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PubMed2005

Characteristics of the development of the sensorimotor cortex in rats aged 7-8 days in roller cultures of free-floating brain slices.

The development and formation of the neuronal architectonics of organotypic structures of the sensorimotor cortex in rats aged 7-8 days were studied in roller cultures. Free-floating slices were cultured for 2-3 weeks. Serial paraffin sections of cultured tissue were stained with fast cresyl violet as described by Nissl. Initially planar sections of the cortex were found to change their configuration during the process of cultivation and were transformed into spherical structures, retaining the major histotypic features of cortical formations. Radially orientated pyramidal cells and fusiform neurons formed a cortical rudiment, not discriminated into layers, over the whole surface of the spherical tissue structures. In free-floating slices of the sensorimotor cortex of rats aged 7-8 days in roller cultivation, histogenetic processes continued, leading to the formation of histotypic cortical structures similar to the phylogenetically more ancient allocortical formations of the forebrain.

Animals↗
PubMed2000

Cloning and expression of the gene encoding thermostable poly(3-hydroxybutyrate) depolymerase.

The gene encoding extracellular poly(3-hydroxybutyrate) depolymerase from a thermophilic poly(3-hydroxybutyrate)-degrading bacterium, strain HS, was cloned and intracellularly expressed in Escherichia coli. The gene was found to consist of 1485-bp nucleotide sequence coding for a 22-amino-acid signal peptide and a 473-amino-acid mature protein. Phylogenetic analysis and domain structure showed that the enzyme was clustered with type II PHB depolymerases. The gene was expressed in E. coli under the control of the tac promoter. A 46-kDa protein was detected in the cell extract by SDS-PAGE. The N-terminal sequence of the protein agreed with that of the original enzyme. The crude enzyme was able to degrade PHB particles at 70 degrees C.

Journal Article↗
PubMed2006

Two faces of the prokaryote concept.

Bacteria had remained undefined when, in 1962, Roger Y. Stanier and C.B. van Niel published their famed paper ''The concept of a bacterium.'' The articulation of the prokaryote-eukaryote dichotomy was a vital moment in the history of biology. This article provides a brief overview of the context in which the prokaryote concept was successfully launched in the 1960s, and what it was meant to connote. Two concepts were initially distinguished within the prokaryote-eukaryote dichotomy at that time. One was organizational and referred to comparative cell structure; the other was phylogenetic and referred to a ''natural'' classification. Here, I examine how the two concepts became inseparable; how the prokaryotes came to signify a monophyletic group that preceded the eukaryotes, and how this view remained unquestioned for 15 years, until the birth of molecular evolutionary biology and coherent methods for bacteria phylogenetics based on 16S rRNA. Today, while microbial phylogeneticists generally agree that the prokaryote is a polyphyletic group, there is no agreement on whether the term should be maintained in an organizational sense.

Archaea↗
PubMed1989

Nucleus limitans thalami--comparative anatomical study.

Cyto- and myeloarchitectonics as well as acetylcholinesterase activity of the nucleus limitans thalami--an important part of the nociceptive system--were studied in insectivora (Sorex araneus, Erinaceus europaeus, Talpa europaeus), rat, rabbit, cat, monkey and man. Our results suggest that nucleus limitans is phylogenetically a rather new structure. In insectivora and in the rat we could not find any evident group of neurons resembling nucleus limitans. In the rabbit and cat nucleus limitans is composed of few layers of elongated cells. In macaca and man it is a larger, more complicated structure possessing various types of neurons, and arranged into many layers.

Acetylcholinesterase↗
PubMed1984

On the variability of the human flocculus and paraflocculus accessorius.

42 human cerebelli were examined macroscopically in order to ascertain the general morphological, conformation of the flocculus and accessory paraflocculus, as well as their variability. The flocculus had a mean number of 14.2 +/- 0.3 (s.e.) folia and 7.0 +/- 0.4 (s.e.) subfolia arranged in a rosette-like cluster of relatively constant shape. The coefficient of variability of the number of folia and subfolia was respectively 22.1% and 47.4%. The accessory paraflocculus had a mean number of 4.2 +/- 0.2 (s.e.) folia and 2.0 +/- 0.2 (s.e.) subfolia, with a variability coefficient of respectively 50.2% and 102.8% which was more than double that of the flocculus. In fact the conformation of the accessory paraflocculus varied from a single small flattened lamella to a rosette-like cluster of folia similar in shape and size to those of the flocculus. There was no correlation between the variability of the flocculus and accessory paraflocculus. The possible reason for this marked variability is discussed in the light of the phylogenetic evolution of the structures examined.

