R. Díaz de la Guardia

A study of the Ag-staining significance in mitotic NOR's

The biological significance of nucleoli and mitotic Ag-staining NORs has been the cause of controversy for several years. Whereas several authors state or assume a direct correlation between transcriptional activity and Ag-staining, others deny this hypothesis or claim a correlation between Ag-staining and decondensation of the NOR chromatin. Our results show that a decondensed state of the NOR chromatin is necessary but not sufficient to permit silver impregnation, allowing us to conclude that transcriptional NOR activity in the previous interphase is additionally required for silver staining.

Inactive ribosomal cistrons are spread throughout the B chromosomes of Rattus rattus (Rodentia, Muridae). Implications for their origin and evolution.

In-situ hybridization with a rDNA probe has demonstrated the presence of non-transcribed ribosomal genes in the B chromosomes of the black rat Rattus rattus. To test whether methylation of ribosomal DNA present in the B chromosomes could account for their inactivation, we performed in-situ digestions and Southern analyses of DNA digested with the isoschizomers MspI and HpaII. Our results suggest that the accessory chromosomes of this species have originated from one of the smaller NOR-carrying chromosome pairs. In the course of evolution, repetitive sequences invaded this supernumerary element and its ribosomal DNA content was dispersed throughout the chromosome and inactivated by heterochromatinization and methylation.

Multiple, polymorphic copies of SRY in both males and females of the vole Microtus cabrerae

In mammals, sex determination is controlled by the Y-linked gene SRY. Although SRY is male-specific in most eutherian and marsupial species, with a single copy on the Y chromosome, several rodent species have multiple Y-linked copies of SRY, and two mole-vole species of the genus Ellobius determine sex without the Y chromosome or the SRY gene. We searched for homologs of SRY in three vole species of the genus Microtus and concluded that this gene is not male-specific in M. cabrerae, as it is present in multiple, polymorphic copies in both males and females. In contrast, SRY is male-specific in the related species M. agrestis and M. nivalis. Up to 15 different partial sequences of the SRY gene were found in M. cabrerae. Southern blots suggest that most of the extra copies of SRY are X-linked. One of the copies observed only in males has a sequence identical to that of the SRY gene in M. agrestis and may represent a functional copy of the gene in this species. The rest are probably nonfunctional pseudogenes.

Heterogeneous heterochromatin and size variation in the sex chromosomes of Microtus cabrerae.

The sex chromosomes of Microtus cabrerae are characterized by large segments of constitutive heterochromatin in both the X and Y. Alkali treatment, enzymatic digestion (trypsin), and fluorochrome staining reveal considerable heterogeneity within these heterochromatic blocks and allow us to distinguish several regions. In addition, variation in the length of these segments is also present, probably as a result of deletions. A possible explanation for the origin of these heterochromatic blocks is discussed.

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the ‘selfish DNA’ hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged (‘giant’) sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.

Multiple mono- and polymorphic Y-linked copies of the SRY HMG-box in Microtidae

Sex determination in mammals is controlled by SRY (sex-determining region of the Y chromosome), a single-copy gene located on the Y-specific region. Several exceptions to this rule have been described: some rodent species present Y-specific multiple copies (either mono- or polymorphic) of this gene, and two Ellobius species and one Tokudaia species determine sex without a Y chromosome or the SRY gene. Recently, we have described multiple polymorphic copies of the SRY gene in both males and females of the vole species Microtus cabrerae. The female location and the presence of stop codons in some copies from males and females also suggest that they are nonfunctional copies of this gene (pseudogenes). We have investigated the SRY HMG-box in nine species of the family Microtidae; we report here the presence, in eight of these species, of multiple mono- or polymorphic copies of the SRY gene located on the Y chromosome.

