Galactose-binding lectins as markers of pregnancy-related glycoproteins

Protein extracts from pregnant mouse endometria were compared with those obtained from non-pregnant and pseudopregnant mice to detect early pregnancy-specific galactose-rich glycoproteins. Gradient gel electrophoresis combined with lectin overlay and lectin histochemistry were used to identify Ricinus communis I (RCA-I), R. communis II (RCA-II) and Cytisus scoparius (CSA) lectin binding glycoproteins. Using this approach, galactose-rich glycoproteins were identified that were maximally expressed in the estrus phase of non-pregnant endometria and also those that had peak expression in pregnancy. Lectin histochemistry revealed pregnancy related changes in three portions of mouse endometrium: endometrial glands, luminal epithelium and its basement membrane. Two major glycoproteins (RCA-I reactive 64 kDa and RCA-II reactive 35 kDa) were specifically expressed in peri-implantation endometrium on days 3 and 4 of pregnancy. The appearance of these glycoproteins during the period of the implantation window in mouse suggests that they could serve as markers of uterine receptivity to the implanting blastocyst.     

Localization of lectins in legume cotyledons.

High-resolution techniques for the localization of lectins are described. Concanavalin A (Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with (FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidney bean cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A and PHA are found in cytoplasmic sites. Lectins with beta-glycoside specificity are detected with red-coloured artificial carbohydrate antigens. The beta-galactosyl and beta-glucosyl antigens bind specifically to clusters of spherical bodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasm associated with the cell membrane.     

Determinants of quaternary association in legume lectins

It is well known that the sequence of amino acids in proteins code for its tertiary structure. It is also known that there exists a relationship between sequence and the quaternary structure of proteins. The question addressed here is whether the nature of quaternary association can be predicted from the sequence, similar to the three-dimensional structure prediction from the sequence. The class of proteins called legume lectins is an interesting model system to investigate this problem, because they have very high sequence and tertiary structure homology, with diverse forms of quaternary association. Hence, we have used legume lectins as a probe in this paper to (1) gain novel insights about the relationship between sequence and quaternary structure; (2) identify the sequence motifs that are characteristic of a given type of quaternary association; and (3) predict the quaternary association from the sequence motif.     

Prediction and comparison of the secondary structure of legume lectins

Secondary structure prediction for the 4 legume lectins: Concanavalin A, soybean agglutinin, favabean lectin and lentil lectin, was done by the method of Chou and Fasman. This prediction shows that these four lectins fall into a structurally distinct class of proteins, containing high amounts of β-sheet and β-turns. There is a notable similarity in the gross structure of these proteins; all four of them contain about 40–50% of β-sheet, 35–45 % β-turn and 0–10% of α-helix. When the secondary structure of corresponding residues in each pair of these lectins was compared, there was a striking similarity in the Concanavalin A-soybean agglutinin and favabean lectin-lentil lectin pairs, and considerably less similarity in the other pairs, suggesting that these legume lectins have probably evolved in a divergent manner from a common ancestor. A comparison of the predicted potential β-turn sites also supports the hypothesis of divergent evolution in this class of lectins.     

Identification of a new quaternary association for legume lectins

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.     

Hyphal polysaccharides as potential phylogenetic markers forEupenicillium species

The hyphal wall polysaccharide fractions of certain species ofEupenicillium have been utilized to study their phylogenetic relationships.E. abidjanum andE. shearii (group 1) cell walls have an alkali-soluble polysaccharide fraction (20%) containing an α-glucan.E. alutaceum, E. baarnense, E. catenatum, andE. erubescens (group 2) contain β-linked polysaccharides in all their polysaccharide fractions. These results suggest that some of the previously assigned taxonomic groups ofEupenicillium may not be natural.     

Mode of molecular recognition of L-fucose by fucose-binding legume lectins

Recognition of cell surface carbohydrate moieties by lectins plays a vital role in many a biological process. Fucosyated residues are often implicated as key recognition markers in many cellular processes. In particular, the aspects of molecular recognition of fucose by fucose-bindinglectins UEA 1 and LTA pose a special case because no crystal structure of these lectins is available. The study was conducted to elucidate the process of recognition of l-fucose by UEA1 and LTA by correlating structure-based sequence alignment and other available biochemical/biophysical data. The study points out that the mode of recognition of l-fucose is coordinated by the invariant triad of residues the asparagine 137, glycine 105, and aspartate 87. The major hydrophobic stacking residue in this case is the tyrosine 220. The study also reiterates the key role of the conserved triad of residues in the combining site which is a common feature for all legume lectins whose crystal structures are known.     

