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

[Abstract] 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 determinan

Heterochromatin and ribosomal genes in Asellus aquaticus (Crust. Isop.)

In the present investigation chromosomal preparations of Asellus aquaticus were sequentially stained with chromomycin A3 to reveal the heterochromatic areas, hybridized in situ with rDNA probes in order to map the ribosomal genes and finally silver stained to check the transcriptional activity of these genes. The results indicate the existence of a substantial correspondence of location and size among the heterochromatic regions and the regions over which the in situ hybridization signals spread. The ribosomal genes, quite independently of their location in the secondary constriction, can be silver stained and thus appear to be transcriptionally active. The ribosomal sequences also hybridize to the entire heterochromatic areas observed on the probable Y chromosome identified in some males of a natural population. These rRNA genes are only rarely transcriptionally active

Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species

Four different units containing three variants of the U1 snRNA gene have been identified in the genome of Asellus aquaticus and only one unit has been identified in the genome of Proasellus coxalis. All four identified U1 snRNA genes can be folded according to the proper secondary structure and possess the functionally useful conserved sequences. Moreover, in the 3 flanking regions, all genes present both the 3 box, a conserved sequence required for 3 processing of mature snRNA, and a polyadenylation signal which is unusual for these genes. The PCR products were used as probes in fluorescent in-situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus and P. coxalis. I.F.: 2.9

Longitudinal differentiation of chromosomes of Asellus aquaticus (Crust. Isop.) by in situ nick translation using restriction enzymes and DNase I

Asellus aquaticus is an isopod crustacean whose chromosomes cannot be differentiated by G- or R-banding techniques. In this work, we have obtained a longitudinal differentiation of these chromosomes by in situ nick translation using restriction enzymes (HaeIII, DraI and BamHI) and DNase I digestions. The four nucleases, with different efficiencies, have produced similar labelling patterns. Staining with DAPI, Giemsa and chromomycin A(3) reveals that the DNA of the nick-translated regions is generally more resistant to extraction from the chromosome. The results obtained on the heteromorphic sex chromosome pair observed in about a quarter of the males of a natural population allow several hypotheses to be advanced on the nature and origin of chromosome dimorphism

Sex chromosome differentiation revealed by comparative genomic hybridization.

In this work, genomic in-situ hybridization (GISH) was used to study the sex chromosome molecular differentiation on chromosomes of male and female individuals of the isopod crustacean Asellus aquaticus. As a composite hybridization probe, we contemporaneously used male and female whole genomic DNA differently labelled in the presence of an excess of unlabelled DNA of the female homogametic sex. The karyotype of A. aquaticus normally displays eight homomorphic chromosome pairs, but a heteromorphic sex chromosome pair is present in about a quarter of the males of a natural population previously identi¢ed by us. GISH did not reveal any sex chromosome molecular differentiation on the male and female homomorphic sex chromosome pair, and the karyotypes of these individuals were equally labelled by the male- and female-derived probe, while the heteromorphic Y chromosome showed a differentially labelled region only with the male-derived probe. This region evidently contains male-speci¢c sequences but, because no similar hybridized region is observed on the male homomorphic chromosome pair, they are probably not important for sex determination but represent a molecular differentiation acquired from the Y chromosome. I.F.: 3.2

Identificazione e caratterizzazione dei geni snRNA U1 di due specie di crostacei isopodi

The U1 small nuclear RNAs (snRNA) gene, together with the U2, U4 and U5 snRNA genes, constitutes a group of class II evolutionary conserved genes, the transcripts of which are involved in pre-mRNA splicing in the nucleus as RNA components of the spliceosome. U1 snRNA genes, isolated from a variety of eukaryotes, were reported to be clustered in a number of species and codified as tandemly repeated units, containing one or more types of U1 snRNA genes, or to be linked but not tandemly repeated. Different types of U1 snRNA were found in some organisms to be tissue-specific and developmentally regulated. Recently, using PCR amplification, in the genome of the crustacean isopod Asellus aquaticus, we found a U1 snRNA gene within a 1842 bp long tandemly repeated unit also containing a 5S rRNA gene. No other snRNA gene had been previously identified in any crustacean species. In the present work, using PCR amplification, we identified three further units containing U1 snDNA in the genome of A. aquaticus. The three units were sequenced and used as probes in FISH experiments to localize them on A. aquaticus chromosomes. Likewise, we investigated the snRNA genes in the genome of Proasellus coxalis and identified only one unit containing U1 snDNA in this organism. P. coxalis and A. aquaticus are two Asellidae species that cohabit in the fresh waters of central and southern Italy. The haploid DNA amount of A. aquaticus is 2.52 pg, its karyotype consists of 2n = 16 homomorphic chromosomes. The haploid DNA amount of P. coxalis is 1.30 pg, its karyotype consists of 2n = 12 homomorphic chromosomes. In A. aquaticus, two intercalary heterochromatic areas are also revealed by CMA on one chromosome, the Y chromosome, of a heteromorphic sex chromosome pair present in about 25% of males from a natural population collected in the Sarno river near Naples (Barzotti et al., 2000 and references cited therein). CONCLUSIONS In this work, we demonstrate that at least three variants of the U1 snRNA gene contained in four different fragments (A,B,C,D) are present in the genome of A. aquaticus, while once again, as for 5S rRNA genes (Barzotti et al., 2000; Pelliccia et al., 1998; Pelliccia et al., 2001), in the genome of P. coxalis we identified only one unit containing one U1 snRNA gene. The genome DNA amount of A. aquaticus (2.52 pg) is about twice that of P. coxalis (1.30 pg) (Rocchi et al., 1989). These values point to a mechanism of genomic duplication as a source of the diversity between the genomes of the two species, while the complex organization of the 5S rRNA genes and of the U1 snRNA genes in genome of A. aquaticus seems to indicate the occurrence of events of duplicative transposition. Undoubtedly a better knowledge of the genome of these organisms will be necessary for more reliable conclusions to be drawn on these matters

5S rRNA and U1 snRNA genes, a new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S rDNA sequences.

We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed. I.F.: 2.2