Polyploidy and epigenetic events in the evolution of Anura.

This article reviews the polyploidy events that have long been demonstrated to play a role in the evolution of Anura, while also discussing the importance of epigenetic control of gene expression and diversity. Findings on Brazilian autopolyploid anurans, mainly of the genus Odontophrynus, obtained in previous studies on their cytogenetics, chromatin ultrastructure, and molecular gene regulation are discussed here. Our data on genome duplication and on epigenetic events were analyzed here regarding phylogenetic trees, including the classic 2R model for vertebrate evolution and the growing evidence of similar epigenetic mechanisms in animal and allopolyploid plants. We propose that polyploidy and epigenetic events led to rapid Anura diversity and speciation. Also, recent advances in molecular studies in other organisms led us to revisit some controversial models of evolution.

The kinin B(2) receptor gene structure, product processing and expression in adult and fetal rats: evidence for gene evolution.

We examined the structure of the rat kinin B2 receptor gene (KB2r) and encoding messenger RNA (mRNA) processing. Differently from the closely related mouse and rabbit genes that have three exons and two introns, the rat gene purportedly consists of four exons and three introns. There are two purported gene products; one of them contains an upstream approximately 180-bp open reading frame region ("exon-X") potentially expressed as a result of alternative processing. To examine the processing of rat KB2r mRNA, cDNA amplicons were generated using primer pairs directed towards 5' or 3' exon or intron flanking regions. Analyses of intron/exon primary cDNA amplicons showed that introns 1 to 3 are removed sequentially and that "exon-X" removal follows that of intron-3. No evidence was found for "exon-X" expression in polyadenylated (mature) mRNA of adult Wistar, Wistar Kyoto, spontaneously hypertensive or Sprague-Dawley rat tissues. Nor was "exon-X" detected in tissues subject to inflammatory stimulus expressing B1 kinin receptor mRNA or in 1- to 21-day-old rat embryos or fetuses. The lack of evidence for the expression of "exon-X" in mature mRNA indicates that the structure of the rat gene is similar to that of the mouse, rabbit and human genes, all consisting of three exons and two introns. The "exon-X" fragment may result from interstitial gene duplication, be a fragment of the ancestral gene, or most likely heterologous transposon insertion of an exon-like fragment into intron-2 of the KB2r gene.

Evolution by polyploidy in Amphibia: new insights.

Polyploidy is an important mechanism of evolution in lower vertebrates, resulting in gene duplication and loci duplication evolving to diploidization. In polyploid anurans DNA is increased, but RNA and protein synthesis is kept at the same levels as in their diploid counterparts. Recent cytogenetic findings in Odontophrynus americanus show: (1) amphiplasty and asynchrony of the cell division cycle in 4n and (2) intra- and interindividual alterations of chromosome 4 morphology in 4n and 2n through breaks at secondary constrictions and at/or around the centromeres, followed by the appearance of changes of centromeres position, dicentrics, bisatellited chromosomes, precocious sister-chromatid segregation, and loss or unequal segregation of the chromosomes. This genome instability suggests the presence of chromosomal aberrations with chromodomain alterations at the centromeres affecting the mechanisms ensuring normal segregation of the chromatids in anaphase of mitosis and meiosis. Cell division asynchrony and chromosome abnormalities, novel findings in polyploids, are discussed respectively in function of gene activity and putative translocations producing polymorphisms in nucleolus organizers and secondary constrictions.

Ribosomal cistrons in Bothrops neuwiedi (Serpentes) subspecies from Brazil.

The ribosomal cistrons of six subspecies of Bothrops neuwiedi (Serpentes) were studied at both the cytogenetic and molecular levels. These subspecies populations occur in several Brazilian regions. The analysis of the nucleolar organizing region banding patterns showed variability in the chromosomal localization of rDNA cistrons. The rDNA clusters were found in two microchromosomes, in chromosome pair Number 6, or even in the one homologue of chromosome 6, and one microchromosome, according to the specimen examined but independent of the population from which it was selected. The organization of the rDNA repeat was studied by Southern blot hybridization using Xenopus laevis ribosomal DNA probes. The size of the repeat is 10.4 kilobases (kb), and the intergenic spacer (IGS), 2.6 kb long, is relatively small compared with the size of other vertebrate IGSs. Restriction mapping using the restriction enzymes EcoRI, BamHI, HindIII, and PvuII showed a highly conserved organization of the ribosomal repeats in the total genomic DNA of the subspecies studied.