Achiasmatic sex chromosomes in Pitymys duodecimcostatus: mechanisms of association and segregation

The meiotic behavior of the sex chromosomes of Pitymys duodecimcostatus was studied by electron microscopy of whole-mount synaptonemal complex preparations. The results established that the sex chromosomes of this species are achiasmatic and remain unassociated throughout meiotic prophase I in most spermatocytes. In other cells, nonspecific association of the X and Y occurred by means of filamentous bridges. Pitymys duodecimcostatus represents an additional example of a mammalian species lacking a homologous pairing segment in its sex chromosomes and extends current knowledge about this controversial subject. In this regard, we suggest that sex-chromosome association is a characteristic that probably followed different evolutionary paths in different mammals, leading to loss of the homologous segment in some species and its conservation in others. It is also suggested that in P. duodecimcostatus, and probably in many other species as well, three mechanisms may act in concert to permit joining of the X and Y chromosomes during meiotic prophase, and, consequently, to ensure proper segregation during anaphase I: (1) joining of the sex-chromosome axes at their ends to the nuclear membrane, (2) formation of fibrillar structures to hold the sex chromosomes together, and (3) cohesiveness due to sex-vesicle formation.

Sex chromosomes, sex determination, and sex-linked sequences in Microtidae

The Arvicolidae is a widely distributed rodent group with several interesting characteristics in their sex chromosomes. Here, we summarize the actual knowledge of some of these characteristics. This mammalian group has species with abnormal sex determination systems. In fact, some species present the same karyotype in both males and females, with total absence of a Y chromosome, and hence of SRY and ZFY genes. Other species present fertile, sex-reversed XY females, generally due to mutations affecting X chromosomes. Furthermore, in Microtus oregoni males and females are gonosomic mosaic (the females are XO in the soma and XX in the germ cells, while the males are XY in the soma and OY in the germ cells). Regarding sex chromosomes, some species present enlarged (giant) sex chromosomes because of the presence of large blocks of constitutive heterochromatin, which have been demonstrated to be highly heterogeneous. Furthermore, we also consider the alterations affecting composition and localization of sex-linked genes or repeated sequences. Finally, this rodent group includes species with synaptic and asynaptic sex chromosomes. In fact, several species with asynaptic sex chromosomes have been described. It is interesting to note that within the genus Microtus both types of sex chromosomes are present.

Recent evolution of NOR-bearing and sex chromosomes of the North African rodent Lemniscomys barbarus

The karyotype and meiotic phases of Lemniscomys barbarus from Morocco were extensively studied with G- and C-banding, Ag-NOR and fluorochrome staining, in situ hybridization with an rDNA probe, and synaptonemal complex analysis. Comparison of the data with those previously published for an Algerian specimen revealed in the Moroccan specimens the presence of large heterochromatic segments in the sex chromosomes, a new nucleolar organizer at chromosome pair 1, and silent NORs on both sex chromosomes — features that are not present in the Algerian specimen. These findings demonstrate that during the very recent evolution of the karyotype of this species a new NOR was acquired by pair 1, possibly by amplification of ribosomal genes after a translocation event. This new NOR changed the preference of activatio n of the NORs in these individuals and became the preferentially activated NOR. Another autosome—sex chromosome translocation led to the presence of NORs on the sex chromosomes, which were then inactivated by the invasion of repetitive sequences. These silent NORs may be involved in the pairing of the two sex chromosomes.

Fluorescence banding in four species of Microtidae: an analysis of the evolutive changes of the constitutive heterochromatin

Three different fluorochrome and specific counterstain combination (DAPI/AMD, DA/DAPI and CMA/DA) treatments were applied to the chromosomes of four Microtidae (Rodentia) species. The results complete the data obtained in our previous paper (Burgos, M., Jiménez, R., & Dìaz de la Guardia, R., Genome 30:540–546, 1988) and prove that the changes in the constitutive heterochromatin in the evolution of the karyotypes of these species are not only due to gain or loss of heterochromatin, but are qualitative with respect to their nucleotide composition, repeated base pair organization or DNA-protein complex modification. These variations lead to the differential response to the fluorescence dye combinations used.