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Weighing in on a Timeless controversy.

Timeless (Tim) and Period (Per) are coordinately synthesized interacting proteins that in response to positional/environmental cues comigrate to the nucleus as obligate heterodimers where they act to suppress their own gene expression as part of the circadian rhythm network in Drosophila. There has been considerable controversy about the structural nature of Tim because of somewhat conflicting results generated by the automated threading algorithm 3D-PSSM. With use of a computer-assisted but largely manual approach, it is shown here that Tim is composed of repetitive structural elements and that those elements comprise two ARM repeat domains, validating the essence of the original 3D-PSSM prediction. Eleven individual ARM structural units are assigned, and a phylogenetic analysis showing their relatedness to one another is performed. In addition, there appears to be a small domain of prenyltransferase-like repeats C-terminal to the second ARM domain, which went undetected in previous analyses. Although we cannot know the precise conformation it adopts until a structure is solved, Tim emerges here as clearly a member of the helical repeat protein superfamily. Given its role in periodic nuclear translocation, Tim may, therefore, have a functional analogy with the photoperiod-responsive protein Phor1 and other karyopherin-like molecules.

ARNTL Transcription Factors↗

Evolution of fragmented mitochondrial ribosomal RNA genes in Chlamydomonas.

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algae Chlamydomonas eugametos and Chlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences of C. eugametos and C. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga, Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genus Chlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the two Chlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between the Chlamydomonas sequences and P. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed for C. eugametos and C. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor of Chlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.

Animals↗

Three-dimensional visualization of insect morphology using confocal laser scanning microscopy.

Cuticular structures of insects are often microscopic and intricately complex; among the most complex structures are male genitalia. Genitalic structures are essential in taxonomic and phylogenetic studies of insects. Using well-described species from two disparate dipteran genera, we demonstrate the utility of confocal laser scanning microscopy for studying the morphological characters of fly genitalia by taking advantage of the autofluorescent properties of cuticle material. Reconstructions of confocal data sets obtained from genitalic structures embedded in two commonly used entomological mounting media (euparal and glycerin jelly) are presented. Aberration artefacts often observed in confocal data obtained from thick specimens were analysed and strategies for their minimization are discussed. Our results indicate that confocal laser scanning microscopy and 3D reconstruction are excellent techniques for visualizing small, complex, autofluorescent structures in flies. These techniques could have a profound impact on the quality of information provided by 3D representations of insect structures over more traditional methods of visualization.

Animals↗

A molecular phylogenetic study of the freshwater talitrid amphipod Orchestia cavimana (Crustacea).

In this paper we performed a molecular phylogenetic study of Orchestia cavimana, the sole talitrid amphipod inhabiting beaches of European freshwater lakes and rivers. For that purpose, we have PCR amplified and sequenced regions of the mitochondrial cytochrome oxidase subunit I (COI) gene, basing our analysis on both nucleotide and amino acid sequences and considering also structural classes of the COI enzyme. Phylogenetic analyses were conducted by neighbour-joining (NJ) and maximum-parsimony (MP) methods comparing homologous sequences of talitrids and other Crustacea. In both NJ and MP trees, O. cavimana shows a basal placement with respect to other talitrid amphipods.

Amino Acid Sequence↗

[Foundations of the new phylogenetics].

