Juan J. Pasantes

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9

A human autosomal XY sex reversal locus, SRA1, associated with the skeletal malformation syndrome campomelic dysplasia (CMPD1), has been placed at distal 17q. The SOX9 gene, a positional candidate from the chromosomal location and expression pattern reported for mouse Sox9, was isolated and characterized. SOX9 encodes a putative transcription factor structurally related to the testis-determining factor SRY and is expressed in many adult tissues, and in fetal testis and skeletal tissue. Inactivating mutations on one SOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal. The 17q breakpoints in three CMPD1 translocation cases map 50 kb or more from SOX9.

A GC-rich satellite DNA and karyology of the bivalve mollusk Donax trunculus: a dominance of GC-rich heterochromatin

We characterized the DTF2 satellite DNA family of the clam Donaxtrunculus and compared its chromosomal localization with cytogenetic data revealed by fluorochrome banding, C-banding, and 28S rDNA FISH. In contrast to the other satellites detected previously in this species, DTF2 is an abundant (2%) GC-rich satellite that exhibits CpG methylation. Sequence characteristics of DTF2 indicate that its evolution is not affected by constraints that might indicate some functional interactions. Fluorescence in situ hybridization revealed subtelomeric location of this satellite on a subset of 14 out of 19 D. trunculus chromosome pairs. The chromomycin A3 (CMA) staining of GC-rich regions on D. trunculus chromosomes revealed a complex banding pattern that overlaps completely with C-bands. In total, only three bands show subtelomeric location, while 13 bands are located interstitially, one of them being coincident with the 28S rDNA hybridization signal. No bands, either CMA positive (GC-rich) or DAPI positive (AT-rich) were detected at centromeric chromosomal positions. Only two of the CMA-positive bands co-localize with the DTF2 satellite, showing a) the presence of small islands of GC-rich repetitive sequences that remained undetected by CMA/C-banding and b) the abundance of DTF2-divergent GC-rich sequences at interstitial chromosomal locations.

NOR activity in larval and juvenile mussels (Mytilus gallopro vincialis Lmk.)

Abstract The silver staining patterns of the nucleolus organizer regions (NORs), an indication of rDNA transcriptional activity, were studied in metaphase chromosomes from larvae and juveniles of a bivalve mussel, Mytilus galloprovincialis. Two submetacentric/subtelocentric pairs of chromosomes, out of the the total complement of 14 pairs, were characterized by the presence of silver-stained NORs. A considerable degree of inter- and intraindividual variation in NOR activity was noted. Mean NOR activity in larvae (3.27 active NORs per metaphase) was demonstrated to be higher than in the gill tissue of the juvenile mussels (1.93).

Analysis of NORs and NOR-associated heterochromatin in the mussel Mytilus galloprovincialis Lmk

The chromosomes of the mussel Mytilus galloprovincialis were analysed by means of chromomycin A3 (CMA), distamycin A/DAPI (DA/DAPI), DAPI/actinomycin D (DAPI/AMD) and chromomycin A3/distamycin A/DAPI(CDD) fluorescence banding techniques, C-banding, silver staining, N-banding and in situ hybridization with 18S+28S rDNA and telomere probes. 18S+28S rDNA clusters were located on the telomeres of two pairs of submeta/subtelocentric chromosomes. The nucleolar organizing regions (NORs) were associated with bright CMA fluorescence, dull DAPI fluorescence and C- and N-positive bands, but not all four NOR-associated heterochromatin bands showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found in this character. Additional non-ribosomal C-bands did not show any differential fluorescent behaviour.

Chromosomal mapping of rRNA genes, core histone genes and telomeric sequences in Brachidontes puniceus and Brachidontes rodriguezi (Bivalvia, Mytilidae)

BackgroundChromosome rearrangements are an important part of the speciation process in many taxa. The study of chromosome evolution in bivalves is hampered by the absence of clear chromosomal banding patterns and the similarity in both chromosome size and morphology. For this reason, obtaining good chromosome markers is essential for reliable karyotypic comparisons. To begin this task, the chromosomes of the mussels Brachidontes puniceus and B. rodriguezi were studied by means of fluorochrome staining and fluorescent in situ hybridization (FISH).ResultsBrachidontes puniceus and B. rodriguezi both have 2n = 32 chromosomes but differing karyotype composition. Vertebrate-type telomeric sequences appear at both ends of every single chromosome. B. puniceus presents a single terminal major rRNA gene cluster on a chromosome pair while B. rodriguezi shows two. Both mussels present two 5S rDNA and two core histone gene clusters intercalary located on the long arms of two chromosome pairs. Double and triple-FISH experiments demonstrated that one of the 5S rDNA and one of the major rDNA clusters appear on the same chromosome pair in B. rodriguezi but not in B. puniceus. On the other hand, the second 5S rDNA cluster is located in one of the chromosome pairs also bearing one of the core histone gene clusters in the two mussel species.ConclusionKnowledge of the chromosomal distribution of these sequences in the two species of Brachidontes is a first step in the understanding of the role of chromosome changes on bivalve evolution.

