Ana Paço

Different evolutionary trails in the related genomes Cricetus cricetus and Peromyscus eremicus (Rodentia, Cricetidae) uncovered by orthologous satellite DNA repositioning

Constitutive heterochromatin comprises a substantial fraction of the eukaryotic genomes and is mainly composed of tandemly arrayed satellite DNAs (satDNA). These repetitive sequences represent a very dynamic and fast evolving component of genomes. In the present work we report the isolation of Cricetus cricetus (CCR, Cricetidae, Rodentia) centromeric repetitive sequences from chromosome 4 (CCR4/10sat), using the laser microdissection and laser pressure catapulting procedure, followed by DOP-PCR amplification and labelling. Physical mapping by fluorescent in situ hybridisation of these sequences onto C. cricetus and another member of Cricetidae, Peromyscus eremicus, displayed quite interesting patterns. Namely, the centromeric sequences showed to be present in another C. cricetus chromosome (CCR10) besides CCR4. Moreover, these almost chromosome-specific sequences revealed to be present in the P. eremicus genome, and most interestingly, displaying a ubiquitous scattered distribution throughout this karyotype. Finally and in both species, a co-localisation of CCR4/10sat with constitutive heterochromatin was found, either by classical C-banding or C-banding sequential to in situ endonuclease restriction. The presence of these orthologous sequences in both genomes is suggestive of a phylogenetic proximity. Furthermore, the existence of common repetitive DNA sequences with a different chromosomal location foresees the occurrence of an extensive process of karyotype restructuring somehow related with intragenomic movements of these repetitive sequences during the evolutionary process of C. cricetus and P. eremicus species.

The involvement of repetitive sequences in the remodelling of karyotypes: the Phodopus genomes (Rodentia, Cricetidae).

In this work we characterize the Phodopus roborovskii and Phodopus sungorus karyotypes, describing the constitutive heterochromatin and the telomeric repeats distribution in these chromosomes. In the two species, (peri)centromeric, interstitial and (sub)telomeric C-bands were revealed, presenting a very high colocalization with evolutionary breakpoint regions identified in these karyotypes. Also both species present telomeric sequences located interstitially (ITS), as short ITS blocks or as large ITS blocks, mainly at the (peri)centromeric heterochromatic regions. The number and degree of ITSs amplification varies greatly in the two hamsters, indicating independent evolutionary events of these repeats in each genome. The combination of the data provided interesting insights about the genome organization of these hamster species, also allowing establishing evolutionary considerations on their chromosomes. The obtained results clearly suggest an involvement of the repetitive genomic fraction in the reshaping of P. roborovskii and P. sungorus karyotypes.

Evolutionary Story of a Satellite DNA from Phodopus sungorus (Rodentia, Cricetidae)

With the goal to contribute for the understanding of satellite DNA evolution and its genomic involvement, in this work it was isolated and characterized the first satellite DNA (PSUcentSat) from Phodopus sungorus (Cricetidae). Physical mapping of this sequence in P. sungorus showed large PSUcentSat arrays located at the heterochromatic (peri)centromeric region of five autosomal pairs and Y-chromosome. The presence of orthologous PSUcentSat sequences in the genomes of other Cricetidae and Muridae rodents was also verified, presenting however, an interspersed chromosomal distribution. This distribution pattern suggests a PSUcentSat-scattered location in an ancestor of Muridae/Cricetidae families, that assumed afterwards, in the descendant genome of P. sungorus a restricted localization to few chromosomes in the (peri)centromeric region. We believe that after the divergence of the studied species, PSUcentSat was most probably highly amplified in the (peri)centromeric region of some chromosome pairs of this hamster by recombinational mechanisms. The bouquet chromosome configuration (prophase I) possibly displays an important role in this selective amplification, providing physical proximity of centromeric regions between chromosomes with similar size and/or morphology. This seems particularly evident for the acrocentric chromosomes of P. sungorus (including the Y-chromosome), all presenting large PSUcentSat arrays at the (peri)centromeric region. The conservation of this sequence in the studied genomes and its (peri)centromeric amplification in P. sungorus strongly suggests functional significance, possibly displaying this satellite family different functions in the different genomes. The verification of PSUcentSat transcriptional activity in normal proliferative cells suggests that its transcription is not stage-limited, as described for some other satellites.