Aged↗
PubMed1993

Third component of trout complement. cDNA cloning and conservation of functional sites.

Of the 30 distinct complement proteins recognized to date, C3 is probably the most versatile and multifunctional molecule known, interacting with at least 20 different proteins. It plays a critical role in both pathways of complement activation and participates in phagocytic and immunoregulatory processes. Structural and functional analysis of C3 from different species, in addition to phylogenetic information, provides insights into the structural elements mediating the various functions. This study describes the cDNA cloning of one of two isoforms of the third complement component, C3-1, of rainbow trout (Salmo gairdneri) and the analysis of its functional sites. By screening a trout liver lambda gt11 library with anti-trout C3 chain-specific antibodies and polymerase chain reaction we have determined the cDNA sequence of trout C3-1. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides, corresponding to different regions of trout C3-1. C3-1 consists of 1640 amino acids with a calculated molecular mass of 181,497 Da. The sequence contains two potential N-glycosylation sites, one on each chain of C3. The deduced protein sequence showed 44.1, 43.3, 44.2, 44.9, 43.1, 43.8, 45.9, 29.9, and 33.1% amino acid identities to human, mouse rat, guinea pig, rabbit, cobra, frog, hagfish, and lamprey C3, whereas the identities to human C4, C5, and alpha 2M are 30.4, 28, and 22.9%, respectively. The trout C3 amino acid sequence shows clusters of high and low similarity to C3 from other species. In the regions of high similarity belong the C3 domains that contain the thiolester site and the properdin binding sites, whereas the regions that correspond to regions of human C3 where CR1 and CR2 bind show low amino acid sequence similarity. The deduced amino acid sequence shows that the C3 convertase cleavage site (Arg-Ser) is conserved in trout C3, whereas the factor I cleavage sites are Arg-Ala and Arg-Thr instead of Arg-Ser, which is found in the C3 of other species. Protein sequencing of the trout C3 fragments fixed on zymosan during complement activation confirmed the cleavage of trout C3 by trout C3 convertase and factor I at Arg-Ser and Arg-Thr, respectively.

Amino Acid Sequence↗
PubMed2002

Both urinary and rectal Escherichia coli isolates are dominated by strains of phylogenetic group B2.

To compare the genetic structures of uropathogenic and commensal Escherichia coli populations, a total of 181 urinary and rectal E. coli isolates were classified into intraspecies phylogenetic groups by PCR amplifications of phylogenetic markers. The genetic variability of these isolates within phylogenetic groups was further assessed by enterobacterial repetitive intergenic consensus (ERIC) typing. The distributions of 10 known virulence factors were also examined. In contrast with most reports, phylogenetic group B2 not only accounted for the majority of urinary isolates from young women with urinary tract infections (69%) but also was the dominant group among the rectal isolates from healthy young women (48%). Such difference may be explained by geographic variation, difference in host population characteristics, or differences in sampling method, or a combination of the three. Strains with known virulence factors most frequently belonged to phylogenetic groups B2 and D. Additionally, group B2 and D rectal isolates were more heterogeneous than urinary isolates. Two subclusters existed within group B2 strains by ERIC typing. These subclusters were not evenly distributed between rectal and urine isolates and differed in virulence gene distribution.

Adolescent↗
PubMed2000

Primary structure of cytochrome c' of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c'-type cytochromes.

Cytochrome c' of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome C555. This cytochrome is spectrally similar to other cytochromes c' but is larger (16,000 Da) and has a lower midpoint potential (-205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c' from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c', only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c' from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c'-type cytochrome which developed a covalent cross-link between a lysine residue and the c'-heme.

Amino Acid Sequence↗
PubMed2003

Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA.

