Ana M. González-Tizón

Molecular evolutionary characterization of the mussel Mytilus histone multigene family: first record of a tandemly repeated unit of five histone genes containing an H1 subtype with "orphon" features.

The present work represents the first characterization of a clustered histone repetitive unit containing an H1 gene in a bivalve mollusk. To complete the knowledge on the evolutionary history of the histone multigene family in invertebrates, we undertake its characterization in five mussel Mytilus species, as an extension of our previous work on the H1 gene family. We report the quintet H4–H2B–H2A–H3–H1 as the major organization unit in the genome of Mytilus galloprovincialis with two 5S rRNA genes with interspersed nontranscribed spacer segments linked to the unit, which is not justified by their cotranscription with histone genes. Surprisingly, 3′ UTR regions of histone genes show two different mRNA termination signals, a stem-loop and a polyadenylation signal, both related to the evolution of histone gene expression patterns throughout the cell cycle. The clustered H1 histones characterized share essential features with “orphon” H1 genes, suggesting a common evolutionary origin for both histone subtypes which is supported by the reconstructed phylogeny for H1 genes. The characterization of histone genes in four additional Mytilus species revealed the presence of strong purifying selection acting among the members of the family. The chromosomal location of most of the core histone genes studied was identified by FISH close to telomeric regions in M. galloprovincialis. Further analysis on nucleotide variation would be necessary to assess if H1 proteins evolve according to the birth-and-death model of evolution and if the effect of the strong purifying selection maintaining protein homogeneity could account for the homologies detected between clustered and “orphon” variants.

Characterization of different chromatin types in Mytilus galloprovincialis L. after C-banding, fluorochrome and restriction endonuclease treatments

In this study, we have carried out the cytogenetical characterization of Mytilus galloprovincialis L. (2n = 28) using conventional staining and banding techniques such as fluorochromes and restriction endonucleases treatment. Chromosome digestion with trypsin enzyme resulted in a G-banding pattern which allowed us to clearly identify and classify the chromosome pairs of M. galloprovincialis. C-banding and chromomycin A3 staining confirmed the existence of small amounts of constitutive heterochromatin. The treatment of samples with AluI, HaeIII, DpnI, MspI, HpaII and HinfI restriction endonucleases produced specific banding patterns which demonstrate the potential of endonucleases for chromosome banding in mussels. The results obtained allow us to describe six different types of chromatin in M. galloprovincialis. The type is determined by the response of the chromosomes to the different treatments. Differential digestion by the enzyme pair HpaII-MspI of specific C-band positive heterochromatic areas in some of the chromosomes suggests the presence of methylation.

DNA content, karyotypes, and chromosomal location of 18S-5.8S-28S ribosomal loci in some species of bivalve molluscs from the Pacific Canadian coast.

The DNA content of 10 species of bivalve molluscs from British Columbia coast was determined by image analysis, and the karyotypes of the horse clam Tressus capax, the bent-nose macoma Macoma nasuta, and the nuttalls mahogany clam Nuttallia nuttallii are described here for the first time. We also have analyzed the location of rDNA loci using a 28S-5.8S-18S probe in four of these species: Mytilus californianus, M. trossulus, Macoma nasuta and N. nuttallii. Results obtained report new data about cytogenetic characteristics of bivalve molluscs.

Chromosomal markers in three species of the genus Mytilus (Mollusca: Bivalvia)

The analysis of C-banding, NOR and fluorochrome staining was carried out in three species of European mussel, Mytilus edulis, M. galloprovincialis and M. trossulus. The results obtained allow us to detect changes in the constitutive heterochromatin within the genus Mytilus. The existence of chromosomal markers permit us to identify and distinguish, at the cytogenetical level, these three types of mussel.

Long-Term Evolution of 5S Ribosomal DNA Seems to Be Driven by Birth-and-Death Processes and Selection in Ensis Razor Shells (Mollusca: Bivalvia)

A study of nucleotide sequence variation of 5S ribosomal DNA from six Ensis species revealed that several 5S ribosomal DNA variants, based on differences in their nontranscribed spacers (NTS), occur in Ensis genomes. The 5S rRNA gene was not very polymorphic, compared with the NTS region. The phylogenetic analyses performed showed a between-species clustering of 5S ribosomal DNA variants. Sequence divergence levels between variants were very large, revealing a lack of sequence homogenization. These results strongly suggest that the long-term evolution of Ensis 5S ribosomal DNA is driven by birth-and-death processes and selection.

