Novel internal regions of fluorescent proteins undergo divergent evolutionary patterns.

Over the past decade, fluorescent proteins (FPs) have become ubiquitous tools in biological research. Yet, little is known about the natural function or evolution of this superfamily of proteins that originate from marine organisms. Using molecular phylogenetic analyses of 102 naturally occurring cyan fluorescent proteins, green fluorescent proteins, red fluorescent proteins, as well as the nonfluorescent (purple-blue) protein sequences (including new FPs from Lizard Island, Australia) derived from organisms with known geographic origin, we show that FPs consist of two distinct and novel regions that have evolved under opposite and sharply divergent evolutionary pressures. A central region is highly conserved, and although it contains the residues that form the chromophore, its evolution does not track with fluorescent color and evolves independently from the rest of the protein. By contrast, the regions enclosing this central region are under strong positive selection pressure to vary its sequence and yet segregate well with fluorescence color emission. We did not find a significant correlation between geographic location of the organism from which the FP was isolated and molecular evolution of the protein. These results define for the first time two distinct regions based on evolution for this highly compact protein. The findings have implications for more sophisticated bioengineering of this molecule as well as studies directed toward understanding the natural function of FPs.

Catching the phylogenic history through the ontogenic hourglass: a phylogenomic analysis of Drosophila body segmentation genes.

The phylogenetic information content of different developmental stages is a long-standing issue in the study of development and evolution. We performed phylogenetic analyses of 51 body segmentation genes in 12 species of Drosophila in order to investigate the impact of the mode of evolution of development on phylogeny inference. Previous studies of these genes in Drosophila using pairwise phenetic comparisons at the species group level revealed the presence of an "hourglass model" (HG), wherein mid-embryonic stages are the most evolutionarily constrained. We utilized two character-based approaches: taxonomic congruence using the relative consensus fork index (RCFI), in which phylogenies are inferred from each gene separately and compared with a total evidence tree (TET), and partitioned simultaneous analysis using several indices such as branch support (BS) and localized incongruence length difference (LILD) test. We also proposed a new index, the recapitulatory index (R), which divides the number of synapomorphies on the total number of informative characters in a data set. Polynomial adjustment of both BS and R indices showed strong support for the hourglass model regardless of the taxonomic level (species subgroup vs. subgenera), showing less phylogenetic information content for mid-developmental stages (mainly the zygotic segment polarity stage). Significant LILD scores were randomly distributed among developmental stages revealing the absence of differential selective constraints, but were significantly related to chromosomal location showing physical (linkage) impact on phylogenetic incongruence. RCFI was the most sensitive measure to taxonomic level, having a convex parabola at the species subgroup level in support of the hourglass model and a concave parabola at the subgeneric level in support of the adaptive penetrance model. This time-dependent discrepancy of best fit developmental model parallels previous conflicting results from the vertebrates. Because of the quasi-phenetic nature of this index, we argue that the discrepancy is due to the evolutionary rate heterogeneity of developmental genes rather than to fundamental differences among organisms. We suggest that simultaneous character-based analyses give better macroevolutionary support to the hourglass model of the developmental constraints on genome evolution than pairwise phenetic comparisons.

Identification of unusual E6 and E7 proteins within avian papillomaviruses: cellular localization, biophysical characterization, and phylogenetic analysis.

Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.

Evidence, content and corroboration and the Tree of Life.

We examine three critical aspects of Popper's formulation of the 'Logic of Scientific Discovery'--evidence, content and degree of corroboration--and place these concepts in the context of the Tree of Life (ToL) problem with particular reference to molecular systematics. Content, in the sense discussed by Popper, refers to the breadth and scope of existence that a hypothesis purports to explain. Content, in conjunction with the amount of available and relevant evidence, determines the testability, or potential degree of corroboration, of a statement; content distinguishes scientific hypotheses from metaphysical assertions. Degree of corroboration refers to the relative and tentative confidence assigned to one hypothesis over another, based upon the performance of each under critical tests. Here we suggest that systematists attempt to maximize content and evidence to increase the potential degree of corroboration in all phylogenetic endeavors. Discussion of this "total evidence" approach leads to several interesting conclusions about generating ToL hypotheses.

Phylogenomic analysis identifies gene gains that define Salmonella enterica subspecies I.

Comparative methods for analyzing whole genome sequence (WGS) data enable us to assess the genetic information available for reconstructing the evolutionary history of pathogens. We used the comparative approach to determine diagnostic genes for Salmonella enterica subspecies I. S. enterica subsp. I strains are known to infect warm-blooded organisms regularly while its close relatives tend to infect only cold-blooded organisms. We found 71 genes gained by the common ancestor of Salmonella enterica subspecies I and not subsequently lost by any member of this subspecies sequenced to date. These genes included many putative functional phenotypes. Twenty-seven of these genes are found only in Salmonella enterica subspecies I; we designed primers to test these genes for use as diagnostic sequence targets and data mined the NCBI Sequence Read Archive (SRA) database for draft genomes which carried these genes. We found that the sequence specificity and variability of these amplicons can be used to detect and discriminate among 317 different serovars and strains of Salmonella enterica subspecies I.

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Description of freshwater bacterial assemblages from the upper paraná river floodpulse system, Brazil.

Bacteria were identified from a large, seasonally flooded river (Paraná River, Brazil) and two floodplain habitats that were part of the same river system yet very different in nature: clearwater Garças Lagoon and the highly humic waters of Patos Lagoon. Bacterioplankton were collected during mid-summer (Jan. 2002) from water samples (2 l) filtered first through a 1.2-microm filter then a 0.2-microm membrane filter representing the particle-attached and free-living sub-communities, respectively. DNA was extracted from filters and purified and a 16S rRNA clone library established for each habitat. Over 300 clones were sequenced and checked for similarity to existing 16S sequences in GenBank using the BLAST algorithm with default parameters. Further classification of clones was done using a species "backbone" attachment followed by parsimony analysis. The majority (85%) of sequences, referred to here as operational taxonomic units (OTUs), were most similar to uncultured bacterium 16S sequences. OTUs from each Proteobacteria sub-phylum (alpha, beta, gamma, delta, epsilon) were present in the Upper Paraná River system, as well as members of the Bacteroidetes. The microbial assemblage from Patos Lagoon was least like other samples in that it had no Firmicutes present and was dominated by Actinobacteria. Verrucomicrobia OTUs were only found in the free-living assemblage. This study documents the presence of globally distributed phyla in Upper Paraná River and taxa unique to habitat and particle attachment.

Reciprocal illumination in the gene content tree of life.

Phylogenies based on gene content rely on statements of primary homology to characterize gene presence or absence. These statements (hypotheses) are usually determined by techniques based on threshold similarity or distance measurements between genes. This fundamental but problematic step can be examined by evaluating each homology hypothesis by the extent to which it is corroborated by the rest of the data. Here we test the effects of varying the stringency for making primary homology statements using a range of similarity (e-value) cutoffs in 166 fully sequenced and annotated genomes spanning the tree of life. By evaluating each resulting data set with tree-based measurements of character consistency and information content, we find a set of homology statements that optimizes overall corroboration. The resulting data set produces well-resolved and well-supported trees of life and greatly ameliorates previously noted inconsistencies such as the misclassification of small genomes. The method presented here, which can be used to test any technique for recognizing primary homology, provides an objective framework for evaluating phylogenetic hypotheses and data sets for the tree of life. It also can serve as a technique for identifying well-corroborated sets of homologous genes for functional genomic applications.