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.

Host humoral immune response to Leishmania lipid-binding protein.

SUMMARY We report on the use of Leishmania donovani lipid-binding proteins (LBPs) as antigens capable of being recognized by serum from immunocompetent patients from southern Spain suffering from visceral leishmaniasis and from Peruvian patients with localized cutaneous leishmaniasis caused by Leishmania braziliensis. The absorbance found by immunoenzymatic techniques gave significantly different results for the serum samples from patients with and without leishmaniasis. Specificity by ELISA testing was 93.2% and sensibility 100%. Dot blots from human patient serum samples or naturally infected dogs from Spain gave similarly significant results. All the human serum samples from individuals with visceral leishmaniasis and the Leishmania-positive canine samples recognized two bands, with molecular weights of 8 and 57 kDa. The serum from individuals with cutaneous leishmaniasis caused by L. braziliensis recognized an additional band of 16 kDa. We discuss the role of Leishmania FABP and compare the immunological reactions found with serum samples from other protozoan infections such as toxoplasma and Chagas as well as bacterial infections such as tuberculosis and syphilis.

Characterization of an EcoRI family of satellite DNA from two species of the genus Eptesicus (Vespertilionidae; Chiroptera).

We have cloned and sequenced a 321 bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321 bp which is composed of two subunits of 160 bp, suggested by the existence of a 160 bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.

Transmission analysis of B chromosomes in Rattus rattus from Northern Africa.

Traditionally, B chromosomes have been classified as parasitic or heterotic, depending of whether or not they show selfish behaviour. Nevertheless, experimental evidence has been found supporting the idea that supernumerary chromosomes may evolve from parasitism to neutrality. In this work, B chromosome transmission in Rattus rattus has been analysed by performing several crosses between individuals carrying different numbers of supernumerary chromosomes. Our results demonstrated a Mendelian transmission rate through males, but slight accumulation of the Bs through females. This parasitic behaviour is shared in populations as distant as Asia and Africa, and even in a related species in Australia, suggesting the possibility of an ancient origin of these supernumerary chromosomes.

X chromosome painting in Microtus: origin and evolution of the giant sex chromosomes.

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as 'giant sex chromosomes') due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.

A repeat DNA sequence from the Y chromosome in species of the genus Microtus.

In most mammals, the Y chromosome is composed of a large amount of constitutive heterochromatin. In some Microtus species, this feature is also extended to the X chromosome, resulting in enlarged (giant) sex chromosomes. Several repeated DNA sequences have been described in the gonosomal heterochromatin of these species, indicating that it has heterogeneous and species-specific composition and distribution. We have cloned an AT-rich, 851-bp long, repeated DNA sequence specific for M. cabrerae Y chromosome heterochromatin. The analysis of other species of the genus Microtus indicated that this sequence is also located on the Y chromosome (male-specific) in three species (M. agrestis, M. oeconomus and M. nivalis), present on both Y and X chromosomes and on some autosomes in M. arvalis and absent in the genome of M. guentheri. Our data also suggest that the mechanism of heterochromatin amplification operating on the sex chromosomes could have been different in each species since the repeated sequences of the gonosomal heterochromatic blocks in M. cabrerae and M. agrestis are different. The absence of this sequence in the mouse genome indicates that its evolutionary origin could be recent. Future analysis of the species distribution, localization and sequence of this repeat DNA family in arvicolid rodent species could help to establish the unsolved phylogenetic relationships in this rodent group.

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.

Pericentric satellite DNA sequences in Pipistrellus pipistrellus (Vespertilionidae; Chiroptera).

This paper reports the molecular and cytogenetic characterization of a HindIII family of satellite DNA in the bat species Pipistrellus pipistrellus. This satellite is organized in tandem repeats of 418 bp monomer units, and represents approximately 3% of the whole genome. The consensus sequence from five cloned monomer units has an A-T content of 62.20%. We have found differences in the ladder pattern of bands between two populations of the same species. These differences are probably because of the absence of the target sites for the HindIII enzyme in most monomer units of one population, but not in the other. Fluorescent in situ hybridization (FISH) localized the satellite DNA in the pericentromeric regions of all autosomes and the X chromosome, but it was absent from the Y chromosome. Digestion of genomic DNAs with HpaII and its isoschizomer MspI demonstrated that these repetitive DNA sequences are not methylated. Other bat species were tested for the presence of this repetitive DNA. It was absent in five Vespertilionidae and one Rhinolophidae species, indicating that it could be a species/genus specific, repetitive DNA family.

Highly repeated DNA sequences in three species of the genus Pteropus (Megachiroptera, Mammalia).

Bat genomes are characterised by an A-T richness and by a small C-value compared with other mammalian groups. It has been suggested that the small C-value is mainly due to the lack of repetitive DNA sequences. However, little information about repetitive DNA sequences in this mammalian group is available at the molecular level. Here we describe a PstI family of repetitive DNA sequences present in three species of the genus Pteropus. These repetitive sequences are 54.97% G-C rich, organised in tandem and with a unit length of 744 bp. Methylation analysis indicates that some of the CCGG target sites present in these repetitive DNA sequences have methylated cytosines and that there are small differences in the methylation pattern between species. Several features of this family of repetitive sequences suggest that they evolved by concerted evolution.

Human secretory immune response to fatty acid-binding protein fraction from Giardia lamblia.

The secretory immune response in humans infected with Giardia lamblia was studied by using saliva samples and an 8-kDa antigen capable of binding fatty acids. This antigen was not recognized by saliva samples from healthy individuals. The antigen may be useful in diagnostic studies of G. lamblia infection.

