chromosome and six syntenic segments in the long arm of P. tullbergi X chromosome. This analysis also revealed the occurrence of a pericentric inversion during the evolution course of P eremicus X chromosome. The characterization of the constitutive heterochromatin performed during this work revealed the existence of C-bands to flanking some syntenic segments, discriminated by the paint RNOX in the X chromosome of the three species analyzed. These results seem to suggest the participation of heterochromatin sequences in the chromosome rearrangements that originated these chromosomes, evidenced by the presence of repetitive sequences at the evolutionary breakpoints regions of these species. Moreover, in this work was performed an in silico analysis among tandem repeats chromosome distribution and the location of evolutionary breakpoints regions in the X chromosome of the índex species M. musculus. In this analysis, the chromosome syntenic regions of M. musculus, R. norvegicus and H- sapiens were used for identification of the breakpoints chromosome regions of M. musculus X chromosome, and bioinformatic tools that allowed the evaluation of tandem repeats density in this chromosome. Preliminary results demonstrate that the regions presenting high accumulation of tandem repeat sequences correspond to evolutionary breakpoints regions, support the important role of these sequences in the reorganization of genome’s architecture along evolution. The X chromosome of C. cricetus, P. eremicus and P. tullbergi show differences in respect to morphology, amount, location and molecular nature of its heterochromatin, suggesting that they arose through the occurrence of different chromosome rearrangements, as inversions, centromeric transpositions, additions/eliminations of heterochromatin and Robertsonian translocations, as is the case of C. cricetus X chromosome, that presents two blocks of constitutive heterochromatin at the (peri)centromeric region. The high number of constitutive heterochromatin subclasses identified in the X chromosome of P. tullbergi, in comparison with the other two analyzed species, suggests that the X chromosome of this species is highly rearranged. However, the analysis of the syntenic segments in the X chromosomes with the painting probe RNOX, suggests P. eremicus X chromosome as the more rearranged. This apparent disagreement is probably the result of different analyses, namely the identification of heterochromatin subclasses and syntenic segments among the chromosomes of different species, requiring a more detailed investigation to clarify the mechanisms underlying the evolution of these chromosomes. Future detailed characterization of constitutive heterochromatin sequences, namely satellite DNA sequences, isolated for exemple by microdissection of chromosome centromeric regions, and the construction of comparative maps for different species of rodents, will certainly contribute to a better understanding of the evolutionary pathways that shaped these genomes and the phylogenetic relationships among species.
The increase of our knowledge concerning the molecular nature of constitutive heterochromatin is now allowing to define these sequences as important genomic components, with functions in the structure and regulation of the genome (Dimitri et al. 2004), besides its involvement in karyotype’s evolution. Nowadays it is accepted that the presence of constitutive heterochromatin can facilitate the occurrence of chromosome rearrangements, being the rich regions in these DNA sequences considered as hotspots for chromosome reshuffling (Yunis and Yasmineh 1971, Peacock et al. 1982, John 1988, Keys et al. 2004) In the present work it was described in detail the constitutive heterochromatin of three rodent species chromosomes, Cricetus cricetus, Peromyscus eremicus (Cricetidae Family), and Praomys tullbergi (Muridae Family), using in situ endonuclease restriction (with a panel of seven restriction enzymes) and sequential C-banding. This work allowed to detect a high molecular nature of the constitutive heterochromatin in the three genomes, demonstrating to be an useful tool in the study of these sequences. The general comparison of the amount, distribution and molecular nature of the constitutive heterochromatin in the three species analyzed, suggests the occurrence of different evolutionary pathways in the origin of these karyotypes. In the species C. cricetus, which presents a nearly meta/submetacentric karyotype, the constitutive heterochromatin seems to be preferentially located in (peri)centromeric regions, presenting the majority of the autosomes two constitutive heterochromatin blocks, what suggests the occurrence, of at least, Robertsonian translocations during the course of these karyotypes evolution. The species P. eremicus, with a submetacentric karyotype, also presents great amounts of constitutive heterochromatin, essentially located in (peri)centromeric regions, being inclusively the short arms of the majority of the chromosomes completely heterochromatic. The species P. tullbergi, with an acrocentric autosome complement, is the one whose chromosomes exhibit less centromeric constitutive heterochromatin. In some chromosomes, interstitial heterochromatin is almost as abundant as the centromeric heterochromatin, in opposition to the observed for most chromosomes of the other two analyzed species. This different distribution patterns and the large number of constitutive heterochromatin subclasses identified in the P. tullbergi chromosomes (52 subclasses), suggest that this species has a more derivative karyotype than the other two genomes analyzed, originated by a great number of complex chromosomal rearrangements. The high heterogeneity of constitutive heterochromatin sequences