Evolutionary idea is the core of the modern biology. Due to this, phylogenetics dealing with historical reconstructions in biology takes a priority position among biological disciplines. The second half of the 20th century witnessed growth of a great interest to phylogenetic reconstructions at macrotaxonomic level which replaced microevolutionary studies dominating during the 30s-60s. This meant shift from population thinking to phylogenetic one but it was not revival of the classical phylogenetics; rather, a new approach emerged that was baptized The New Phylogenetics. It arose as a result of merging of three disciplines which were developing independently during 60s-70s, namely cladistics, numerical phyletics, and molecular phylogenetics (now basically genophyletics). Thus, the new phylogenetics could be defined as a branch of evolutionary biology aimed at elaboration of "parsimonious" cladistic hypotheses by means of numerical methods on the basis of mostly molecular data. Classical phylogenetics, as a historical predecessor of the new one, emerged on the basis of the naturphilosophical worldview which included a superorganismal idea of biota. Accordingly to that view, historical development (the phylogeny) was thought an analogy of individual one (the ontogeny) so its most basical features were progressive parallel developments of "parts" (taxa), supplemented with Darwinian concept of monophyly. Two predominating traditions were diverged within classical phylogenetics according to a particular interpretation of relation between these concepts. One of them (Cope, Severtzow) belittled monophyly and paid most attention to progressive parallel developments of morphological traits. Such an attitude turned this kind of phylogenetics to be rather the semogenetics dealing primarily with evolution of structures and not of taxa. Another tradition (Haeckel) considered both monophyletic and parallel origins of taxa jointly: in the middle of 20th century it was split into phylistics (Rasnitsyn's term; close to Simpsonian evolutionary taxonomy) belonging rather to the classical realm, and Hennigian cladistics that pays attention to origin of monophyletic taxa exclusively. In early of the 20th century, microevolutionary doctrine became predominating in evolutionary studies. Its core is the population thinking accompanied by the phenetic one based on equation of kinship to overall similarity. They were connected to positivist philosophy and hence were characterized by reductionism at both ontological and epistemological levels. It led to fall of classical phylogenetics but created the prerequisites for the new phylogenetics which also appeared to be full of reductionism. The new rise of phylogenetic (rather than tree) thinking during the last third of the 20th century was caused by lost of explanatory power of population one and by development of the new worldview and new epistemological premises. That new worldview is based on the synergetic (Prigoginian) model of development of non-equilibrium systems: evolution of the biota, a part of which is phylogeny, is considered as such a development. At epistemological level, the principal premise appeared to be fall of positivism which was replaced by post-positivism argumentation schemes. Input of cladistics into new phylogenetics is twofold. On the one hand, it reduced phylogeny to cladistic history lacking any adaptivist interpretation and presuming minimal evolution model. From this it followed reduction of kinship relation to sister-group relation lacking any reference to real time scale and to ancestor-descendant relation. On the other hand, cladistics elaborated methodology of phylogenetic reconstructions based on the synapomorphy principle, the outgroup concept became its part. The both inputs served as premises of incorporation of both numerical techniques and molecular data into phylogenetic reconstruction. Numerical phyletics provided the new phylogenetics with easily manipulated algorithms of cladogram construing and thus made phylogenetic reconstructions operational and repetitive. The above phenetic formula "kinship = similarity" appeared to be a keystone for development of the genophyletics. Within numerical phyletics, a lot of computer programs were elaborated which allow to manipulate with evolutionary scenario during phylogenetic reconstructions. They make it possible to reconstruct both clado- and semogeneses based on the same formalized methods. Multiplicity of numerical approaches indicates that, just as in the case of numerical phenetics, choice of adequate method(s) should be based on biologically sound theory. The main input of genophyletics (= molecular phylogenetics) into the new phylogenetics was due to completely new factology which makes it possible to compare directly such far distant taxa as prokaryotes and higher eukaryotes. Genophyletics is based on the theory of neutral evolution borrowed from microevolutionary theory and on the molecular clock hypothesis which is now considered largely inadequate. The future developments of genophyletics will be aimed at clarification of such fundamental (and "classical" by origin) problems as application of character and homology concepts to molecular structures. The new phylogenetics itself is differentiated into several schools caused basically by diversity of various approaches existing within each of its "roots". Cladistics makes new phylogenetics splitted into evolutionary and parsimonious ontological viewpoints. Numerical phyletics divides it into statistical and (again) parsimonious methodologies. Molecular phylogenetics is opposite by its factological basis to morphological one. The new phylogenetics has significance impact onto the "newest" systematics. From one side, it gives ontological status back to macrotaxa they have lost due to "new" systematics based on population thinking. From another side, it rejects some basical principles of classical phylogenetic (originally Linnean) taxonomy such as recognitions of fixed taxonomic ranks designated by respective terms and definition of taxic names not by the diagnostic characters but by reference to the ancestor. The latter makes the PhyloCode overburdened ideologically and the "newest" systematics self-controversial, as concept of ancestor has been acknowledged non-operational from the very beginning of cladistics. Relation between classical and new phylogenetics is twofold. At the one hand, general phylogenetic hypothesis (in its classical sense) can be treated as a combination of cladogenetic and semogenetic reconstructions. Such a consideration is bound to pay close attention to the uncertainty relation principle which, in case of the phylogenetics, means that the general phylogenetic hypothesis cannot be more certain than any of initial cladogenetic or semogenetic hypotheses. From this standpoint, the new phylogenetics makes it possible to reconstruct phylogeny following epistemological principle "from simple to complex". It elaborates a kind of null hypotheses about evolutionary history which are more easy to test as compared to classical hypotheses. Afterward, such hypotheses are possible to be completed toward the classical, more content-wise ones by adding anagenetic information to the cladogenetic one. At another hand, reconstructions elaborated within the new phylogenetics could be considered as specific null hypotheses about both clado- and semogeneses. They are to be tested subsequently by mean of various models, including those borrowed from "classical" morphology. The future development of the new phylogenetics is supposed to be connected with getting out of plethora of reductionism inherited by it from population thinking and specification of object domain of the phylogenetics. As the latter is a part of an evolutionary theory, its future developments will be adjusted with the latter. Lately predominating neodarwinism is now being replaced by the epigenetic evolutionary theory to which phylistics (one of the modern versions of classical phylogenetics) seems to be more correspondent.

