Basal divergences in birds and the phylogenetic utility of the nuclear RAG-1 gene.

The single-copy RAG-1 gene is found throughout higher vertebrates and consists of a single 3.1-kb exon without intervening introns. A 2.9-kb region of the RAG-1 locus was sequenced for 14 basal taxa of birds plus the crocodylian outgroups Alligator and Gavialis. Phylogenetic analysis of the sequences supported the hypothesis that the deepest evolutionary split in extant birds separates paleognaths from neognaths. A deep division among neognaths separates the chicken- and duck-like birds ("galloanserines") from a clade consisting of all other birds ("plethornithines"). The relationships of these three basal clades in Aves were supported by high bootstrap (98 to 100%) and large decay index values (above 14). Additionally, the plethornithine clade is characterized by a 15-bp (five-codon) synapomorphic deletion relative to all other birds. RAG-1 evolves slowly, with a number of properties favoring its phylogenetic utility, including rarity of indels, minimal saturation of transition changes at 3rd positions of codons, nearly constant base composition across taxa, and no asymmetry in directional patterns of reconstructed change. However, RAG-1 does not evolve in a clocklike manner, suggesting that this gene cannot easily be used for estimating ages of ancient lineages.     

The pattern and timing of diversification of Philippine endemic rodents: evidence from mitochondrial and nuclear gene sequences

The 22 genera and 64 species of rodents (Muridae: Murinae) distributed in the Philippine Islands provide a unique opportunity to study patterns and processes of diversification in island systems. Over 90% of these rodent species are endemic to the archipelago, but the relative importance of dispersal from the mainland, dispersal within the archipelago, and in situ differentiation as explanations of this diversity remains unclear, as no phylogenetic hypothesis for these species and relevant mainland forms is currently available. Here we report the results of phylogenetic analyses of the endemic Philippine murines and a wide sampling of murine diversity from outside the archipelago, based on the mitochondrial cytochrome b gene and the nuclear-encoded IRBP exon 1. Analysis of our combined gene data set consistently identified five clades comprising endemic Philippine genera, suggesting multiple invasions of the archipelago. Molecular dating analyses using parametric and semiparametric methods suggest that colonization occurred in at least two stages, one ca. 15 Mya, and another 8 to 12 million years later, consistent with the previous recognition of "Old" and "New" endemic rodent faunas. Ancestral area analysis suggests that the Old Endemics invaded landmasses that are now part of the island of Luzon, whereas the three New Endemic clades may have colonized through either Mindanao, Luzon, or both. Further, our results suggest that most of the diversification of Philippine murines took place within the archipelago. Despite heterogeneity between nuclear and mitochondrial genes in most model parameters, combined analysis of the two data sets using both parsimony and likelihood increased phylogenetic resolution; however, the effect of data combination on support for resolved nodes was method dependent. In contrast, our results suggest that combination of mitochondrial and nuclear data to estimate relatively ancient divergence times can severely compromise those estimates, even when specific methods that account for rate heterogeneity among genes are employed. [Biogeography; divergence date estimation; mitochondrial DNA; molecular systematics; Murinae; nuclear exon; Philippines; phylogeny.].     

RAG-1 and RAG-2 are not sufficient to direct all phases of immunoglobulin gene rearrangement in pre-B-cell lines.

To probe the factors controlling immunoglobulin heavy-chain gene rearrangement, we analyzed Abelson virus-transformed pre-B-cell lines that fail to undergo VH-to-DJH joining at an appreciable frequency. Despite this feature, some of these cell lines (rechi) rearrange an extrachromosomal recombination substrate at levels normal for transformed pre-B cells. Others (reclo) rearrange these substrates at levels characteristic of nonlymphoid hematopoietic cells. The DJH rearrangements from a representative rechi cell line were aberrant, suggesting that these cells probably fail to complete heavy-chain gene assembly because some of the necessary cis-acting signals are missing. In contrast, both DJH rearrangements from a reclo cell line appeared normal in structure, indicating that trans-acting factors necessary for recombination might be missing. Introduction of the RAG-1 and RAG-2 genes, genes encoding two such factors, failed to confer a rechi phenotype to these cells. However, fusion of the reclo cells to a rechi cell line generated a high frequency of rechi hybrids. In addition, most of the hybrids rearranged the endogenous kappa light-chain locus. Neither the rechi phenotype nor kappa-chain rearrangement correlated with levels of RAG-1 and RAG-2 expression in all of the hybrids. Thus, both gene transfer and cell fusion experiments indicate that RAG-1 and RAG-2 are not sufficient to activate immunoglobulin gene recombination in at least some pre-B-cell lines. In addition, the fusion experiments suggest that two gene products in addition to RAG-1 and RAG-2 may be required for kappa-gene rearrangement.     

