The small non-coding RNA processing machinery of two living fossil species, lungfish and coelacanth, gives new insights into the evolution of the Argonaute protein family

Argonaute (AGO) family proteins play many roles in epigenetic programming, genome rearrangement, mRNA breakdown, inhibition of translation, and transposon silencing. Despite being a hotspot in current scientific research, their evolutionary history is still poorly understood and consequently the identification of evolutionary conserved structural features should also generate useful information for better understanding their functions. We report here for the first time the transcript sets of the two subfamilies, Ago and Piwi, in the West African lungfish Protopterus annectens and in the Indonesian coelacanth Latimeria menadoensis, two key species in the evolutionary lineage leading to tetrapods. The phylogenetic analysis of 142 inferred protein sequences in 22 fully sequenced species and the analysis of microsynteny performed in the major vertebrate lineages indicate an intricate pattern for the evolution of both subfamilies that has been shaped by whole genome duplications and lineage specific gains and losses. The argonaute subfamily was additionally expanded by local gene duplications at the base of the jawed vertebrate lineage. The subfamily of Piwi proteins is involved in several processes such as spermatogenesis, piRNA biogenesis, and transposon repression. Expression assessment of AGO genes and genes coding for proteins involved in small RNA biogenesis suggests a limited activity of the Piwi pathway in lungfish in agreement with the lungfish genome containing mainly old and inactive transposons

A comparative view on sex differentiation and gametogenesis genes in lungfish and coelacanths

none8siGonadal sex differentiation andreproductionare the keys totheperpetuationof favorable gene combinations andpositively selected traits. In vertebrates, several gonad development features that differentiate tetrapods and fishes are likely to be, at least in part, related to the water-to-land transition. The collection of information from basal sarcopterygians, coelacanths, and lungfishes, is crucial to improve our understanding of the molecular evolution of pathways involved in reproductive functions, since these organisms are generally regarded as “living fossils” and as the direct ancestors of tetrapods. Here, we report for the first time the characterization of >50 genes related to sex differentiation and gametogenesis in Latimeria menadoensis and Protopterus annectens. Although the expression profiles of most genes is consistent with the intermediate position of basal sarcopterygians between actinopterygian fish and tetrapods, their phylogenetic placement and presence/absence patterns often reveal a closer affinity to the tetrapod orthologs. On the other hand, particular genes, for example, the male gonad factor gsdf (Gonadal Soma-Derived Factor), provide examples of ancestral traits sharedwith actinopterygians,which disappeared in the tetrapod lineage.openMaria Assunta Biscotti, Mateus Contar Adolfi, Marco Barucca, Mariko Forconi, Alberto Pallavicini, Marco Gerdol, Adriana Canapa, Manfred SchartlBiscotti, Maria Assunta; Contar Adolfi, Mateus; Barucca, Marco; Forconi, Mariko'; Pallavicini, Alberto; Gerdol, Marco; Canapa, Adriana; Schartl, Manfre

Relaxation of natural selection in the evolution of the giant lungfish genomes

Nonadaptive hypotheses on the evolution of eukaryotic genome size predict an expansion when the process of purifying selection becomes weak. Accordingly, species with huge genomes, such as lungfish, are expected to show a genome-wide relaxation signature of selection compared with other organisms. However, few studies have empirically tested this prediction using genomic data in a comparative framework. Here, we show that 1) the newly assembled transcriptome of the Australian lungfish, Neoceratodus forsteri, is characterized by an excess of pervasive transcription, or transcriptional leakage, possibly due to suboptimal transcriptional control, and 2) a significant relaxation signature in coding genes in lungfish species compared with other vertebrates. Based on these observations, we propose that the largest known animal genomes evolved in a nearly neutral scenario where genome expansion is less efficiently constraine

Analysis of the African coelacanth genome sheds light on tetrapod evolution

It was a zoological sensation when a living specimen of the coelacanth was first discovered in 1938, as this lineage of lobe-finned fish was thought to have gone extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features . Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain, and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues demonstrate the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution

Molecular and cytogenetic characterization of repetitive DNA in species belonging to Mollusca phylum

My thesis is focused on the identification and characterization of repetitive DNA sequences in two molluscan species, the bivalve Pecten maximus and the Antarctic polyplacophoran Nuttallochiton mirandus. Two recombinant DNA libraries were constructed after partial digestion of genomic DNA from Pecten maximus with PstI and ApaI restriction enzymes. Colonies showing strong hybridisation were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and southern hybridisation with all repeat families to genomic DNA of scallop digested with six different restriction enzymes showed characteristic ladders. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. In Nuttallochiton mirandus clear bands indicating the presence of highly repeated DNA were detected in all three digestions carried out with HindIII, PstI, and EcoRV. Sequence analysis revealed four repeated DNAs (NmH, NmP, NmE250, and NmE700) showing no similarity between them with the exception of NmE700 and NmE250 families. In fact NmE700 family displayed an inserted sequence which might arise from a transposable element. Moreover subrepeat structures were identified in the monomers belonging to NmH and NmP families. Southern blotting analysis showed a ladder banding pattern in HindIII digest indicating a tandem organization of NmH family, while suggested a more complex arrangement for the other families identified. Finally, dot blot analyses carried out in several molluscan species revealed a different degree of conservation of the repeated DNAs isolated with EcoRV. Furthermore, fluorescent in situ hybridisation (FISH) on metaphase chromosomes revealed that NmE700 family is located mainly at centromeric regions on all chromosomes with the exception of the pair of macrochromosomes. The study of the structure and organization of repetitive DNAs may contribute to understanding not only the genetic diversity but also mechanisms of DNA sequence evolution

