The evolution of coloration and toxicity in the poison frog family (Dendrobatidae)

The poison frogs (family Dendrobatidae) are terrestrial anuran amphibians displaying a wide range of coloration and toxicity. These frogs generally have been considered to be aposematic, but relatively little research has been carried out to test the predictions of this hypothesis. Here we use a comparative approach to test one prediction of the hypothesis of aposematism: that coloration will evolve in tandem with toxicity. Recently, we developed a phylogenetic hypothesis of the evolutionary relationships among representative species of poison frogs, using sequences from three regions of mitochondrial DNA. In our analysis, we use that DNA-based phylogeny and comparative analysis of independent contrasts to investigate the correlation between coloration and toxicity in the poison frog family (Dendrobatidae). Information on the toxicity of different species was obtained from the literature. Two different measures of the brightness and extent of coloration were used. (i) Twenty-four human observers were asked to rank different photos of each different species in the analysis in terms of contrast to a leaf-littered background. (ii) Color photos of each species were scanned into a computer and a computer program was used to obtain a measure of the contrast of the colors of each species relative to a leaf-littered background. Comparative analyses of the results were carried out with two different models of character evolution: gradual change, with branch lengths proportional to the amount of genetic change, and punctuational change, with all change being associated with speciation events. Comparative analysis using either method or model indicated a significant correlation between the evolution of toxicity and coloration across this family. These results are consistent with the hypothesis that coloration in this group is aposematic.

A neural system for human visual working memory

Working memory is the process of actively maintaining a representation of information for a brief period of time so that it is available for use. In monkeys, visual working memory involves the concerted activity of a distributed neural system, including posterior areas in visual cortex and anterior areas in prefrontal cortex. Within visual cortex, ventral stream areas are selectively involved in object vision, whereas dorsal stream areas are selectively involved in spatial vision. This domain specificity appears to extend forward into prefrontal cortex, with ventrolateral areas involved mainly in working memory for objects and dorsolateral areas involved mainly in working memory for spatial locations. The organization of this distributed neural system for working memory in monkeys appears to be conserved in humans, though some differences between the two species exist. In humans, as compared with monkeys, areas specialized for object vision in the ventral stream have a more inferior location in temporal cortex, whereas areas specialized for spatial vision in the dorsal stream have a more superior location in parietal cortex. Displacement of both sets of visual areas away from the posterior perisylvian cortex may be related to the emergence of language over the course of brain evolution. Whereas areas specialized for object working memory in humans and monkeys are similarly located in ventrolateral prefrontal cortex, those specialized for spatial working memory occupy a more superior and posterior location within dorsal prefrontal cortex in humans than in monkeys. As in posterior cortex, this displacement in frontal cortex also may be related to the emergence of new areas to serve distinctively human cognitive abilities.

Flower color variation: A model for the experimental study of evolution

We review the study of flower color polymorphisms in the morning glory as a model for the analysis of adaptation. The pathway involved in the determination of flower color phenotype is traced from the molecular and genetic levels to the phenotypic level. Many of the genes that determine the enzymatic components of flavonoid biosynthesis are redundant, but, despite this complexity, it is possible to associate discrete floral phenotypes with individual genes. An important finding is that almost all of the mutations that determine phenotypic differences are the result of transposon insertions. Thus, the flower color diversity seized on by early human domesticators of this plant is a consequence of the rich variety of mobile elements that reside in the morning glory genome. We then consider a long history of research aimed at uncovering the ecological fate of these various flower phenotypes in the southeastern U.S. A large body of work has shown that insect pollinators discriminate against white phenotypes when white flowers are rare in populations. Because the plant is self-compatible, pollinator bias causes an increase in self-fertilization in white maternal plants, which should lead to an increase in the frequency of white genes, according to modifier gene theory. Studies of geographical distributions indicate other, as yet undiscovered, disadvantages associated with the white phenotype. The ultimate goal of connecting ecology to molecular genetics through the medium of phenotype is yet to be attained, but this approach may represent a model for analyzing the translation between these two levels of biological organization.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions.

Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila. Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2 alpha, a human homologue of tra-2. Two alternative types of htra-2 alpha cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2 alpha isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2 alpha to regulate dsx is female-specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2 alpha has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression.

Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein. HUMSIAH is localized on human chromosome 16q12-q13. This gene is activated during the physiological program of cell death in the intestinal epithelium. Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed by the p21waf1. These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.

Translocation events in the evolution of aminoacyl-tRNA synthetases.