Animals↗

Molecular homology assessment and phylogeny in the lizard family opluridae (Squamata: Iguania).

The effect of utilizing secondary structure on multiple alignment and phylogeny reconstruction was examined for the mitochondrial 12S ribosomal RNA and valine transfer RNA genes in the lizard family Opluridae. Computer-generated multiple alignments were performed under parsimony using gap costs of 1-10 both with and without reference to secondary structure. Alignments performed without secondary structure resulted in disruption of positional homology in some stems at all gap costs. Number of misaligned stem positions increased with gap cost. Phylogenetic analyses of multiple alignments without secondary structure recovered a well-supported morphological tree for five gap costs, and this was the single most parsimonious tree only for a gap cost of 1. Computer-generated multiple alignments using secondary structure to maintain positional homologs in stems recovered the morphological tree over all gap costs, and this was the single most parsimonious tree for 8 of 10 gap costs. The combined morphological and molecular data produced a single most parsimonious oplurid tree that was congruent with the morphological tree over all gap costs. Chalaradon madagascariensis is the sister taxon of Oplurus, which itself contains two clades: one with the arboreal Oplurus cuvieri and Oplurus cyclurus and the other with the saxicolous Oplurus grandidieri, Oplurus fierinensis, Oplurus quadrimaculatus, and Oplurus saxicola.

Animals↗

An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression.

RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTRs are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity.

5' Untranslated Regions↗

House cleaning, a part of good housekeeping.

Cellular metabolism constantly generates by-products that are wasteful or even harmful. Such compounds are excreted from the cell or are removed through hydrolysis to normal cellular metabolites by various 'house-cleaning' enzymes. Some of the most important contaminants are non-canonical nucleoside triphosphates (NTPs) whose incorporation into the nascent DNA leads to increased mutagenesis and DNA damage. Enzymes intercepting abnormal NTPs from incorporation by DNA polymerases work in parallel with DNA repair enzymes that remove lesions produced by modified nucleotides. House-cleaning NTP pyrophosphatases targeting non-canonical NTPs belong to at least four structural superfamilies: MutT-related (Nudix) hydrolases, dUTPase, ITPase (Maf/HAM1) and all-alpha NTP pyrophosphatases (MazG). These enzymes have high affinity (Km's in the micromolar range) for their natural substrates (8-oxo-dGTP, dUTP, dITP, 2-oxo-dATP), which allows them to select these substrates from a mixture containing a approximately 1000-fold excess of canonical NTPs. To date, many house-cleaning NTPases have been identified only on the basis of their side activity towards canonical NTPs and NDP derivatives. Integration of growing structural and biochemical data on these superfamilies suggests that their new family members cleanse the nucleotide pool of the products of oxidative damage and inappropriate methylation. House-cleaning enzymes, such as 6-phosphogluconolactonase, are also part of normal intermediary metabolism. Genomic data suggest that house-cleaning systems are more abundant than previously thought and include numerous analogous enzymes with overlapping functions. We discuss the structural diversity of these enzymes, their phylogenetic distribution, substrate specificity and the problem of identifying their true substrates.