The linked units of 5S rDNA and U1 snDNA of razor shells (Mollusca: Bivalvia: Pharidae)

The linkage between 5S ribosomal DNA and other multigene families has been detected in many eukaryote lineages, but whether it provides any selective advantage remains unclear. In this work, we report the occurrence of linked units of 5S ribosomal DNA (5S rDNA) and U1 small nuclear DNA (U1 snDNA) in 10 razor shell species (Mollusca: Bivalvia: Pharidae) from four different genera. We obtained several clones containing partial or complete repeats of both multigene families in which both types of genes displayed the same orientation. We provide a comprehensive collection of razor shell 5S rDNA clones, both with linked and nonlinked organisation, and the first bivalve U1 snDNA sequences. We predicted the secondary structures and characterised the upstream and downstream conserved elements, including a region at −25 nucleotides from both 5S rDNA and U1 snDNA transcription start sites. The analysis of 5S rDNA showed that some nontranscribed spacers (NTSs) are more closely related to NTSs from other species (and genera) than to NTSs from the species they were retrieved from, suggesting birth-and-death evolution and ancestral polymorphism. Nucleotide conservation within the functional regions suggests the involvement of purifying selection, unequal crossing-overs and gene conversions. Taking into account this and other studies, we discuss the possible mechanisms by which both multigene families could have become linked in the Pharidae lineage. The reason why 5S rDNA is often found linked to other multigene families seems to be the result of stochastic processes within genomes in which its high copy number is determinant.

Polyploidy in a natural population of mussel, Mytilus trossulus.

We have analyzed natural polyploidy in a population of Mytilus trossulus from Vancouver Island (British Columbia, Canada) by means of cytogenetic techniques. Results obtained are the first reporting on this type of numerical chromosome aberrations in mussels.

Heterogeneous nature and distribution of interruptions in dinucleotides may indicate the existence of biased substitutions underlying microsatellite evolution.

Some aspects of microsatellite evolution, such as the role of base substitutions, are far from being fully understood. To examine the significance of base substitutions underlying the evolution of microsatellites we explored the nature and the distribution of interruptions in dinucleotide repeats from the human genome. The frequencies that we inferred in the repetitive sequences were statistically different from the frequencies observed in other noncoding sequences. Additionally, we detected that the interruptions tended to be towards the ends of the microsatellites and 5′-3′ asymmetry. In all the estimates nucleotides forming the same repetitive motif seem to be affected by different base substitution rates in AC and AG. This tendency itself could generate patterning and similarity in flanking sequences and reconcile these phenomena with the high mutation rate found in flanking sequences without invoking convergent evolution. Nevertheless, our data suggest that there is a regional bias in the substitution pattern of microsatellites. The accumulation of random substitutions alone cannot explain the heterogeneity and the asymmetry of interruptions found in this study or the relative frequency of different compound microsatellites in the human genome. Therefore, we cannot rule out the possibility of a mutational bias leading to convergent or parallel evolution in flanking sequences.

The 5S rDNA Gene Family in Mollusks: Characterization of Transcriptional Regulatory Regions, Prediction of Secondary Structures, and Long-Term Evolution, with Special Attention to Mytilidae Mussels

Several reports on the characterization of 5S ribosomal DNA (5S rDNA) in various animal groups have been published to date, but there is a lack of studies analyzing this gene family in a much broader context. Here, we have studied 5S rDNA variation in several molluskan species, including bivalves, gastropods, and cephalopods. The degree of conservation of transcriptional regulatory regions was analyzed in these lineages, revealing a conserved TATA-like box in the upstream region. The evolution of the 120 bp coding region (5S) was also studied, suggesting the occurrence of paralogue groups in razor clams, clams, and cockles. In addition, 5S rDNA sequences from 11 species and 7 genus of Mytilidae Rafinesque, 1815 mussels were sampled and studied in detail. Four different 5S rDNA types, based on the nontranscribed spacer region were identified. The phylogenetic analyses performed within each type showed a between-species gene clustering pattern, suggesting ancestral polymorphism. Moreover, some putative pseudogenized 5S copies were also identified. Our report, together with previous studies that found high degree of intragenomic divergence in bivalve species, suggests that birth-and-death evolution may be the main force driving the evolution of 5S rDNA in these animals, even at the genus level.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection

BackgroundThe 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context.ResultsThe nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types.ConclusionsThese results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.