The evolution of South American endemic canids: a history of rapid diversification and morphological parallelism.

The origin of endemic South American canid fauna has been traditionally linked with the rise of the Isthmus of Panama, suggesting that diversification of the dog fauna on this continent occurred very rapidly. Nevertheless, despite its obvious biogeographic appeal, the tempo of Canid evolution in South America has never been studied thoroughly. This issue can be suitably tackled with the inference of a molecular timescale. In this study, using a relaxed molecular clock method, we estimated that the most recent common ancestor of South American canids lived around 4 Ma, whereas all other splits within the clade occurred after the rise of the Panamanian land bridge. We suggest that the early diversification of the ancestors of the two main lineages of South American canids may have occurred in North America, before the Great American Interchange. Moreover, a concatenated morphological and molecular analysis put some extinct canid species well within the South American radiation, and shows that the dental adaptations to hypercarnivory evolved only once in the South American clade.

A molecular study on the evolution of a subtype B variant frequently found in Brazil.

In spite of the remarkable diversity of HIV-1 env genes, several amino acids are extremely conserved, probably due to functional constraints. One example is the proline found at the second position of the GPGR motif. Several viruses, however, bear substitutions at this site, for instance, GWGR subtype B variant. GWGR viruses are described in Brazil since the beginning of the epidemics, but the extent of their dispersion or the geographical origin of the variant remains unknown. In the present study, phylogenetic trees were constructed in order to study the origin and spread of this variant. All GWGR sequences as well as a subset of subtype B sequences available were included in the analyses. Analyses of differential selection were also performed on GWGR and non-GWGR sequences in order to unveil evolutionary novelties due to the action of positive selection. Although the GWGR variant was found at least in 23 countries, its expansion probably has a single origin, and Brazil is the epicenter.

Genetic variation and population structure of two species of neo-tropical mud-mussels (Mytella spp)

Mytella guyanensis Lamarck (1819) and Mytella charruana d'Orbigny (1846) are widespread euryhaline bivalves that have become commercially important in Brazil. Despite their importance, however, no genetic information that would be useful to orient governmental policies is available for these species. We analyzed, through allozyme electrophoresis, populations of M. guyanensis and M. charruana along 3,500 km of Brazilian coast. Pairwise comparisons among gene frequencies in M. guyanensis resulted in high levels of pairwise gene identity (I = 0.976 to 0.998). Conversely, significant levels of population structure were found in both M. guyanensis (FST = 0.089) and M. charruana (FST = 0.102). Heterozygosity levels for both species were high (H(e) = 0.090 to 0.134 in M. guyanensis and H(e) = 0.191 to 0.228 in M. charruana). The larger population size of M. charruana could explain, at least partially, the higher levels of genetic variability for this species. These levels of genetic variability yield an effective population size estimate of about 300,000 for M. guyanensis, and 540,000 for M. charruana, based on neutralist expectations. Remarkably, these numbers are much smaller than the estimated actual population sizes. This distortion might be explained by unstable population sizes and it suggests that long-term genetic variability studies are crucial to prevent artifactual viability analysis data for these commercially exploited species.

Molecular phylogeny of penaeid shrimps inferred from two mitochondrial markers

Penaeid shrimps are an important resource in crustacean fisheries, representing more than the half of the gross production of shrimp worldwide. In the present study, we used a sample of wide-ranging diversity (41 shrimp species) and two mitochondrial markers (758 bp) to clarify the evolutionary relationships among Penaeidae genera. Three different methodologies of tree reconstruction were employed in the study: maximum likelihood, neighbor joining and Bayesian analysis. Our results suggest that the old Penaeus genus is monophyletic and that the inclusion of the Solenocera genus within the Penaeidae family remains uncertain. With respect to Metapenaeopsis monophyly, species of this genus appeared clustered, but with a nonsignificant bootstrap value. These results elucidate some features of the unclear evolution of Penaeidae and may contribute to the taxonomic characterization of this family.

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny.

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.

