[Optimization of the conditions for RAPD-PCR of Candida spp. and Cryptococcus spp]. / Optimización de las condiciones RAPD-PCR en Candida spp. y Cryptococcus spp.

In this study, we determined the optimal RAPD amplification conditions to obtain genetic molecular markers for the rapid and accurate identification of Cryptococcus spp. and Candida spp. The following parameters are modified: template DNA, DNA polymerase, magnesium cloride and primer concentration; denaturation, annealing and extension time, temperature of annealing and thermal cycles. After the optimization, reliable and reproducible RAPD patterns are obtained.

Genetic variability in the Iberian imperial eagle (Aquila adalberti) demonstrated by RAPD analysis.

RAPD analysis was used to estimate the genetic diversity in an Iberian imperial eagle (Aquila adalberti) population, one of the most threatened bird species in the world. Forty-five of 60 arbitrarily designed primers amplified 614 loci in 25 individual eagles, 59.7% of which were polymorphic. In contrast to the traditional allozyme analysis performed in a previous study, the RAPD method has revealed a high level of heterozygosity in this species (H = 0.267+/-0.008). The genetic distances estimated between 25 eagles can serve to establish more adequate mating in order to preserve genetic variability. Conservation efforts being carried out in Spain in this species might be successful based on the results obtained in the present work.

The karyotype of the Iberian imperial eagle (Aquila adalberti) analyzed by classical and DNA replication banding.

We report here for the first time the karyotype of the Iberian imperial eagle (Aquila adalberti). All eagles examined had a diploid number of 82 chromosomes and a greater number of microchromosomes (12 pairs) than has been found in all other species of the Accipitridae family. This karyotypic evidence corroborates the recent separation of A. adalberti from A. heliaca on the basis of molecular data. RB-FPG banding induced a specific banding pattern that allowed us to identify homologous chromosome pairs and revealed features about late and early replicating regions. Several chromosome banding techniques (C-, CMA3-, and restriction endonuclease banding and silver staining) were used to characterize the karyotype more accurately. Two GC-rich, late-replicating heterochromatin regions were found in the W chromosome. These regions are AluI resistant and can be used for sex determination in this species. All microchromosomes were heterochromatic, GC rich, and late replicating. Silver staining revealed active nucleolus organizing regions on a pair of microchromosomes that were entirely heterochromatic and stained intensely after CMA3-banding. Different chromosome rearrangements are discussed in order to establish the phylogenetic relationship between A. adalberti and its most closely related species, A. heliaca.

Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts.

The mucopolysaccharide capsule of Cryptococcus neoformans and other pathogenic yeasts prevent the extraction of DNA from these important zoonotic agents. We report that the use of a lysis buffer containing a high concentration of urea is an easy, efficient and time-saving technique to obtain high yields of good-quality DNA for molecular diagnosis. The use of urea also prevents the degradation of DNA during storage of samples at room temperature for up to 6 months.

Characterization of the heterochromatic chromosome regions in sheep.

In order to elucidate the structural chromosome organization of the heterochromatic regions in sheep, we have used C-banding, silver-staining, sequential CDD technique and restriction endonuclease banding. By these banding techniques we obtained four fractions of repetitive DNA, the autosomal fractions A and B, the C fraction in the X chromosome, and the D fraction in the Y chromosome. Silver staining revealed active nucleolus organizer regions (NOR's) on the telomeric GC-rich areas of chromosomes 1, 2, 3, 4, and 25 which were digested with HaeIII restriction endonuclease.

Characterization of the karyotype of the tench (Tinca tinca L.) and analysis of its chromosomal heterochromatic regions by C-banding, Ag-staining, and restriction endonuclease banding.

Beginning with a description of the conventional Giemsa-stained karyotype of the tench (Tinca tinca L.), the structure and variability of the chromosomal heterochromatic regions in this cyprinid species were analyzed by means of C-, silver-, and restriction endonuclease banding. Silver staining revealed active nucleolus organizer regions (NORs) on the secondary constriction of chromosome pair 3. Constitutive heterochromatin was associated with NOR regions detected by C-banding. Restriction endonuclease digestion with AluI, TaqI, and HaeIII induced specific banding patterns that allowed identification of homologous chromosome pairs and revealed features about the sequence composition of several chromosomal heterochromatic regions and of the NOR-associated heterochromatin.