Transfer RNA (tRNA) is structurally unique among nucleic acids in harboring an astonishing diversity of post-transcriptionally modified nucleoside. Two of the most radically modified nucleosides known to occur in tRNA are queuosine and archaeosine, both of which are characterized by a 7-deazaguanosine core structure. In spite of the phylogenetic segregation observed for these nucleosides (queuosine is present in Eukarya and Bacteria, while archaeosine is present only in Archaea), their structural similarity suggested a common biosynthetic origin, and recent biochemical and genetic studies have provided compelling evidence that a significant portion of their biosynthesis may in fact be identical. This review covers current understanding of the physiology and biosynthesis of these remarkable nucleosides, with particular emphasis on the only two enzymes that have been discovered in the pathways: tRNA-guanine transglycosylase (TGT), which catalyzes the insertion of a modified base into the polynucleotide with the concomitant elimination of the genetically encoded guanine in the biosynthesis of both nucleosides, and S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), which catalyzes the penultimate step in the biosynthesis of queuosine, the construction of the carbocyclic side chain.

Catalysis↗
PubMed2006

Extensive gene flow blurs phylogeographic but not phylogenetic signal in Olea europaea L.

Genetic structure and evolutionary patterns of the wild olive tree (Olea europaea L.) were investigated with AFLP fingerprinting data at three geographic levels: (a) phylogenetic relationships of the six currently recognized subspecies in Eurasia and Africa; (b) lineage identification in subsp. europaea of the Mediterranean basin; and (c) phylogeography in the western Mediterranean. Two statistical approaches (Bayesian inference and analysis of molecular variance) were used to analyse the AFLP fingerprints. To determine the congruency and transferability of results across studies previous RAPD and ISSR data were analysed in a similar manner. Comparisons proved that qualitative results were mostly congruent but quantitative values differed, depending on the method of analysis. Neighbour-Joining analysis of AFLP phenotypes supported current classification of subspecies. At a Mediterranean scale no clear cut phylogeographic pattern was recovered, likely due to extensive gene flow between populations of subsp. europaea. Gene flow estimates calculated with conventional F-statistics showed that reproductive barriers separated neither populations nor lineages of O. europaea. Genetic divergence between eastern and western parts of the Mediterranean basin was observed only when geographical and population information were incorporated into the analyses through hierarchical analysis of molecular variance (AMOVA). Within the western Mediterranean, the highest genetic diversity was found in two regions: on both sides of the Strait of Gibraltar and in the Balearic archipelago. Additionally, long-lasting isolation of the northern-most populations of the Iberian Peninsula appeared to be responsible for a significant divergence.

DNA Fingerprinting↗
PubMed2006

Red blood with blue-blood ancestry: intriguing structure of a snail hemoglobin.

The phylogenetic enigma of snail hemoglobin, its isolated occurrence in a single gastropod family, the Planorbidae, and the lack of sequence data, stimulated the present study. We present here the complete cDNA and predicted amino acid sequence of two hemoglobin polypeptides from the planorbid Biomphalaria glabrata (intermediate host snail for the human parasite Schistosoma mansoni). Both isoforms contain 13 different, cysteine-free globin domains, plus a small N-terminal nonglobin "plug" domain with three cysteines for subunit dimerization (total M(r) approximately 238 kDa). We also identified the native hemoglobin molecule and present here a preliminary 3D reconstruction from electron microscopical images (3 nm resolution); it suggests a 3 x 2-mer quaternary structure (M(r) approximately 1.43 MDa). Moreover, we identified a previously undescribed rosette-like hemolymph protein that has been mistaken for hemoglobin. We also detected expression of an incomplete hemocyanin as trace component. The combined data show that B. glabrata hemoglobin evolved from pulmonate myoglobin, possibly to replace a less-efficient hemocyanin, and reveals a surprisingly simple evolutionary mechanism to create a high molecular mass respiratory protein from 78 similar globin domains.

Amino Acid Sequence↗
PubMed1991

Predicting common foldings of homologous RNAs.

A new approach is proposed for determining common RNA secondary structures within a set of homologous RNAs. The approach is a combination of phylogenetic and thermodynamic methods which is based on the prediction of optimal and suboptimal secondary structures, topological similarity searches and phylogenetic comparative analysis. The optimal and suboptimal RNA secondary structures are predicted by energy minimization. Structural comparison of the predicted RNA secondary structures is used to find conserved structures that are topologically similar in all these homologous RNAs. The validity of the conserved structural elements found is then checked by phylogenetic comparison of the sequences. This procedure is used to predict common structures of ribonuclease P (RNAase P) RNAs.