Patterns of speciation and limits to phylogenetic resolution

The practice of phylogenetic systematics frequently Includes the assumption that cladogenesis occurs by a series of bifurcations. Consequently, a phylogenetic tree that includes one or more polytomous nodes is generally viewed as unresolved. However, while some polytomles surely represent a failure of resolution, others may be real or the best resolution that can be achieved. Therefore, polytomies should be considered as phylogenetic hypotheses in the same way as bifurcating topologies.     

Role of lectins (and rhizobial exopolysaccharides) in legume nodulation.

The lectin recognition hypothesis proposes that plant lectins mediate specificity in the Rhizobium-legume symbiosis. Although the hypothesis was developed eight years before nod genes were identified in rhizobia and sixteen years before Nod factor was shown to be a major determinant of host specificity, experiments performed recently using transgenic lectin plants support its main tenets.     

Signature of quaternary structure in the sequences of legume lectins.

Legume lectins exhibit a wide variety of oligomerization and sugar specificity while retaining the characteristic jelly-roll tertiary fold. An attempt has been made here to find whether this diversity is reflected in their primary structures by constructing phylogenetic trees. Dendrograms based on sequence alignment showed clustering related to the oligomeric nature of legume lectins. Though the clustering primarily follows the oligomeric states, it also appears to correlate with different sugar specificities indicating an interdependence of these two properties. Analysis of the structure-based alignment and the alignment of the sequences of the carbohydrate-binding loops alone also revealed the same features. By a close examination of the interfaces of the various oligomers it was also possible, in some cases, to pinpoint a few key residues responsible for the stabilization of the interfaces.     

Nested gene fusions as markers of phylogenetic branchpoints in prokaryotes

Phylogenetic trees for prokaryotic microorganisms are being assembled at a rapid pace, primarily through sequence comparisons of ribosomal RNA genes. For lineages that diverged from the ancestral stem at nearly the same time, the order of branching may be uncertain. The problem applies both to minor branches that separated very recently and to major branches that diverged long ago. Bifunctional proteins produced by gene fusion provide the clarity of a plus-or-minus character state, and analysis of the distribution of genefusion patterns can reveal the order of phylogenetic branching.     

New partial sequences of phosphoenolpyruvate carboxylase as molecular phylogenetic markers

To better understand the evolution of the enzyme phosphoenolpyruvate carboxylase (PEPC) and to test its versatility as a molecular character in phylogenetic and taxonomic studies, we have characterized and compared 70 new partial PEPC nucleotide and amino acid sequences (about 1100 bp of the 3' side of the gene) from 50 plant species (24 species of Bryophyta, 1 of Pteridophyta, and 25 of Spermatophyta). Together with previously published data, the new set of sequences allowed us to construct the up to now most complete phylogenetic tree of PEPC, where the PEPC sequences cluster according to both the taxonomic positions of the donor plants and the assumed specific function of the PEPC isoforms. Altogether, the study further strengthens the view that PEPC sequences can provide interesting information for the reconstruction of phylogenetic relations between organisms and metabolic pathways. To avoid confusion in future discussion, we propose a new nomenclature for the denotation of PEPC isoforms.     

Mitochondrial genomes in the Hymenoptera and their utility as phylogenetic markers

Abstract.  In this study, we assessed the ability of mitochondrial genome sequences to recover a test phylogeny of five hymenopteran taxa from which phylogenetic relationships are well accepted. Our analyses indicated that the test phylogeny was well recovered in all nucleotide Bayesian analyses when all the available holometabolan (i.e. outgroup) taxa were included, but only in Bayesian analyses excluding third codon positions when only the hymenopteran representatives and a single outgroup were included. This result suggests that taxon sampling of the outgroup might be as important as taxon sampling of the ingroup when recovering hymenopteran phylogenetic relationships using whole mitochondrial genomes. Parsimony analyses were more sensitive to both taxon sampling and the analytical model than Bayesian analyses, and analyses using the protein dataset did not recover the test phylogeny. In general, mitochondrial genomes did not resolve the position of the Hymenoptera within the Holometabola with confidence, suggesting that an increased taxon sampling, both within the Holometabola and among outgroups, is necessary.     

