Accessing transcriptomic data for ecologically important genes in the goose barnacle (Pollicipes pollicipes), with particular focus on cement proteins.

In this study 4310 expressed sequence tags (ESTs) were used to identify potentially useful transcripts for future studies in the gooseneck barnacle Pollicipes pollicipes (Gmelin, 1789). 119 ESTs were obtained in this work and 4191 were taken from Meusemann et al. (2010). The gooseneck barnacle is a sessile pedunculate cirripede of great economic importance that occurs in dense aggregations, and is harvested for human consumption. The assembly of these ESTs yielded 1805 unigenes (461 contigs and 1344 singlets). The identification of cement proteins in our data is particularly interesting for cirripedes. Only a small part of the assembled unigenes could be functionally annotated. However, our results greatly improve our understanding of the biological features of P. pollicipes. In addition to this, a large number of potentially interesting genes were identified in order to serve as the base for future evolutionary studies in P. pollicipes.

Karyological characterization of the endemic Iberian rock lizard, Iberolacerta monticola (Squamata, Lacertidae): insights into sex chromosome evolution.

Rock lizards of the genus Iberolacerta constitute a promising model to examine the process of sex chromosome evolution, as these closely related taxa exhibit remarkable diversity in the degree of sex chromosome differentiation with no clear phylogenetic segregation, ranging from cryptic to highly heteromorphic ZW chromosomes and even multiple chromosome systems (Z1Z1Z2Z2/Z1Z2W). To gain a deeper insight into the patterns of karyotype and sex chromosome evolution, we performed a cytogenetic analysis based on conventional staining, banding techniques and fluorescence in situ hybridization in the species I. monticola, for which previous cytogenetic investigations did not detect differentiated sex chromosomes. The karyotype is composed of 2n = 36 acrocentric chromosomes. NORs and the major ribosomal genes were located in the subtelomeric region of chromosome pair 6. Hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes and interstitially in 5 chromosome pairs. C-banding showed constitutive heterochromatin at the centromeres of all chromosomes, as well as clear pericentromeric and light telomeric C-bands in several chromosome pairs. These results highlight some chromosomal markers which can be useful to identify species-specific diagnostic characters, although they may not accurately reflect the phylogenetic relationships among the taxa. In addition, C-banding revealed the presence of a heteromorphic ZW sex chromosome pair, where W is smaller than Z and almost completely heterochromatic. This finding sheds light on sex chromosome evolution in the genus Iberolacerta and suggests that further comparative cytogenetic analyses are needed to understand the processes underlying the origin, differentiation and plasticity of sex chromosome systems in lacertid lizards.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans.

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12,766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

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.

Origin and evolution of Mytilus mussel satellite DNAs.

A phylogenetic reconstruction based on the amplification of 3 satellite DNAs (stDNAs) was carried out in 1 crustacean species and 15 bivalve species of the subclass Pteriomorphia (10, subfamily Mytilinae; 1, subfamily Litophaginae; 1, subfamily Modiolinae, all belonging to family Mytilidae; 1, family Arcidae; and 2, family Pectinidae). The sequences obtained showed motifs with high similarity to those of A and B boxes of tRNA promoter regions. Dot-blot hybridizations revealed that the 3 stDNAs are present mainly in high copy numbers for each species of the genus Mytilus, whereas for the other species they appear in low copy numbers. Maximum-parsimony trees evidenced a tendency to group Mytilus clones together, and species containing these sequences as a single copy were distributed among the different mytilids. Finally, the possible origin and evolution of these stDNAs is discussed.

Cytogenetics of the razor clam Solen marginatus (Mollusca: Bivalvia: Solenidae).

The razor clam Solen marginatus has a diploid chromosome number of 38. The karyotype consists of one metacentric/submetacentric, three submetacentric/metacentric, five submetacentric, one submetacentric/subtelocentric, one subtelocentric/submetacentric, six subtelocentric and two telocentric chromosome pairs. Staining with chromomycin A3 revealed bright positive bands subcentromerically in the long arms of one medium-sized subtelocentric pair, while DAPI staining showed uniform fluorescence in all chromosomes of the complement. Fluorescence in situ hybridization using an 18S-5.8S-28S rDNA probe locates these loci at the subcentromeric region of one subtelocentric pair and at the subtelomeric region of another subtelocentric pair.

Comparative analysis of different satellite DNAs in four Mytilus species.

We report the characterization of three satellite DNAs in four species of mussel: Mytilus edulis, Mytilus galloprovincialis, Mytilus trossulus, and Mytilus californianus. The monomers of the Apa I satellite DNAs were 173, 161, and 166 bp long. These satellite monomers were used to construct phylogenetic trees to infer relationships among these species. The topologies obtained clearly indicate that M. californianus is the most divergent species with respect to the other three. Furthermore, localization of satellite DNAs on metaphase chromosomes was performed using fluorescent in situ hybridization (FISH). Fluorescent signals revealed a different organization and distribution of these three satellite DNAs.

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.

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 nuttall's 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.

Analysis of the influence of chromosomal condensation on the activity of restriction endonucleases.

The different responses of metaphase chromosomes to restriction endonuclease activity depend on the stage of chromosomal condensation. Results obtained established that this factor plays a remarkable role in obtaining high resolution banding patterns which facilitated visualization of a large number of sub-bands. It is evident that various restriction endonucleases, which are able to induce a coincident G-like banding pattern on Don cell line chromosomes, act in very different ways.

Chromosomal DNA extraction after restriction endonuclease treatments: a study by microdensitometry and electrophoresis.

The appearance of G- and C-banding patterns on metaphase chromosomes from the Don cell line (Cricetulus griseus) was accompanied by extraction of chromosomal DNA. A microdensitometric technique was employed to measure the loss of DNA produced by Hin dIII, Hae III and Eco RI endonuclease digestion on fixed chromosomes, from 0.5 to 24 h of treatment. Agarose gel electrophoresis was carried out to estimate the size of the DNA fragments extracted after digestion by Alu I and the restriction endonucleases already mentioned. Results obtained show that Alu I and Hae III, which possess 4 base pair recognition sequences, caused higher DNA extraction than enzymes with recognition targets of 6 base pairs (Hin dIII and Eco RI). Incubation buffers induced G-banding patterns which were accompanied by DNA extraction, although this loss was lower than that produced by endonuclease treatments.

Denaturing effect of acridine orange and adriamycin.

Acridine orange and adriamycin have a very low denaturing effect on DNA isolated from Chinese hamster cells but strongly increase heat-induced denaturation. When these intercalating agents are added to the growth media they modify the sensitivity to heat denaturation of DNA extracted from treated cultures when compared with untreated cultures. This effect correlates with a considerable increase in the disruption of chromosomal ultrastructure.