Binding Sites↗

Resting and stimulated respiration in vitro in the central nervous system. Regional distribution and relation to cell density.

1. The respiratory rates in vitro of ten structures of the rabbit brain were measured in the presence of a normal (5mm) and an elevated (50mm) concentration of K(+) ions. The results were expressed on a dry-weight basis and in terms of cell density. 2. On a dry-weight basis, with a normal concentration of K(+), there was a steady decrease in respiratory rate on passing from the cerebral cortex through successively lower centres to the brain-stem, i.e. respiration was negatively correlated with phylogenetic age. 3. When the resting respiratory rates were expressed in terms of cell density there was no correlation with phylogenetic age. The neuron-containing structures of the cerebrum and brain-stem had identical respiratory rates with the exception of the cerebral cortex which had a higher rate. 4. The K(+)-stimulated respiratory rates/cell also showed a negative correlation with phylogenetic age; the higher centres responded with a greater percentage increase than the lower. 5. The results from the cerebellar cortex were anomalous. 6. There were variations of respiratory rate within the diencephalon. The thalamus had a relatively high respiratory rate, the anterior and ventral regions relatively low and the hypothalamus intermediate. 7. The oxygen consumption of white matter was not increased by a high external concentration of K(+).

Animals↗

Protein structural influences in rhodopsin evolution.

Incorporating specific structural information can be important for developing a realistic model of evolution for phylogenetic reconstruction of protein-coding genes. We analyzed 62 sequences of vertebrate rhodopsin. The bovine rhodopsin structure was used to label residue sites by surface accessibility, secondary structure, and transmembrane (TM) location. Residue sites with amino acid differences were identified; using maximum parsimony (MP), homoplasious residues were identified. Residues were analyzed for patterns that would indicate correlation of rate with secondary structure, surface accessibility, or position relative to the lipid bilayer. Surface residues, especially those residing in one of the seven TM helices, were significantly correlated with high rates of amino acid substitution. This category of residues, defined solely by protein structural characteristics, potentially defined a class enriched in homoplasious residues. MP analysis using all sites led to a tree with anomalies in the relationships of amphibian, mammalian, bird, and alligator species. Analysis excluding the structurally defined residue class recovered a more accurate phylogeny. A model is presented for including structural influences on rate in phylogenetic inference.

Amino Acid Substitution↗

Genetic heterogeneity in the foot-and-mouth disease virus Leader and 3C proteinases.

The Leader and 3C proteinases of foot-and-mouth disease virus (FMDV) are responsible for almost all the proteolytic processing events of the viral polyprotein precursor. Investigation into the genetic heterogeneity of the regions encoding these proteins from isolates of six FMDV serotypes revealed the 3C proteinase to be more conserved than the Leader proteinase. Maximum likelihood analysis indicated similar phylogenetic groupings for the non-structural protein coding regions of both the Leader and 3C. These groupings were different from the structural VP1 protein coding region which, as shown previously, grouped according to serotype. Two distinct clades were apparent for both the Leader and 3C coding regions: one comprising of serotypes A, O and C together with SAT (South African Territories) isolates from eastern Africa. The other clade consisted of SAT isolates originating from southern Africa. Only one virus isolate, obtained from a buffalo in Uganda, did not conform to this phylogenetic pattern. This SAT 1 virus grouped with types A, O and C in the Leader analysis, but with the southern African SAT types in the 3C analysis, implicating intertypic recombination. The leader proteinases of southern African SAT type isolates differed from those present in European type isolates, particularly in the self-processing region. A three-dimensional structure was modeled for the Leader proteinase of one of the SAT type viruses, ZIM/7/83/2, and compared with the previously elucidated crystal structure of O(1)Kaufbeuren Leader proteinase. The active sites of the two leaders were found to superimpose closely, despite the observed sequence variation between the two molecules. Comparison of the 3C proteinase P1 cleavage sites suggested that the FMDV 3C proteinase may possess a broader substrate specificity, as observed in hepatitis A virus 3C proteinase.

3C Viral Proteases↗

[Partial sequence homology of FtsZ in phylogenetics analysis of lactic acid bacteria].