Can a protein influence the fate of its own coding sequence?: the amino- and carboxyl-terminal regions of H1 histone.

The amino- and carboxyl-terminal regions of H1 histone are afforded an opportunity to bind to their own coding segments. While such binding is of no consequence to the type of H1 histone that is expressed only in terminally differentiated cells (here referred to as H5), the same binding occurring in H1 of proliferating cells might affect the course of evolution of its amino- and carboxyl-terminal regions. With the above in mind, amino acid sequences of H5 from three species were compared with those of H1 from five species. The spherical core was more conserved in H1 than in H5, whereas the amino- and carboxyl-terminal regions of both were hypervariable. A distinct evolutionary pattern, however, was observed only in these regions of H1 but not of H5. Tandem repeats of short palindromes often generated nearly identical long palindromes in distantly related species. Thus, as far as the 37-residue-long amino-terminal region was concerned, the greatest homology was between the rat and human rather than between the rat and the mouse. With regard to the last 25 residues in the carboxyl-terminal region, the greatest homology was between the chicken and the rainbow trout; the next was between the mouse and the trout. Successions of long palindromes populating the amino- and carboxyl-terminal regions of H1 might be the direct consequence of the autologous binding between peptides and their coding sequence.

Behaviour of the ZW sex bivalent in the snake Bothrops jararaca.

The behavior of the ZW sex bivalent was investigated in female meiosis of the poisonous snake Bothrops jararaca. The Z is euchromatic and synapses end to end with the W. The W chromosome shows a heterochromatic segment distally in the short arm. Pairing occurs between the long arm of the W and the slightly longer arm of the mediocentric Z. A sex vesicle, similar to the one found in the XY placental mammals, does not occur in snakes. The Z and W chromosomes segregate reductionally in the first meiotic division and equationally in the second.

Behaviour of the ZW sex bivalent in the snake Bothrops jararaca

The behavior of the ZW sex bivalent was investigated in female meiosis of the poisonous snake Bothrops jararaca. The Z is euchromatic and synapses end to end with the W. The W chromosome shows a heterochromatic segment distally in the short arm. Pairing occurs between the long arm of the W and the slightly longer arm of the mediocentric Z. A sex vesicle, similar to the one found in the XY placental mammals, does not occur in snakes. The Z and W chromosomes segregate reductionally in the first meiotic division and equationally in the second.

Chromatin circles in amphibian provitellogenic oocytes.

Previtellogenic oocytes of Odontophrynus americanus display hundreds of chromatin circles. Electron microscopy of spread preparations of isolated nuclei shows that the circles originate from the chromatin. The circles change their morphology and form new copies. The length of the DNA packed in the nucleosomal circles is about 2.5--3.5 micrometer or multiples of this value. Assuming that histones need not be removed from chromatin before DNA replication we suggest that the circles might belong to the process of rDNA amplification.

Arrangement of nucleosomes in condensed chromatin fibres.

Condensed chromatin shows globules of 300 A formed by approximately 8 to 10 nucleosomes. Each globule might be an uncoiled turn of a supercoil. This supercoil forms major coils along the fibre.

Circles in spermatocyte chromatin loops. Electron microscopy and AgAs-NORs studies.

We describe the production of circles in chromomeric loops during the pachytene stage of the spermatocytes. These circles are found attached to chromatin or already free in the nucleoplasm. Each circle measures an average of 3700 A in circunference. We suggest that such circles might indicate the presence of tandem repetitions.

Chromatin ultrastructure of lower vertebrates.

Meiotic and mitotic chromosomes from amphibians and snakes were studied by electron microscopy. By using water spreading, preceded by a mild NaCl pretreatment, we showed: 1. 'Beads on a string' arrangement of the chromatin fibres; 2. The presence of loops at pachytene chromomeres as well as during metaphase of both mitosis and first meiosis; 3. Transcriptional activity for non-ribosomal RNA on peripheral loops during the middle pachytene.

Mouse sex vesicle. C-band and pairing at the light and electron microscope.

C banded mouse pachytene chromsomes were studied with the light and electron microscopes by the whole mount technique. The X and Y chromosomes show pairing by the long, by the short or by both long and short arms. Assuming Lyon's hypothesis, the latter suggests that the Y segment transferred to the X is intercalar. With the light microscope, a negative image of the synaptonemal complex is evidenced.