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Simple DNA repeats and sex chromosome differentiation in Asellus aquaticus (Crustacea, Isopoda)

We tested the reroductive compatibility existing between four geographically different populations of Proaselis coxalis (Crustacea Isopoda) collected in the river Sarno (near Naples) and on three Sicilian sites: San Domenico; Villa Grazia di Carini and Enna. By means of laboratory hybridization experiments (“no-choice” method) we investigated and characterized the reproductive isolation established between these populations under allopatric conditions. The populations San Domenico and Enna are reproductively isolated from Villa Grazia di Carini and Sarno, so that they may be considered as belonging to different species. The mechanism of reproductive isolation are of postzygotic nature only: embronic death, hbrid inviability and sterility of the surviving hybrids. These results agree with tie theory of aioatric speciation, according to which in the first phase of speciation only post-zygotic mechanisms occur as an accidental product of genetic divergence.

In this investigation we analysed the 5S rRNA genes of the isopod crustacean Proasellus coxalis. 5S rDNA hybridization of digested genomic DNA and amplification by PCR demonstrate that these genes are organized in tandem repeats of 589 bp, 120 of which represent the coding sequence and 469 the spacer sequence. Proasellus coxalis is the first crustacean species in which 5S rRNA genes have been found tandemly arranged without being linked to other repeated genes. The PCR product has been used as a probe in FISH to locate the 5S rRNA genes on two chromosome pairs of the P. coxalis karyotype. Comparison of the 5S rDNA sequence of this species with previously published sequences of six other crustacean species shows the existence of a good correlation between phylogenetic relationships and sequence identity.

Protocadherin X (PCDHX) and Protocadherin Y (PCDHY) are cell-surface adhesion molecules expressed predominantly in brain. The human PCDH11X/Y gene pair is located in the non-pseudoautosomal X-Y homologous region (Xq21.3/Yp11.2). The possible existence of PCDH11 gene dosage differences between human and non-human primates is of evolutionary significance with respect to species differences and escape from X inactivation, and has been repeatedly debated. Previous investigations on the X/Y homologous status of PCDH11 and adjacent sequences in non-human primates have highlighted the complexity of the molecular pattern and evolutionary history of this genomic region. This paper provides for the first time direct evidence for the absence of the PCDH11 genefrom the Y chromosome of chimpanzee (Pan troglodytes) as well as gorilla (Gorilla gorilla). By confirmingthe suspected lack of X-Y homologous status for PCDH11 in non-human primates, our results reinforce the hypothesis of a hominid-specific role for this gene in brain development.

Retrospective analysis of chromosomal changes in endometrial carcinoma was performed by fluorescence in situ hybridization on free nuclei isolated from formalin-fixed paraffin-embedded tissue. We examined 23 archival samples for numerical aberrations of chromosomes 1 and 10 with the use of specific DNA probes for the pericentromeric and centromeric regions of these two chromosomes. Numerical aberrations of chromosomes 1 and 10 were detected in 39% of the case analyzed, and the frequency of trisomy 10 tended to increase as the histological grade worsened. Our findings confirm the association of cytogenetic anomalies involving chromosomes 1 and 10 with endometrial carcinoma, as reported by other studies, and suggest that changes in centromere 10 copy number may correlate with the degree of tumor differentiation.

Specific chromosomal aberrations might indicate the position of genes responsible for a particular disease. Neuroblastoma is characterized by frequent deletions and/or rearrangements of the subtelomeric 1p region which, accordingly, is believed to host one or more oncosuppressor gene(s) directly or indirectly involved in the development of this and other tumors. Identification of these genes could be facilitated if cell lines with well characterized interstitial deletions or reciprocal translocations could be available for application of positional cloning strategies. In the present report we present additional and novel molecular data on three well established neuroblastoma cell lines (NLF, NMB and NGP). In one of these we have identified two sites that might be good candidates for hosting oncosuppressor genes; one of these is flanked by the D1S47 and ENO1 loci while the other is distal to the A12M2 locus.