New insights into the genome repetitive fraction of the Antarctic bivalve Adamussium colbecki

Repetitive DNA represents the major component of the genome in both plant and animal species. It includes transposable elements (TEs), which are dispersed throughout the genome, and satellite DNAs (satDNAs), which are tandemly organized in long arrays. The study of the structure and organization of repetitive DNA contributes to our understanding of genome architecture and the mechanisms leading to its evolution. Molluscs represent one of the largest groups of invertebrates and include organisms with a wide variety of morphologies and lifestyles. To increase our knowledge of bivalves at the genome level, we analysed the Antarctic scallop Adamussium colbecki. The screening of the genomic library evidenced the presence of two novel satDNA elements and the CvA transposon. The interspecific investigation performed in this study demonstrated that one of the two satDNAs isolated in A. colbecki is widespread in polar molluscan species, indicating a possible link between repetitive DNA and abiotic factors. Moreover, the transcriptional activity of CvA and its presence in long-diverged bivalves suggests a possible role for this ancient element in shaping the genome architecture of this clade

Rex Retroelements and Teleost Genomes: An Overview

Repetitive DNA is an intriguing portion of the genome still not completely discovered and shows a high variability in terms of sequence, genomic organization, and evolutionary mode. On the basis of the genomic organization, it includes satellite DNAs, which are organized as long arrays of head-to-tail linked repeats, and transposable elements, which are dispersed throughout the genome. These repeated elements represent a considerable fraction of vertebrate genomes contributing significantly in species evolution. In this review, we focus our attention on Rex1, Rex3 and Rex6, three elements specific of teleost genomes. We report an overview of data available on these retroelements highlighting their significative impact in chromatin and heterochromatin organization, in the differentiation of sex chromosomes, in the formation of supernumerary chromosomes, and in karyotype evolution in teleosts

Isolation of Hox and ParaHox genes in bivalve Pecten maximus.

The Hox cluster genes encode a set of transcription factors that have been shown to control spatial patterning mechanisms in bilaterian organism development. The ParaHox cluster is the evolutionary sister of the Hox cluster. The two are believed to descend from an ancestral ProtoHox cluster of four genes from which the three ParaHox genes (Gsx, Xlox, and Cdx) and the four Hox gene classes are believed to have originated. Although molluscs are among the most successful lophotrochozoan groups, very little work has been devoted to the characteristics of their homeotic genes. Using polymerase chain reaction-based approaches, we isolated 13 different Pecten maximus (Bivalvia: Pteriomorphia) sequences corresponding to all the genes of the four Hox cluster classes and to genes Xlox and Cdx of the ParaHox cluster. Comparison of results with those obtained in other lophotrochozoans seems to confirm the considerable homogeneity of the Hox and ParaHox genes in these taxa both as regards the presence of nearly all the genes of the two clusters and the marked sequence resemblance among orthologous genes. © 2005 Elsevier B.V. All rights reserved

Investigation of the activity of transposable elements and genes involved in their silencing in the newt Cynops orientalis, a species with a giant genome

Caudata is an order of amphibians with great variation in genome size, which can reach enormous dimensions in salamanders. In this work, we analysed the activity of transposable elements (TEs) in the transcriptomes obtained from female and male gonads of the Chinese fire-bellied newt, Cynops orientalis, a species with a genome about 12-fold larger than the human genome. We also compared these data with genomes of two basal sarcopterygians, coelacanth and lungfish. In the newt our findings highlighted a major impact of non-LTR retroelements and a greater total TE activity compared to the lungfish Protopterus annectens, an organism also characterized by a giant genome. This difference in TE activity might be due to the presence of young copies in newt in agreement also with the increase in the genome size, an event that occurred independently and later than lungfish. Moreover, the activity of 33 target genes encoding proteins involved in the TE host silencing mechanisms, such as Ago/Piwi and NuRD complex, was evaluated and compared between the three species analysed. These data revealed high transcriptional levels of the target genes in both newt and lungfish and confirmed the activity of NuRD complex genes in adults. Finally, phylogenetic analyses performed on PRDM9 and TRIM28 allowed increasing knowledge about the evolution of these two key genes of the NuRD complex silencing mechanism in vertebrates. Our results confirmed that the gigantism of the newt genomes may be attributed to the activity and accumulation of TEs