Algorithms↗
PubMed2005

Reevaluation of the predicted gene structure of Dictyostelium cystatin A3 (cpiC) by nucleotide sequence determination of its cDNA* and its phylogenetic position in the cystatin superfamily.

Cystatins, cysteine protease inhibitors, are widely distributed among eukaryotes. We reevaluated the structure of the gene cpiC, a gene encoding the third identified member of cystatin family (cystatin A3) that was predicted in the genome database of the social amoeba Dictyostelium discoidium (dictyBase) but remained controversial. We determined the sequences of cDNA and PCR-amplified genomic DNA fragment and found a critical error in the registered nucleotide sequence. The corrected cystatin A3 gene has an open reading frame (ORF) without intron sequence interruption and encodes 94 amino acids (aa), in contrast to the previously predicted sequence of either 80, 82 or 118 aa. The cDNA has an unusual internal poly(A) sequence of 31 adenines, which immediately follows the translation termination codon (TAA) located 146 nucleotides upstream of the post-transcriptional polyadenylation site. The amino acid sequence of Dictyostelium cystatin A3 shows a high similarity to those of previously reported Dictyostelium cystatins as well as Family I cystatins of higher eukaryotes.

Amino Acid Sequence↗
PubMed2026

Genome-wide SNP data support species boundaries in sympatric Polylepis Ruiz & Pav. (Rosaceae) species from Bolivia and Ecuador.

Species delimitation in the South American genus Polylepis is notoriously challenging due to high morphological similarity and phenotypic plasticity, likely driven by hybridization and gene flow. Previous phylogenetic studies suggested that genetic structure aligns more strongly with geography than with taxonomy, questioning existing species concepts and hampering conservation efforts. We used double-digest RAD sequencing (ddRADseq) to generate genome-wide SNP data for 11 Polylepis species sampled across multiple localities in Bolivia and Ecuador. Population genetic analyses, phylogenetic inference, and network approaches were combined to assess whether genetic structure aligns more closely with taxonomy or geography. Morphologically defined species formed largely cohesive genetic lineages across regions, with species identity explaining substantially more genetic variation than locality. While localized admixture and reticulation were detected among closely related taxa, widespread species showed strong genetic cohesion and clear separation from congeners. Our results indicate that the sampled Polylepis species from Bolivia and Ecuador maintain distinct genetic identities despite localized signals consistent with gene flow. This genome-wide support for current taxonomy highlights Polylepis as a valuable model for studying speciation under gene flow and indicates that multiple geographic sampling will be essential in reconstructing a robust phylogeny of the genus, with important implications for conservation planning in Andean montane forests.

Bolivia↗
PubMed2002

Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal.

The packaging signal (Psi) of the human immunodeficiency virus type 1 (HIV-1) enables encapsidation of the full-length genomic RNA against a background of a vast excess of cellular mRNAs. The core HIV-1 Psi is approximately 109 nucleotides and contains sequences critical for viral genomic dimerisation and splicing, in addition to the packaging signal. It consists of a series of stem-loops (termed SL-1 to SL-4), which can be arranged in a cloverleaf secondary structure. Using a combination of NMR spectroscopy, UV melting experiments, molecular modeling and phylogenetic analyses, we have explored the structure of two conserved internal loops proximal to the palindromic sequence of SL-1. Internal loop A, composed of six purines, forms a flexible structure that is strikingly similar to the Rev responsive element motif when bound to Rev protein. This result suggests that it may function as a protein-binding site. The absolutely conserved four-purine internal loop B is instead conformationally and thermodynamically unstable, and exhibits multiple conformations in solution. By introducing a double AGG to GGA mutation within this loop, its conformation is stabilised to form a new intra-molecular G:A:G base-triplet. The structure of the GGA mutant explains the relative instability of the wild-type loop. In a manner analogous to SL-3, we propose that conformational flexibility at this site may facilitate melting of the structure during Gag protein capture or genomic RNA dimerisation.