New C-band protocol by heat denaturation in the presence of formamide.

C-banding techniques detect the presence of constitutive heterochromatin, which is usually located in centromeric regions of chromosomes in the majority of analysed species. The common method for C-banding used over the last 30 years involves treatment with a mild alkali barium hydroxide 5% Ba(OH)2 at 50 degrees C for 5-15 min and subsequent incubation in salt solution (2 x SSC at 60 degrees C for 1 h). We here present a new, easy and reliable technique for C-banding, which basically involves heat denaturation of chromosomal DNA in the presence of formamide and incubation in 2 x SSC at room temperature.

Repeated DNA sequences in the microbat species Miniopterus schreibersi (Vespertilionidae; Chiroptera).

Repetitive DNA sequences represent a substantial component of eukaryotic genomes. These sequences have been described and characterized in many mammalian species. However, little information about repetitive DNA sequences is available in bat species. Here we describe an EcoRI family of repetitive DNA sequences present in the species Miniopterus schreibersi. These repetitive sequences are 57.85%, A-T rich, organized in tandem, and with a monomer unit length of 904 bp. Methylation analysis using the isoesquizomer pair MspI and HpaII indicates that the cytosines present in the sequences CCGG are partially methylated. Furthermore, Southern blot analysis demonstrated that these DNA sequences are absent in the genomes of four related microbat species and suggest that it could be specific to the M. schreibersi genome.

Silent ribosomal cistrons are located at the pairing segment of the postreductional sex chromosomes of Apodemus sylvaticus (Rodentia, Muridae).

We analysed the karyotype of the rodent species Apodemus sylvaticus by G- and C-banding, Ag-NOR-staining and in situ hybridization, with special attention to the sex chromosomes. NOR-bearing chromosome pairs were identified with simultaneous staining of G bands and NORs. In situ hybridization with an rDNA probe revealed the presence of silent ribosomal cistrons in both sex chromosomes. Studies of meiosis demonstrated that these inactive ribosomal cistrons are located in the pairing segment, which occupies the proximal portion of the telocentric sex chromosomes, and may thus be involved in postreduction caused by an obligatory chiasma in this position.

Molecular and cytogenetic characterization of highly repeated DNA sequences in the vole Microtus cabrerae.

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.

Sex-chromosome pairing through heterochromatin in the African rodent Lemniscomys barbarus (Rodentia, Muridae). A synaptonemal complex study.

Giemsa-stained spread preparations and microspread preparations of Lemniscomys barbarus spermatocytes were made to investigate the meiotic behaviour of the peculiar sex chromosomes of this species. A typical sex vesicle is absent, as the X and Y chromosomes appear unfolded at zygotene and pachytene. In most cells, the sex chromosomes are associated at distal segments at metaphase I, probably as a consequence of a distal chiasma. The pairing segment is located in the heterochromatic regions of both sex chromosomes, which include silent ribosomal cistrons interspersed throughout the heterochromatin. This may suggest a possible involvement of ribosomal genes in both pairing and recombination processes. X-Y pairing proceeds beyond the pseudoautosomal region, thus involving heterologous segments of the differential regions, a fact that is clearly evident at the Y centromeric region.

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.

The SRY gene HMG-box in micro- and megabats.

Sex determination in mammals is controlled by the Y-linked SRY gene, which encodes a transcription factor with a DNA-binding motif of the HMG type. The only conserved region in this gene is the HMG-box, whose nucleotide sequence is currently available in a number of mammalian taxa. However, nothing is known about this gene in bats. Here, we report partial sequences of the SRY HMG-box from four microbat and four megabat species. We used the SRY HMG- box sequences from micro- and megabats to test the phylogenetic relationships between microbats, megabats, and primates. In maximum parsimony and maximum-likelihood trees, mega- and microbat branches start in the same internal node, which is consistent with a monophyletic origin of this mammalian group.

HMG-box sequences from microbats homologous to the human SOX30 HMG-box.

SOX genes are a family of genes that encode for proteins which are characterised by the presence of a HMG-domain related to that of the mammalian sex-determining gene (SRY). By definition, the DNA binding domain of SOX genes is at least 50% identical to the 79 amino acid HMG domain of the SRY gene. We report here two HMG-box sequences from two microbat species (R. ferrumequinum and P. Pipistrellus) which were PCR amplified using a primer pair specific to the mouse Sry HMG-box. The high percentage of identity of this sequences with the human and mouse SOX30 HMG-box suggests that they are the SOX30 HMG-box for these two bat species.

The relative rDNA content of a NOR determines its level of expression and its probability of becoming active. A sequential silver staining and in-situ hybridization study.

Silver staining was used to estimate the expression of nucleolar organizing regions (NORs), and in-situ hybridization (ISH) with rDNA probes was used to estimate the relative content of rDNA in each NOR in chromosome preparations of the dormouse, Eliomys quercinus, a species with two NOR-bearing chromosome pairs. Both types of signals were sequentially investigated on every NOR by using an Ag-ISH sequential staining method, which made it possible to demonstrate that the relative amount of rDNA in a NOR in comparison with the other chromosomes of the complement determines its level of expression and its likelihood of becoming active, regardless of whether the NORs are homologous or not. We suggest that the NORs in each cell are activated in accordance with an established hierarchy. We propose a two-stage model to relate NOR structure and function, which is consistent with these results and with current knowledge on the molecular regulation of NOR transcription.

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.