The anoxic fibroblast response is an early-stage wound healing program.

When fibroblasts experience anoxia, a cellular program is initiated which results in stepwise activation of an endogenous SVL30 retroelement, metabolic adaptation to reliance on glycolysis, protease secretion, and endonuclease induction. This response is reversible and occurs with no reduction in cellular viability. Examination of experimental wounds reveals that this same response is physiologically activated during the early phase of wound healing when near-anoxic conditions prevail and wound debridement predominates. Procathepsin D has been identified as one of three major anoxia-inducible secretory proteins; its secretion is the result of altered routing within the cell, with no apparent change in the amount of the protein synthesized. A delayed cellular contractile response occurs approximately 1 week after fibroblasts experience as little as 16 hr of anoxia; this response appears to contribute to wound contraction.

Quantifying c-myc expression in c-myc antisense phosphorothioate oligodeoxynucleotide-treated leukemic and colon cancer cell lines.

Antisense oligodeoxynucleotides (oligo) have been used to inhibit oncogene expression and have potential therapeutic applications. Using a 15-mer antisense phosphorothioate oligo (S-oligo), inhibition of c-myc oncogene expression and cellular proliferation is studied in two cell lines with c-myc overexpression: a colon cancer cell line (Colo 320 DM) and a promyelocytic leukemic cell line(HL-60). Quantitative analysis of c-myc mRNA transcript is performed by competitive reverse transcription-polymerase chain reaction (RT-PCR). This utilizes an RNA competitive reference standard (CRS RNA) template that is identical to the native c-myc mRNA except for a short segment deletion to allow for differentiation of the two by gel electrophoresis. A fixed quantity of test mRNA and a series of known concentrations of CRS RNA template placed in the same test tubes under identical conditions are reverse transcribed and amplified by PCR. Since the reaction is competitive, the ratio of the PCR products reflects the ratio of the initial concentrations of the two templates. After gel electrophoresis, the two PCR products are quantified densitometrically. Treating Colo 320 DM and HL-60 cells with c-myc antisense oligo and S-oligo results in a 20- to 100-fold decrease in c-myc mRNA transcripts, respectively. This inhibition is dose dependent and sequence specific (c-myc sense and missense oligo have no effects). The quantitative decrease in c-myc mRNA is associated with corresponding inhibition of c-myc oncoprotein synthesis as demonstrated by flow cytometry and Western blots. Furthermore, there is inhibition of cellular proliferation of the respective cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular phylogeny and divergence times of drosophilid species.

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.

An anoxia inducible endonuclease and enhanced DNA breakage as contributors to genomic instability in cancer.

Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery period show genomic instability in the form of highly elevated CAD gene amplification rates. As revealed by flow cytometric analysis this is associated with DNA breakage in vivo, followed by repair during the recovery period. Such genomic instability parallels expression of a M(r) 29,000/31,000 endonuclease; this enzyme requires no added divalent metal ion and has a pH optimum of about 6.5. The same endonuclease was found to be expressed within healing wounds and in four of ten human colorectal cancers but was not seen in eight normal colorectal tissue samples. Our results indicate that DNA breakage resulting from endogenous endonuclease activity can have a substantial effect in modulating genomic instability.

Anoxia-inducible endonuclease activity as a potential basis of the genomic instability of cancer cells.

Normal rat fibroblasts exhibit a staged response to anoxia which in several respects parallels processes activated in malignant tumor cells. We describe here a new element of the anoxic response, the induction by anoxia of a sequestered endonuclease activity. Such activity is elevated approximately 3-fold within anoxic fibroblasts and during Hirt DNA isolation is able to digest chromatin to produce a nucleosomal ladder. However, DNA is not measurably affected within intact cells, and cells retain complete viability as the endonuclease is induced. The anoxia-inducible endonuclease acts without specificity for DNA sequence. Trace leakage of this endonuclease into the nucleus has obvious potential to underlie the known propensity of anoxic cells to undergo amplification and may be associated with the break-related genomic instability of cancer cells.