Impacts of gene essentiality, expression pattern, and gene compactness on the evolutionary rate of mammalian proteins

Understanding the determinants of the rate of protein sequenceevolution is of fundamental importance in evolutionary biology.Many recent studies have focused on the yeast because of theavailability of many genome-wide expressional and functionaldata. Yeast studies revealed a predominant role of gene expressionlevel and a minor role of gene essentiality in determining therate of protein sequence evolution. Whether these rules applyto complex organisms such as mammals is unclear. Here we assemblea list of 1,138 essential and 2,341 nonessential mouse genesbased on targeted gene deletion experiments and report a significantimpact of gene essentiality on the rate of mammalian proteinevolution. Gene expression level has virtually no effect, althoughtissue specificity in expression pattern has a strong influence.Unexpectedly, gene compactness, measured by average intron sizeand untranslated region length, has the greatest influence.Hence, the relative importance of the various factors in determiningthe rate of mammalian protein evolution is gene compactness> gene essentiality tissue specificity > expression level.Our results suggest a considerable variation in rate determinantsbetween unicellular organisms such as the yeast and multicellularorganisms such as mammals.     

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.     

Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish

Background

Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.

Methodology/Principal Findings

We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.

Conclusions/Significance

These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.     

Roles of the "dispensable" portions of RAG-1 and RAG-2 in V(D)J recombination

V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispensable" regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.     

Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1.

An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG-1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10-fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X-100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8-9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 degrees C, with maximal activity observed at 55 degrees C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG-1 lipase offers potential for use as a biocatalyst.     

Sequences from 14 mitochondrial genes provide a well-supported phylogeny of the Charadriiform birds congruent with the nuclear RAG-1 tree

Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morphological characters, recent studies have turned to DNA sequences to resolve the systematic uncertainties of family-level relationships in this group. However, trees constructed using nuclear genes or the mitochondrial Cytochrome b gene suggest deep-level relationships of shorebirds that differ from previous studies based on morphology or DNA-DNA hybridization distances. To test phylogenetic hypotheses based on nuclear genes (RAG-1, myoglobin intron-2) and single mitochondrial genes (Cytochrome b), approximately 13,000 bp of mitochondrial sequence was collected for one exemplar species of 17 families of Charadriiformes plus potential outgroups. Maximum likelihood and Bayesian analyses show that trees constructed from long mitochondrial sequences are congruent with the nuclear gene topologies [Chardrii (Lari, Scolopaci)]. Unlike short mitochondrial sequences (such as Cytochrome b alone), longer sequences yield a well-supported phylogeny for shorebirds across various taxonomic levels. Examination of substitution patterns among mitochondrial genes reveals specific genes (especially ND5, ND4, ND2, and COI) that are better suited for phylogenetic analyses among shorebird families because of their relatively homogeneous nucleotide composition among lineages, slower accumulation of substitutions at third codon positions, and phylogenetic utility in both closely and distantly related lineages. For systematic studies of birds in which family and generic levels are examined simultaneously, we recommend the use of both nuclear and mitochondrial sequences as the best strategy to recover relationships that most likely reflect the phylogenetic history of these lineages.     

Phylogeny, diversity, and classification of the Accipitridae based on DNA sequences of the RAG-1 exon

The avian family Accipitridae has historically been divided into subfamilies or tribes based on features such as general resemblance, feeding ecology, and behavior. Consequently, the monophyly of those groups has been questionable. Recently, three phylogenetic analyses of a majority of the genera have appeared, one based on osteology, one on DNA sequences from a single mitochondrial gene, and the third on mitochondrial plus nuclear DNA sequences, and the resulting phylogenies were in substantial disagreement concerning the composition and basal branching patterns of the clades and hence require further analysis and confirmation. Here we use DNA sequences from the large nuclear RAG-1 exon to investigate the phylogenetic relationships of these birds. Our results largely corroborated the prior study that included nuclear genes. We found strong support for a monophyletic clade comprising the secretarybird Sagittarius serpentarius , the osprey Pandion haliaetus , and the traditional accipitrids. However, every one of the traditionally recognized subfamilies of accipitrids was found to be polyphyletic. The most basal nodes in the phylogeny separate small clades of insectivorous and scavenger species, such as kites and Old World vultures, from the rest of the family. The speciose genera of bird and mammal predators are all relatively derived (terminal) in the phylogeny. Many of the basal clades are cosmopolitan in their distributions, consistent with the great mobility of these raptors. A new classification is proposed that eliminates the problem of polyphyletic intrafamilial taxa.     