FtsZ is a structurally conserved protein, which is universal among the prokaryotes. It plays a key role in prokaryote cell division. A partial fragment of the ftsZ gene about 800bp in length was amplified and sequenced and a partial FtsZ protein phylogenetic tree for the lactic acid bacteria was constructed. By comparing the FtsZ phylogenetic tree with the 16S rDNA tree, it was shown that the two trees were similar in topology. Both trees revealed that Pediococcus spp. were closely related with L. casei group of Lactobacillus spp. , but less related with other lactic acid cocci such as Enterococcus and Streptococcus. The results also showed that the discriminative power of FtsZ was higher than that of 16S rDNA for either inter-species or inter-genus and could be a very useful tool in species identification of lactic acid bacteria.

Bacterial Proteins↗

Differences in structure and dynamics of Polynucleobacter communities in a temperate and a subtropical lake, revealed at three phylogenetic levels.

The results of ecological studies investigating bacteria by cultivation-independent methods are expected to be influenced by the phylogenetic resolution of the applied molecular tools. This potential influence was investigated in a comparative community study on Polynucleobacter cluster bacteria (Betaproteobacteria) inhabiting the pelagic of the large, shallow, partially hypertrophic Taihu Lake located in subtropical East Asia, and the deep oligo-mesotrophic Lake Mondsee located in temperate Central Europe. The two contrasting habitats were sampled over the same period of 12 months. The community dynamics were investigated at three phylogenetic levels by fluorescent in situ hybridizations with a set of nested probes specific for the beta II clade (including the Polynucleobacter cluster), the genus-like monophyletic Polynucleobacter cluster, and four species-like subclusters. Subcluster B was the numerically dominating subcluster in both lakes over the investigation periods, but demonstrated different population dynamics in the two habitats. Interhabitat comparisons of the Polynucleobacter community structure in the two lakes in the study, and a previously investigated acidic pond indicated ecological diversification within the phylogenetically narrow Polynucleobacter cluster. These results could be obtained by help of the subcluster-specific probes, but would have been missed with probes of a lower phylogenetic resolution.

Austria↗

Evolutionary history and higher order classification of AAA+ ATPases.

The AAA+ ATPases are enzymes containing a P-loop NTPase domain, and function as molecular chaperones, ATPase subunits of proteases, helicases or nucleic-acid-stimulated ATPases. All available sequences and structures of AAA+ protein domains were compared with the aim of identifying the definitive sequence and structure features of these domains and inferring the principal events in their evolution. An evolutionary classification of the AAA+ class was developed using standard phylogenetic methods, analysis of shared sequence and structural signatures, and similarity-based clustering. This analysis resulted in the identification of 26 major families within the AAA+ ATPase class. We also describe the position of the AAA+ ATPases with respect to the RecA/F1, helicase superfamilies I/II, PilT, and ABC classes of P-loop NTPases. The AAA+ class appears to have undergone an early radiation into the clamp-loader, DnaA/Orc/Cdc6, classic AAA, and "pre-sensor 1 beta-hairpin" (PS1BH) clades. Within the PS1BH clade, chelatases, MoxR, YifB, McrB, Dynein-midasin, NtrC, and MCMs form a monophyletic assembly defined by a distinct insert in helix-2 of the conserved ATPase core, and additional helical segment between the core ATPase domain and the C-terminal alpha-helical bundle. At least 6 distinct AAA+ proteins, which represent the different major clades, are traceable to the last universal common ancestor (LUCA) of extant cellular life. Additionally, superfamily III helicases, which belong to the PS1BH assemblage, were probably present at this stage in virus-like "selfish" replicons. The next major radiation, at the base of the two prokaryotic kingdoms, bacteria and archaea, gave rise to several distinct chaperones, ATPase subunits of proteases, DNA helicases, and transcription factors. The third major radiation, at the outset of eukaryotic evolution, contributed to the origin of several eukaryote-specific adaptations related to nuclear and cytoskeletal functions. The new relationships and previously undetected domains reported here might provide new leads for investigating the biology of AAA+ ATPases.

Adenosine Triphosphatases↗

Gene structure of a chlorophyll a/c-binding protein from a brown alga: presence of an intron and phylogenetic implications.

