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Arvicolid rodents present both synaptic and asynaptic sex chromosomes. We analyzed the pairing behaviour of sex chromosomes in two species belonging to this rodent group (Microtus nivalis and Arvicola sapidus). At pachynema, the sex chromosomes of both species paired in a small region while the rest remain unsynapsed. Consequently at metaphase I, sex chromosomes present end-to-end association. Thus, the pairing behaviour of sex chromosomes in these species is very similar to that previously described for other arvicolid rodents and for most mammals. According to this, we propose that synaptic sex chromosomes were the ancestral condition in the family Arvicolidae, including the genus Microtus. The phylogenetic origin of the asynaptic sex chromosomes in the genus Microtus would have arisen once in the lineage that originated the species M. arvalis/agrestis and related species, while the lineage that originated the species M. oeconomous and related species conserved synaptic chromosomes. Furthermore, the phylogenetic relationships between the genus Microtus, Chionomys and Pitymys are discussed in relation to the synaptic behaviour of sex chromosomes.

The SRY gene is a single-copy, male-specific gene, located on the Y chromosome in most mammals. However, recently we have described the presence of multiple polymorphic copies of this gene in both males and females of the vole species Microtus cabrerae. Here, we present the chromosomal localization of SRY gene copies in this species by fluorescent in situ hybridization (FISH). This technique localized these gene copies in the short arm, and hence in the euchromatic region, of the Y chromosome. Furthermore, several copies of the SRY gene are located on the X chromosome. These copies are spread along the entire heterochromatic region of the X chromosome, occupying the whole short arm, the centromeric region, and the pericentromeric region of the long arm.Key words: FISH mapping, Micotus cabrerae, SRY gene, X chromosome, Y chromosome.

The genus Microtus presents several species with extremely large sex chromosomes that contain large blocks of constitutive heterochromatin. Several cytogenetic and molecular studies of the repetitive sequences in species of the genus Microtus have demonstrated that the heterochromatin is highly heterogeneous. We have cloned and characterized a family of repetitive DNA sequences from M. cabrerae, a species with large heterochromatic blocks on the giant sex chromosomes. These repetitive sequences are 65.84% A–T rich, organized in tandem, with a 161-bp unit and are located on the centromeric region of autosomes and the X chromosome. In addition, this repetitive DNA is located throughout the entire heterochromatic block of the X chromosome and on three interstitial bands in the heterochromatic block of the Y chromosome. Comparative analysis of this family of repetitive sequences from three Microtus species revealed that the development of these sequences has occurred by concerted evolution. Our results support the hypothesis that the heterochromatic blocks from the sex chromosomes of different species are evolving independently and they probably have the genetic capacity to amplify and retain different satellite DNAs. For a topic related to the location of these repetitive DNA sequences on the Y chromosome of M. cabrerae, we propose a model to explain the origin of a length polymorphism previously described for this chromosome.

RAFAEL JIMI~NEZ*, ANTONIO S,6NCHEZ :t:, MIGUEL BURGOS AND RAFAEL DiAZ DE LA GUARDIA mburgos@goliat.ugr.es DEPART&~IEYFO DE GENETIC.A, FACb'LTAD DE CIE.XClM, UNIVERSIDAD DE GP,&'~ADA, r UENqF2~a:'EVA S/N, E-18071 GR~'~ADA, SPAIN. * DEPARTa.',IEVfO DE BIOLOGiA EXPEPL~IEN'rAL Y CIENCIAS DE LA SALUD, FA~'LTAD DE CIENC1AS F.2(PERL'*~N'rALES, UNIVERSIDAD DE JAEN, Mammals possess an XX/XY chromo- somal system for sex determination. The presence of SRY, a master- regulatory gene on the Y chromosome, is necessary to induce the undifferen- tiated, bipotential gonadal primor- dium (the embryonic genital ridge) to develop as a testis. In the absence of SRY, it develops as an ovary I. Once the gonads differentiate, their male- or female-specific endocrine function is responsible for the rest of the events involved in the pheno~pic ~_~..'.=l dif- fcrenuation process 2. Accordingly, in mammals, sex determination can be equated with testis determination. SRY is the only gene currently known to be involved in the process of sex determination. A number of cloned genes probably participate in gonadal development: the Mtillerian inhibiting substance gene (MIS; also called the antimiillerian hormone gene, AMH)3, the SRY-related gene, SOX9 (ReL 4), the mouse gene en- coding steroidogenic factor (SF1), FtzF1 (Ref. 5), the X-linked gene, DAXI (Refs 6, 7), and the Wilm's tumor suppressor gene, WT1 (Ref. 8). Whereas MIS and SOX9 are probably involved in the male pathway, the moment and site of action of the rest of these genes in the sex-differ- entiation process is unclear. FtzF1 and WT1 might be involved in the formation of the bipotential gonad, thus acting before SRY. The female pathway remains almost completely unknown. An X-linked, dosage-sen- sitive gene (DSS), which might be identical to DAXI, causes male-to- female sex reversal when duplicated, anti has been proposed to be a member of the female pathway 9.1°, but the findings on this point are inconclusive (see below). Therefore, mammalian sex-determination is, in fact, an unsolved genetic puzzle from which a number of pieces are prob- at~ly still missing.

In the rodent species Microtus cabrerae, males as well as females present several copies of the SRY gene, a single-copy gene located on the Y chromosome in most mammals. Using different PCR approaches, we have characterized the sequence, structure, and organization of the SRY copies and their flanking regions distributed on the X and Y chromosomes of this species. All copies of SRY analyzed, including those from the Y chromosome, proved to be nonfunctional pseudogenes, as they have internal stop codons. In addition, we demonstrated the association of SRY pseudogenes with different fragments of L1 and LTR retroelements in both sex chromosomes of M. cabrerae. Examining the possible origin of SRY pseudogene and retroposons association, we propose that retroposons could have been involved in the mechanism of SRY gene amplification on the Y chromosome and in the transference of the Y-linked SRY copies to the X-chromosome heterochromatin.