Cytogenetics in Brachidontes rodriguezi d'Orb (Bivalvia, Mytilidae)

The chromosomes of Brachidontes rodriguezi were analysed by means of direct Giemsa staining, silver staining, fluorescent in-situ hybridization (FISH) with 18S + 28S rDNA probes, replication banding and chromomycin A3 (CMA) and DAPI fluorescence banding techniques. The diploid chromosome number in this species is 32 and the karyotype is composed of two pairs of metacentric chromosomes, 2 pairs of telo/subtelocentric chromosomes and 12 pairs of subtelocentric chromosomes. 18S + 28S rDNA clusters were located on the short arms of the two pairs of telo/subtelocentric chromosomes. The replication band pattern induced in this species facilitates chromosome pairing and differentiation. The nucleolar organizing regions (NORs) replicate late in the S phase and were associated with bright CMA fluorescence and dull DAPI fluorescence, but not all the four NORs showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found for this character.

Proliferation kinetics of mussel (Mytilus galloprovincialis) gill cells

A technique of sister chromatid differentiation (SCD) using bromodeoxyuridine (BrdU) incorporation and a modification of the fluorescence plus Giemsa (FPG) method was employed to determine cell-proliferation kinetics in gill tissue of the mussel Mytilus galloprovincialis. Dose-dependent proliferation inhibition was examined. In vivo administration of BrdU for 12, 24, 36, 48, 60, 72, 84, and 96 h was studied. Our data show that the highest yield of second-generation metaphase plates is obtained after 60 h BrdU treatment; the duration of a cell cycle is 24 to 30 h. On the basis of these data, a BrdU incorporation period of 48 to 60 h would seem to be most appropriate for the sister chromatid exchange (SCE) tests carried out in gill cells of M. galloprovincialis, whereas the 12 to 24 h exposure would give the best results for replication band analysis.

Comparative mapping of Xp22 genes in hominoids – evolutionary linear instability of their Y homologues

Several genes located within or proximal to the human PAR in Xp22 have homologues on the Y chromosome and escape, or partly escape, inactivation. To study the evolution of Xp22 genes and their Y homologues, we applied multicolour fluorescence in situ hybridization (FISH) to comparatively map DNA probes for the genes ANT3, XG, ARSD, ARSE (CDPX), PRK, STS, KAL and AMEL to prometaphase chromosomes of the human species and hominoid apes. We demonstrate that the genes residing proximal to the PAR have a highly conserved order on the higher primate X chromosomes but show considerable rearrangements on the Y chromosomes of hominoids. These rearrangements cannot be traced back to a simple model involving only a single or a few evolutionary events. The linear instability of the Y chromosomes gives some insight into the evolutionary isolation of large parts of the Y chromosomes and thus might reflect the isolated evolutionary history of the primate species over millions of years.

Banding pattern of mussel (Mytilus galloprovincialis) chromosomes induced by 2 × SSC/Giemsa-stain treatment

Mytilus galloprovincialis were collected from an intertidal population in NW Spain in 1988, and chromosomes from the gill tissue of 37 individuals were studied. The present paper describes the banding pattern of aM. galloprovincialis population which enabled us to identify all pairs of chromosomes. Banding was induced by means of a 2 × SSC (0.3M sodium chloride:0.03M sodium citrate)/Giemsa-staining technique, and a diagrammatic representation was constructed based on mean number of bands. The application of this banding technique may prove useful in future research related to the cytogenetics and cytotaxonomy of mussels and other molluscs.

Surface spreading of synaptonemal complexes in the clam Dosinia exoleta (Mollusca, Bivalvia)

There are only a few reports on the chromosomal location of DNA sequences in bivalve species, none of them using meiotic chromosomes. Mitotic chromosomes of the clam Dosinia exoleta were analysed by means of Giemsa, silver and fluorochrome staining and fluorescent in situ hybridization (FISH) with 18S + 28S rDNA and telomeric probes. A technique for surface spreading of synaptonemal complexes (SCs) of Dosinia exoleta was developed for the first time in a bivalve species. Silver and DAPI/PI staining and SC-FISH were also applied to the study of the meiotic chromosomes of this clam. The diploid chromosome number in this species is 38 and the karyotype is composed of 11 pairs of metacentric and eight pairs of submetacentric chromosomes. 18S + 28S rDNA clusters map to the subtelomeric region of the short arm of one metacentric chromosome pair whereas telomeric signals appear at both ends of every chromosome.