Hidden heterochromatin: characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.

LINE-1 retrotransposons: from 'parasite' sequences to functional elements.

Long interspersed nuclear elements-1 (LINE-1) are the most abundant and active retrotransposons in the mammalian genomes. Traditionally, the occurrence of LINE-1 sequences in the genome of mammals has been explained by the selfish DNA hypothesis. Nevertheless, recently, it has also been argued that these sequences could play important roles in these genomes, as in the regulation of gene expression, genome modelling and X-chromosome inactivation. The non-random chromosomal distribution is a striking feature of these retroelements that somehow reflects its functionality. In the present study, we have isolated and analysed a fraction of the open reading frame 2 (ORF2) LINE-1 sequence from three rodent species, Cricetus cricetus, Peromyscus eremicus and Praomys tullbergi. Physical mapping of the isolated sequences revealed an interspersed longitudinal AT pattern of distribution along all the chromosomes of the complement in the three genomes. A detailed analysis shows that these sequences are preferentially located in the euchromatic regions, although some signals could be detected in the heterochromatin. In addition, a coincidence between the location of imprinted gene regions (as Xist and Tsix gene regions) and the LINE-1 retroelements was also observed. According to these results, we propose an involvement of LINE-1 sequences in different genomic events as gene imprinting, X-chromosome inactivation and evolution of repetitive sequences located at the heterochromatic regions (e.g. satellite DNA sequences) of the rodents’ genomes analysed.

The puzzling character of repetitive DNA in Phodopus genomes (Cricetidae, Rodentia)

Three novel repetitive DNA sequences are described, presenting a similar heterochromatic chromosomal location in two hamster species: Phodopus roborovskii and Phodopus sungorus (Cricetidae, Rodentia). Namely, two species-specific repetitive sequences (PROsat from P. roborovskii and PSUchr1sat from P. sungorus) surrounding a third one (PsatDNA), that is shared by both hamster genomes. Fiber-FISH analyses revealed that PROsat intermingles with PsatDNA in P. roborovskii and PSUchr1sat intermingles with PsatDNA in P. sungorus. A model explaining the evolution of this intricate chromosomal distribution is proposed, which can explain better the evolution of these very derivative genomes (in comparison to the ancestral Muroidea). The most plausible evolutionary scenario seems to be the expansion of a number of repeats into other’s domain, most probably resulting in its intermingling, followed by the subsequent spread of these complex repeats from a single chromosomal location to other chromosomes. Evidences of an association between repetitive sequences and the chromosome evolution process were observed, namely for PROsat. Most probably, the evolutionary breakpoints that shaped PRO and PSU chromosomes (pericentric inversions and fusions) occurred within the boundaries of PROsat blocks in the ancestor. The repeats high diversity at the heterochromatic regions of Phodopus chromosomes, together with its complex organization, suggests that these species are important models for evolutionary studies, namely in the investigation of a possible relationship between repetitive sequences and the occurrence of chromosomal rearrangements and consequently, in genome evolution.

Nanomaterials and Microplastics

Nanomaterials and microplastics have been identified as contaminants of major environmental concern and a global problem. Many efforts have been made, in the recent years, in order to understand the nanomaterials and microplastics fate and behavior in the environment, but significant gaps have been identified concerning exposure and risk assessment to these contaminants. These gaps rely mainly in the specific analytical methods and standard protocols for the characterization and quantification of those particles. This work briefly overviews the nanomaterials and microplastics sources and effects in the environment and review the analytical methodologies currently used and developed for their characterization and quantification.