Base Pairing↗
PubMed1987

Structural diversity of eukaryotic small subunit ribosomal RNAs. Evolutionary implications.

The phylogenetic diversity of the eukaryotic kingdom was assessed by comparing the structural and evolutionary diversity of 18-20S ribosomal RNA genes. The coding regions for cytoplasmic small subunit ribosomal RNA genes vary in length from 1753 to 2305 nucleotides, and they appear to be evolutionary mosaics in which highly and partially conserved sequences are interspersed among regions that display very high rates of genetic drift. Structural similarities between these gene sequences were used to establish a phylogenetic framework for the eukaryotes. The extent of sequence variation within the eukaryotes exceeds that displayed within the eubacterial or archaebacterial lines of descent. The kinetoplastids and euglenoids represent the earliest branchings among the eukaryotes. These branchings preceded the divergence of lineages leading to the slime molds and apicomplexans and far antedate a radiative period that gave rise to the plants, animals, fungi, and other protists.

Animals↗
PubMed2001

Gene structure and chromosomal location of a human bHLH transcriptional factor DEC1 x Stra13 x SHARP-2/BHLHB2.

DEC1/BHLHB2 is a novel cAMP-inducible basic helix-loop-helix (bHLH) transcriptional factor isolated from human chondrocyte cultures by the subtraction method [Shen et al. (1997) Biochem. Biophys. Res. Commun. 236, 294--298]. DEC1 seems to be involved in controlling the proliferation/differentiation of some cell lineages. We determined the structure of the human DEC1 gene and its chromosomal locus. Phylogenetic analysis and comparison of the gene structure showed that the DEC1 protein is a member of a new subgroup of the proline bHLH protein family that diverged earlier than other proline bHLH proteins including HES, hairy and E(spl). The human DEC1 gene spans approximately 5.7 kb and contains 5 exons. The putative promoter region contains multiple GC boxes but no TATA box. A primer extension study showed multiple transcriptional initiation sites. In the 5'-flanking region of the DEC1 gene, several transcriptional factor binding sites, including a cAMP-responsive element (CRE), were found using the transcription factor database. The DEC1 gene locates at Chromosome 3p25.3--26 by the FISH method. This is the first study to determine the genomic structure of the DEC1 gene subgroup.

Amino Acid Sequence↗
PubMed1997

Effects of nucleotide sequence alignment on phylogeny estimation: a case study of 18S rDNAs of apicomplexa.

The reconstruction of phylogenetic history is predicated on being able to accurately establish hypotheses of character homology, which involves sequence alignment for studies based on molecular sequence data. In an empirical study investigating nucleotide sequence alignment, we inferred phylogenetic trees for 43 species of the Apicomplexa and 3 of Dinozoa based on complete small-subunit rDNA sequences, using six different multiple-alignment procedures: manual alignment based on the secondary structure of the 18S rRNA molecule, and automated similarity-based alignment algorithms using the PileUp, ClustalW, TreeAlign, MALIGN, and SAM computer programs. Trees were constructed using neighboring-joining, weighted-parsimony, and maximum-likelihood methods. All of the multiple sequence alignment procedures yielded the same basic structure for the estimate of the phylogenetic relationship among the taxa, which presumably represents the underlying phylogenetic signal. However, the placement of many of the taxa was sensitive to the alignment procedure used; and the different alignments produced trees that were on average more dissimilar from each other than did the different tree-building methods used. The multiple alignments from the different procedures varied greatly in length, but aligned sequence length was not a good predictor of the similarity of the resulting phylogenetic trees. We also systematically varied the gap weights (the relative cost of inserting a new gap into a sequence or extending an already-existing gap) for the ClustalW program, and this produced alignments that were at least as different from each other as those produced by the different alignment algorithms. Furthermore, there was no combination of gap weights that produced the same tree as that from the structure alignment, in spite of the fact that many of the alignments were similar in length to the structure alignment. We also investigated the phylogenetic information content of the helical and nonhelical regions of the rDNA, and conclude that the helical regions are the most informative. We therefore conclude that many of the literature disagreements concerning the phylogeny of the Apicomplexa are probably based on differences in sequence alignment strategies rather than differences in data or tree-building methods.

Algorithms↗