A reciprocal t(1;15)(p36.1-36.3;q25-26) has been identified in an established neuroblastoma cell line (NGP) that earlier studies had shown to carry, among others, a rearrangement at the 1p subtelomeric region. Though it has not been possible to establish whether this translocation was constitutional, it is of interest to note that one of the breakpoints is located within the well-known 1p consensus site of tumor-associated chromosomal rearrangements where, as a result of the reciprocal translocation, the FES oncogene has been transferred from autosome 15. It is to be expected that the molecular cloning of the 1p and 15q translocation breakpoints may yield crucial data for understanding the association between specific chromosomal rearrangements and malignant tumor progression.

The repeated sequence TTAGGG is present at all tested vertebrate telomeres including those of humans and at the telomeres of evolutionarily very distant organisms such as trypanosomes and slime moulds. We tested for the presence of this sequence in the genome of the crustacean isopod Asellus aquaticus. Fluorescence in situ hybridization and BAL31 nuclease digestion demonstrate that the (TTAGGG)n sequence occurs at the extreme termini of the chromosomes and also at an interstitial site.

With this fast and simple MULTIPRINS protocol we show simultaneous mapping of multiple repetitive sequences by sequential cycles of primed in situ (PRINS) labelling with various primers and reporter molecules. Differentially labelled DNA sequences synthesized in situ were visualized by directly incorporated rhodamine-4-dUTP, by fluorescein isothiocyanate and Cy5 using digital imaging microscopy. This further development of the PRINS method enhances its potential as an alternative to traditional in situ hybridization.

The karyotype of the isopod crustacean Asellus aquaticus does not normally display any heteromorphic sex chromosome pair. Some of the males in a wild population of A. aquaticus collected in the Sarno river near Naples do display a heteromorphic chromosome pair. The hetero-morphism is due to the presence of two intercalary heterochromatic areas on one chromosome. This chromosome is inherited through the male line as a normal Y chromosome. The heteromorphic pair has retained the capacity to recombine during meiosis. In-situ hybridization of ribosomal probes, labelled with digoxigenin-dUTP, reveals that ribosomal sequences are associated with both intercalary heterochromatic areas of the heterochromosome. The ribosomal genes are normally telomeric and associated with heterochromatin. After digestion of genomic DNA with BamHI EcoRI and HindIII restriction endonucleases and hybridization with ribosomal probes, the hybridization patterns of the males with the heterochromosome differ from those of the males without the heterochromosome, as well as from those of the females, which are identical. The possible origin of the morphological differentiation of the heterochromosome, and the causes of the differences in the ribosomal DNA restriction patterns linked to the presence of this chromosome, are discussed.

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A3 staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic areas of the male heterochromosome are not labelled.

The circadian variation of the mitotic index during spermiohistogenesis was studied in Asellus aquaticus (L.). The actual number of metaphases and prometaphases was determined at the end of each hour of light or darkness over a 24 h period in animals bred under LD 12:12. The number of the metaphases and prometaphases decreases during the light period and sharply increases in the last 3 hrs of the dark period. This variation in the proliferative activity suggests that photoperiod can play a role in the synchronization of mitosis.

SUMMARYThe genome of Proasellus meridianus (2 n= 10) contains two kinds of constitutive heterochromatin: one, polymorphic and stainable with Chromomycin A3 located on certain telomeres, and the other centromeric and stainable with Hoechst 33258, visible only at pachytene. The NORs stainable with silver nitrate are terminal and located on at least three pairs of chromosomes. In diakinesis and first meiotic metaphase, usually the silver technique differentially stains the centromeric area of only one of the two sister chromatids of each homologue. This indicates a direct relationship between silver staining and kinetochore function. Surface spread of testicular tissue displays five synaptonemal complexes which are homogeneous throughout their length.