M. Ruiz Rejón

Cytogenetic and molecular analysis of the multiple sex chromosome system of Rumex acetosa

A repeated sequence of 180 bp in tandem array has been isolated from the dioecious plant species Rumex acetosa which has a multiple sex chromosome system, XX♀/XY1Y2♂. There are two or three thin C-bands on the X while the Ys are almost entirely heteropycnotic and DAPI-positive but contain no C-band material. The Ys thus represent massive blocks of facultative heterochromatin. The 180 bp sequence has been localized to the X and both Y chromosomes by in situ hybridization. The molecular data support an origin of the sex-multiple from a homomorphic chromosome pair rather than from a sex chromosome/autosome translocation.

Cloning and characterization of a fish centromeric satellite DNA

A highly repetitive DNA sequence family from the genome of Sparus aurata has been cloned and characterized. The family is composed of repeat units of 186 bp in length, and it accounts for 2% of the fish genome. Data from Southern blots and in situ hybridization demonstrate that repeating units are tandemly arranged at the centromeres of all the chromosomes in this species. The repetitive sequence is AT rich (67%) and is characterized by short stretches of consecutive AT base pairs and by short direct and inverted repeats. Sequence analysis of six cloned monomers of the family reveals some variation among clones at random positions and also distinguishes two subfamilies of repeats that differ in a highly divergent block of 31 bp. These two subfamilies do not seem to be located in separate domains but occur together in the centromere of each chromosome pair. The presence of this repeat family in the genome of other Sparidae species, some of which are relatively distant from S. aurata, indicates that this repetitive sequence could be an important component of the centromere in this fish family.

A satellite DNA of the Sparidae family (Pisces, perciformes) associated with telomeric sequences

This paper reports on the isolation and localization of the subtelomeric DraI satellite DNA in the Sparidae family. Gene cloning determined that the DraI satellite DNA is present in only 3 species (Pagrus pagrus, P. auriga, and Pagellus erythrinus) of the 10 Sparidae species analyzed. The results were confirmed by PCR amplification. This satellite DNA is located in a subtelomeric position in all 48 acrocentric chromosomes of these species. However, interstitial loci are also observed. Sequence analysis of monomers of this repetitive family indicates that the satellite DNA is associated with telomeric sequences, (TTAGGG)n, in at least one species, P. erythrinus. This is the first direct demonstration of the existence of the consensus telomere sequences of vertebrates in fish. Likewise, this report also demonstrates that the ends of fish chromosomes have a structure similar to those of most eukaryote chromosomes, viz., telomere sequences and subtelomeric sequences associated by a boundary in which both types of sequences are interspersed. The recent origin of the DraI satellite DNA and its use as a phylogenetic marker is discussed.

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.

The EcoRI centromeric satellite DNA of the Sparidae family (Pisces, Perciformes) contains a sequence motive common to other vertebrate centromeric satellite DNAs

By means of cloning, sequencing, and fluorescence in situ hybridization, we have determined that the EcoRI satellite DNA family is conserved in the 10 sparid species analyzed here. Its conservation, its chromosomal location at the centromere of each chromosome, and its structural features could make this satellite DNA family an important structural and/or functional element of the centromeres of these species. Monomeric units of this satellite DNA have a consensus length of 187 bp. Its sequence is characterized by a high AT content and the presence of short runs of consecutive AT base pairs. These monomeric EcoRI repeats also contain three to four copies, depending on the species, of a short sequence reflecting the repetitive duplication and subsequent divergence of an ancestral 9-bp sequence in this family. This sequence motive is conserved in some parts of the monomeric units of the different species studied at the same positions, and, precisely, surrounding the area in which the curvature of the monomeric molecule is greatest. The 9-bp sequence motive is similar to other direct-repeat sequences of the centromeric satellite DNAs of other vertebrates, including those of amphibians and mammals.

Molecular characterization of the ribosomal RNA gene region of Perkinsus atlanticus: its use in phylogenetic analysis and as a target for a molecular diagnosis.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S-5S-18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.

Chromosomal structure of populations of Scilla autumnalis in the Iberian Peninsula

Four cytological races of Scilla autumnalis, Liliaceae, were found in a study of 31 populations from Spain and Portugal. Two are diploids with 2n = 14, designated AA and B7B7, which differ by 70 per cent in DNA content, one is an allotetraploid AAB7B7 and one an autotetraploid of the B7 genome. Races AA and AAB7B7 are Iberian endemics, B7B7 is widespread throughout the Mediterranean, while the autotetraploid is the common European race. In Iberia, the races are parapatric and distribution is not related to climatic conditions. The populations are chromosomally heterogeneous. Polymorphisms include B-chromosomes (five types) and supernumerary segments. Large euchromatic segments occur on the homoeologues Al and Bl in both diploids and the allotetraploid, converting these acrocentrics to metacentrics. A wide spectrum of non-polymorphic numerical and structural variants was also found. The chromosomal structure of this species complex is discussed.

A method for increasing the number of mitoses available for cytogenetic analysis in rainbow trout.

A new method for the cytogenetic analysis of fishes with special interest for the rainbow trout (Salmo gairdneri Rich.) is described. This reliable method that includes treatment with a yeast solution provides high quality spreads of a great number of metaphases.

Cytogenetic analysis of gilthead seabream Sparus aurata (Pisces, Perciformes), a deletion affecting the NOR in a hatchery stock

We have cytogenetically characterized a hatchery stock of gilthead seabream, Sparus aurata. The study included larvae, juveniles and adults. In S. aurata (diploid chromosome number 2n = 48), a pair of NORs is located at the ends of the short arms of the first submetacentric pair of chromosomes. In this stock we discovered a polymorphism which affects the NORs, and, by means of several cytogenetic and molecular techniques, we demonstrate that this polymorphism is due to the complete deletion of one of the two NORs in a high number of individuals. The significance of these cytogenetic characteristics for this species are discussed since they may be the source of aquaculture problems.

An analysis of coho salmon chromatin by means of C-banding, AG- and fluorochrome staining, and in situ digestion with restriction endonucleases

The chromosome complement of the coho salmon (Oncorhynchus kisutch) has been analysed by means of C-banding, silver and fluorochrome staining, and in situ digestion with restriction endonucleases. C-banding shows heterochromatic regions in the centromeres of most chromosomes but not in the telomeric areas. The fifteenth metacentric chromosome pair contains a large block of constitutive heterochromatin, which occupies almost all of one chromosome arm. This region is also the site where the ribosomal cistrons are located and it reacts positively to CMA3/DA fluorochrome staining. The NORs are subject to chromosome polymorphism, which might be explicable in terms of an amplification of ribosomal cistrons. The digestion banding patterns produced by four types of restriction endonucleases on the euchromatic and heterochromatic regions are described. Two kinds of highly repetitive DNAs can be distinguished and the role of restriction endonucleases as a valuable tool in chromosome characterization studies, as well as in the analysis of the structure and organization of fish chromatin, are also discussed.