We have characterized hisS, the gene encoding the histidyl-tRNA synthetase (HisRS) from the tetraodontoid fish Fugu rubripes. The hisS gene is about 3.5 kbp long and contains 13 exons and 12 introns of 172 bp, on average. The Fugu hisS gene encodes a putative protein of 519 amino acids with the three motifs identified as signatures of class 2 aminoacyl-tRNA synthetases. A model for the shifting of intron 8 between Fugu and hamster is proposed based on the successive appearance of a cryptic splicing site followed by an insertion mutation that created a new acceptor site. In addition, sequence comparisons suggest that the hisS gene has undergone a translocation through the first intron. As a result, the Fugu HisRS has an N-terminal sequence markedly different from that in the human and hamster enzymes. We propose that similar events have been responsible for variations at the N-terminal end of other aminoacyl-tRNA synthetases. Our analysis suggests that this involves exchanges through introns of two exons encoding an ancestral 32-amino acid motif.

Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees.

Evolutionary theory predicts the recent spread of primate immunodeficiency viruses (PIVs) to new human populations to be accompanied by positive selection in response to new host environments and/or by random genetic drift. I assess evidence for positive selection in human and chimpanzee PIVs type I (PIV1s), using ratios of synonymous to nonsynonymous nucleotide change based on branch lengths and outgroup rooting. Ratios are smaller for PIV1s from humans than for PIV1 from a chimpanzee for the pol, gag, and env glycoprotein 120 (gp120) regions, indicating greater effects of positive selection in PIV1s from humans. Parsimony-based relative rate tests for amino acid changes showed significant differences between PIV1s from humans and chimpanzees in 18 of 48 pairwise comparisons, with all 18 showing faster rates of change in PIV1s from humans. This study indicates that in some instances, the recent evolution of human PIV1s follows a speciational pattern, in which increased diversification of taxa is correlated with greater amounts of character change appearing and being maintained through time. This extends the generality of the speciational pattern to a group of organisms (viruses) having the fastest known rates of anagenetic change for nucleotide characters and indicates that comprehensive understanding of PIV1 evolution requires consideration of both anagenetic change within viral lineages and the relative historical success of different viral clades. Phylogenetic analyses show that neither PIV1s infecting humans nor those infecting chimpanzees represent monophyletic groups and suggest multiple host-species shifts for PIV1s.

Human cooperation in the simultaneous and the alternating Prisoner's Dilemma: Pavlov versus Generous Tit-for-Tat.

The iterated Prisoner's Dilemma has become the paradigm for the evolution of cooperation among egoists. Since Axelrod's classic computer tournaments and Nowak and Sigmund's extensive simulations of evolution, we know that natural selection can favor cooperative strategies in the Prisoner's Dilemma. According to recent developments of theory the last champion strategy of "win--stay, lose--shift" ("Pavlov") is the winner only if the players act simultaneously. In the more natural situation of players alternating the roles of donor and recipient a strategy of "Generous Tit-for-Tat" wins computer simulations of short-term memory strategies. We show here by experiments with humans that cooperation dominated in both the simultaneous and the alternating Prisoner's Dilemma. Subjects were consistent in their strategies: 30% adopted a Generous Tit-for-Tat-like strategy, whereas 70% used a Pavlovian strategy in both the alternating and the simultaneous game. As predicted for unconditional strategies, Pavlovian players appeared to be more successful in the simultaneous game whereas Generous Tit-for-Tat-like players achieved higher payoffs in the alternating game. However, the Pavlovian players were smarter than predicted: they suffered less from defectors and exploited cooperators more readily. Humans appear to cooperate either with a Generous Tit-for-Tat-like strategy or with a strategy that appreciates Pavlov's advantages but minimizes its handicaps.

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.

Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display.

Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor-derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes.

Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.

Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen.

Because of variations in tRNA sequences in evolution, tRNA synthetases either do not acylate their cognate tRNAs from other organisms or execute misacylations which can be deleterious in vivo. We report here the cloning and primary sequence of a 958-aa Saccharomyces cerevisiae alanyl-tRNA synthetase. The enzyme is a close homologue of the human and Escherichia coli enzymes, particularly in the region of the primary structure needed for aminoacylation of RNA duplex substrates based on alanine tRNA acceptor stems with a G3.U70 base pair. An ala1 disrupted allele demonstrated that the gene is essential and that, therefore, ALA1 encodes an enzyme required for cytoplasmic protein synthesis. Growth of cells harboring the ala1 disrupted allele was restored by a cDNA clone encoding human alanyl-tRNA synthetase, which is a serum antigen for many polymyositis-afflicted individuals. The human enzyme in extracts from rescued yeast was detected with autoimmune antibodies from a polymyositis patient. We conclude that, in spite of substantial differences between human and yeast tRNA sequences in evolution, strong conservation of the G3.U70 system of recognition is sufficient to yield accurate aminoacylation in vivo across wide species distances.