Primate phylogeny, evolutionary rate variations, and divergence times: a contribution from the nuclear gene IRBP

The first third (ca. 1200 bp) of exon 1 of the nuclear gene encoding the interstitial retinoid-binding protein (IRBP) has been sequenced for 12 representative primates belonging to Lemuriformes, Lorisiformes, Tarsiiformes, Platyrrhini, and Catarrhini, and combined with available data (13 other primates, 11 nonprimate placentals, and 2 marsupials). Phylogenetic analyses using maximum likelihood on nucleotides and amino acids robustly support the monophyly of primates, Strepsirrhini, Lemuriformes, Lorisiformes, Anthropoidea, Catarrhini, and Platyrrhini. It is interesting to note that 1) Tarsiidae grouped with Anthropoidea, and the support for this node depends on the molecular characters considered; 2) Cheirogaleidae grouped within Lemuriformes; and 3) Daubentonia was the sister group of all other Lemuriformes. Study of the IRBP evolutionary rate shows a high heterogeneity within placentals and also within primates. Maximum likelihood local molecular clocks were assigned to three clades displaying significantly contrasted evolutionary rates. Paenungulata were shown to evolve 2.5-3 times faster than Perissodactyla and Lemuriformes. Six independent calibration points were used to estimate splitting ages of the main primate clades, and their compatibility was evaluated. Divergence ages were obtained for the following crown groups: 13.8-14.2 MY for Lorisiformes, 26.5-27.2 MY for Lemuroidea, 39.6-40.7 MY for Lemuriformes, 45.4-46.7 MY for Strepsirrhini, and 56.7-58.4 MY for Haplorrhini. The incompatibility between some paleontological and molecular estimates may reflect the incompleteness of the placental fossil record, and/or indicate that the variable IRBP evolutionary rates are not fully accommodated by local molecular clocks.     

The RAG-1 exon in the avian order Caprimulgiformes: phylogeny, heterozygosity, and base composition

We sequenced 2.8 kb of the RAG-1 exon for most of the extant genera in the avian order Caprimulgiformes to investigate monophyly of the order and phylogeny within the traditional families. The order is not monophyletic: the Aegothelidae (owlet-nightjars) were the sister group of the Apodiformes (swifts and hummingbirds). There was no support for the monophyly of a clade containing the remaining families of Caprimulgiformes. However, the RAG-1 data strongly supported a relationship between the Podargidae (frogmouths) and Caprimulgidae (nightjars). Within the Caprimulgidae, the Australasian genus Eurostopodus was sister to the rest of the family, which in turn was composed of four major clades, three of which were restricted to the New World and primarily to the Neotropics. The Old World caprimulgids form a monophyletic clade embedded within the New World taxa; consequently, most Old World nightjars are probably the result of a single expansion out of the Neotropics. The genus Caprimulgus was not found to be monophyletic. Several species in the Caprimulgidae have both elevated heterozygosity and high GC3 content; it is likely that these are causally related.     

Expression pattern, genomic cloning and RFLP analyses of the swine PIT-1 gene

The swine PIT-1 POU domain cDNA was used to study PIT-1 expression and to clone the PIT-1 genomic region to identify additional PIT-1 polymorphisms. PIT-1 was expressed only in the pituitary, confirming the swine cDNA identity. To study the PIT-1 genomic region, a clone containing 13.7 kb DNA was isolated. EcoRI fragments hybridizing to PIT-1 were sequenced, and the 3′ portion of PIT-1 gene identified. Primers for polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis were then designed. Restriction analysis of PCR products identified a RsaI RFLP. In contrast to other PIT-1 RFLPs, the RsaI PCR-RFLP was widely distributed in American breeds. Thus, the RsaI RFLP and the previously reported PIT-1 RFLPs allow detection of PIT-1 alleles in most, if not all, breeds currently used in the US and European reference/resource pig mapping families.