A Laminaria saccharina genomic library in the phage EMBL 4 was used to isolate and sequence a full-length gene encoding a fucoxanthin-chlorophyll a/c-binding protein. Contrary to diatom homologues, the coding sequence is interrupted by an intron of about 900 bp which is located in the middle of the transit peptide. The deduced amino acid sequence of the mature protein is very similar to those of related proteins from Macrocystis pyrifera (Laminariales) and, to a lesser extent, to those from diatoms and Chrysophyceae. Seven of the eight putative chlorophyll-binding amino acids determined in green plants are also present. Alignments of different sequences related to the light-harvesting proteins (LHC) demonstrate a structural similarity among the three transmembrane helices and suggest a unique ancestral helix preceded by two beta-turns. The beta-turns are conserved in front of the second helices of the chlorophyll a/c proteins more so than in chlorophyll a/b proteins. Phylogenetic trees generated from sequence data indicate that fucoxanthin-chlorophyll-binding proteins diverged prior to the separation of photosystem I and photosystem II LHC genes of green plants. Among the fucoxanthin-containing algae, LHC I or II families could not be distinguished at this time.

Amino Acid Sequence↗

Phylogenetic signal in the song of crests and kinglets (Aves: Regulus).

Territorial song structures are often the most prominent characters for distinguishing closely related taxa among songbirds. Learning processes may cause convergent evolution of passerine songs, but phylogenetic information of acoustic traits can be investigated with the help of molecular phylogenies, which are not affected by cultural evolutionary processes. We used a phylogeny based on cytochrome b sequences to trace the evolution of territorial song within the genus Regulus. Five discrete song units are defined as basic components of regulid song via sonagraphic measurements. Traits of each unit are traced on a molecular tree and a mean acoustic character difference between taxon pairs is calculated. Acoustic divergence between regulid taxa correlates strongly with genetic distances. Syntax features of complete songs and of single units are most consistent with the molecular data, whereas the abundance of certain element types is not. Whether song characters are innate or learned was interpreted using hand-reared birds in aviary experiments. We found that convergent character evolution seems to be most probable for learned acoustic traits. We conclude that syntax traits of whole verses or subunits of territorial song, especially innate song structures, are the most reliable acoustic traits for phylogenetic reconstructions in Regulus.

Animals↗

Forced evolution of a regulatory RNA helix in the HIV-1 genome.

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.

Base Sequence↗

Molecular phylogeny and phylogeography of free-living Bryozoa (Cupuladriidae) from both sides of the Isthmus of Panama.

Genetic data were used to identify Recent species of free-living bryozoans (Cupuladriidae) from both sides of the Isthmus of Panama, and to examine their phylogenetic relationships, species richness, and population structures. An approximately 480bp fragment of the 16S mitochondrial rRNA gene was sequenced from 182 individuals from Panama, the Gulf of Mexico, and El Salvador. Ten haplotype groups (Cupuladria 4, 5, and 6; Discoporella 1, 2, 3A, 3B, 3C, 7, and 8) were identified. Genetic distances between haplotype groups (3.2-26.5%; K2P+Gamma) were 1-2 orders of magnitude greater than within groups (0.1-1.4%). Seven of the haplotype groups represent morphologically distinct species; Discoporellas 3A-C appear to be cryptic species. Phylogenetic analyses identified two pairs of transisthmian sister clades. An average divergence rate derived from other taxa suggests that Cupuladrias 4 and 5 diverged approximately 7Ma, a Discoporella 7 clade diverged from a 3A-C clade approximately 11Ma, and the 3A-C clade radiated approximately 6-4Ma; these events all predated final closure of the isthmus? 3Ma. The Caribbean side of the isthmus, with 5 species, is only marginally richer in cupuladriids than the Pacific side, with 4, but has greater phylogenetic depth. The Caribbean retains lineages stemming from a New World Miocene radiation that are not represented in the eastern Pacific; extant eastern Pacific cupuladriids share most recent common ancestry with only two of the Caribbean lineages. Species in the eastern Pacific tend to show shallow population structures, with high levels of gene flow between geographically separate populations, whereas Caribbean species tend to show deeper populations structures, with indications of restricted gene flow between Bocas del Toro/Gulf of Mosquitos and Costa Arriba/San Blas. The population structures derive from Pleistocene histories and may be of limited value in interpreting the macroevolutionary pattern, as our results provide no evidence of speciation on either side of the isthmus following closure in the late Pliocene.

Animals↗