identified, suggests the coexistence of different DNA satellite families, or variants of these families in these three analyzed genomes The in silico analysis of LINE-1 sequences (Long Interspersed Elements-1) allowed to establish phylogenetic relationships among species, since they are homoplasy-free characters (Verneau et al. 1998, Waters et al. 2007). The comparison among LINE-1 sequences from C. cricetus, P. eremicus, P. tullbergi (isolated for the first time in this work), M. musculus, R. norvegicus, and of 10 more species from the Cricetidae Family (available in the NCBI Nucleotide database), demonstrated that all the sequences are similar to a certain region of ORFII LINE-1 sequence of the índex species M. musculus and R. norvegicus. In this way, it is suggested that this region of the sequence is conserved in the rodent species analyzed, presenting similarity values above 75% among the sequences. The alignment of all the sequences allowed obtaining a dendogram that relates phylogenetically the analyzed species, specifically it was observed a larger phylogenetic proximity among rodents belonging to the same family. The physical mapping of C. cricetus, P. eremicus and P. tullbergi LINE-1 sequences, in the respective chromosomes, demonstrated that these sequences are interspersed along all the chromosomes, not presenting any preferential location in the X chromosome. These results do not support the Lyon hypothesis (1998), which suggests the involvement of LINE-1 sequences in the X chromosome inactivation, after observing a preferential location of these sequences in man and mouse X chromosomes. Two hypotheses can thus be drawn to justify the obtained results. It might have happened loss of LINE-1 sequences in the X chromosome of the three species during the evolution course, being this event previously suggested for other rodent species by Casavant et al. (2000), although in this case, the loss of these sequences was considered for the whole chromosome complement. Another possible scenario is the existence of different mechanisms of X chromosome inactivation (at least in these rodents), without the participation of LINE-1 sequences, and in this case, the genome wouldn’t need to conserve these sequences highly located in the X chromosome, being alternatively observed an undifferentiated distribution by the whole chromosome complement. Comparative Chromosome Painting using a R. norvegicus X chromosome (RNOX) specific probe in chromosome preparations of the three species, revealed three syntenic segments in one arm of C. cricetus X chromosome, eight syntenic segments in P. eremicus X chromosome and six syntenic segments in the long arm of P. tullbergi X chromosome. This analysis also revealed the occurrence of a pericentric inversion during the evolution course of P eremicus X chromosome. The characterization of the constitutive heterochromatin performed during this work revealed the existence of C-bands to flanking some syntenic segments, discriminated by the paint RNOX in the X chromosome of the three species analyzed. These results seem to suggest the participation of heterochromatin sequences in the chromosome rearrangements that originated these chromosomes, evidenced by the presence of repetitive sequences at the evolutionary breakpoints regions of these species. Moreover, in this work was performed an in silico analysis among tandem repeats chromosome distribution and the location of evolutionary breakpoints regions in the X chromosome of the índex species M. musculus. In this analysis, the chromosome syntenic regions of M. musculus, R. norvegicus and H- sapiens were used for identification of the breakpoints chromosome regions of M. musculus X chromosome, and bioinformatic tools that allowed the evaluation of tandem repeats density in this chromosome. Preliminary results demonstrate that the regions presenting high accumulation of tandem repeat sequences correspond to evolutionary breakpoints regions, support the important role of these sequences in the reorganization of genome’s architecture along evolution. The X chromosome of C. cricetus, P. eremicus and P. tullbergi show differences in respect to morphology, amount, location and molecular nature of its heterochromatin, suggesting that they arose through the occurrence of different chromosome rearrangements, as inversions, centromeric transpositions, additions/eliminations of heterochromatin and Robertsonian translocations, as is the case of C. cricetus X chromosome, that presents two blocks of constitutive heterochromatin at the (peri)centromeric region. The high number of constitutive heterochromatin subclasses identified in the X chromosome of P. tullbergi, in comparison with the other two analyzed species, suggests that the X chromosome of this species is highly rearranged. However, the analysis of the syntenic segments in the X chromosomes with the painting probe RNOX, suggests P. eremicus X chromosome as the more rearranged. This apparent disagreement is probably the result of different analyses, namely the identification of heterochromatin subclasses and syntenic segments among the chromosomes of different species, requiring a more detailed investigation to clarify the mechanisms underlying the evolution of these chromosomes. Future detailed characterization of constitutive heterochromatin sequences, namely satellite DNA sequences, isolated for exemple by microdissection of chromosome centromeric regions, and the construction of comparative maps for different species of rodents, will certainly contribute to a better understanding of the evolutionary pathways that shaped these genomes and the phylogenetic relationships among species.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Genoma , Heterocromatina , Biologia molecular