Highly repeated DNA sequences as phylogenetic markers among the galaginae

Highly repeated DNA sequences were investigated as potential phylogenetic indicators among five species of galagines. One lorisine, one cheirogaleid, and two lemurid species were also investigated as progressively more distant outgroups. The lorisids displayed strong conservatism with regard to these sequences, to the point where the galegine species proved difficult to differentiate. When restriction fragment differences were observed, the galagine species fell into two groups: one containing the greater galagos and G. alleni and the other comprising the lesser galagos. The sequences of the cheirogaleid Microcebus murinus were found to be highly species-specific, bearing little resemblance to those of the galagines or the lemurids. Common sequences detected between M. murinus and G. senegalensis may be ancient sequences shared by all strepsirhines. © 1994 Wiley-Liss, Inc.     

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants). However, the best performance was delivered by adsorbents featuring coupled tentacular dextran chains displaying a maximum binding capacity of 238 mg g(-1) and a dissociation constant of 0.13 microM. Adsorbents derivatized with mixed mode or hydrophobic charge induction ligands likewise demonstrated very high capacities for both concanavalin A and Lens culinaris agglutinin (> or = 250 mg g(-1)) with dissociation constants in the micromolar range, though neither of these systems showed any selectivity for lectins in leguminous extracts. When the affinity supports were applied to carbohydrate containing legume extracts only the dextran-linked adsorbents supplied sufficient competition to dissolved sugars to selectively bind concanavalin A in an extract of jack beans. The dextran-linked supports were employed in a high-gradient magnetic fishing experiment, in which concanavalin A was purified to near homogeneity from a crude, unclarified extract of jack beans.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

Comparing the rates of speciation and extinction between phylogenetic trees

Over the past decade or so it has become increasingly popular to use reconstructed evolutionary trees to investigate questions about the rates of speciation and extinction. Although the methodology of this field has grown substantially in its sophistication in recent years, here I will take a step back to present a very simple model that is designed to investigate the relatively straightforward question of whether the tempo of diversification (speciation and extinction) differs between two or more phylogenetic trees, without attempting to attribute a causal basis to this difference. It is a likelihood method, and I demonstrate that it generally shows type I error that is close to the nominal level. I also demonstrate that parameter estimates obtained with this approach are largely unbiased. As this method can be used to compare trees of unknown relationship, it will be particularly well‐suited to problems in which a difference in diversification rate between clades is suspected, but in which these clades are not particularly closely related. As diversification methods can easily take into account an incomplete sampling fraction, but missing lineages are assumed to be missing at random, this method is also appropriate for cases in which we have hypothesized a difference in the process of diversification between two or more focal clades, but in which many unsampled groups separate the few of interest. The method of this study is by no means an attempt to replace more sophisticated models in which, for instance, diversification depends on the state of an observed or unobserved discrete or continuous trait. Rather, my intention is to provide a complementary approach for circumstances in which a simpler hypothesis is warranted and of biological interest.     

Properties of phylogenetic trees generated by Yule-type speciation models

We investigate some discrete structural properties of evolutionary trees generated under simple null models of speciation, such as the Yule model. These models have been used as priors in Bayesian approaches to phylogenetic analysis, and also to test hypotheses concerning the speciation process. In this paper we describe new results for three properties of trees generated under such models. Firstly, for a rooted tree generated by the Yule model we describe the probability distribution on the depth (number of edges from the root) of the most recent common ancestor of a random subset of k species. Next we show that, for trees generated under the Yule model, the approximate position of the root can be estimated from the associated unrooted tree, even for trees with a large number of leaves. Finally, we analyse a biologically motivated extension of the Yule model and describe its distribution on tree shapes when speciation occurs in rapid bursts.     

Weighting indels as phylogenetic markers of 18S rDNA sequences in Diptera and Strepsiptera

Opinions split when it comes to the significance and thus the weighting of indel characters as phylogenetic markers. This paper attempts to test the phylogenetic information content of indels and nucleotide substitutions by proposing an a priori weighting system of non-protein-coding genes. Theoretically, the system rests on a weighting scheme which is based on a falsificationist approach to cladistic inference. It provides insertions, deletions and nucleotide substitutions weights according to their specific number of identical classes of potential falsifiers, resulting in the following system: nucleotide substitutions weight = 3, deletions of n nucleotides weight = (2ⁿ–1), and insertions of n nucleotides weight = (5ⁿ–1). This weighting system and the utility of indels as phylogenetic markers are tested against a suitable data set of 18S rDNA sequences of Diptera and Strepsiptera taxa together with other Metazoa species. The indels support the same clades as the nucleotide substitution data, and the application of the weighting system increases the corresponding consistency indices of the differentially weighted character types. As a consequence, applying the weighting system seems to be reasonable, and indels appear to be good phylogenetic markers.     

Inverse PCR-based site-directed mutagenesis of nucleotide sequences coding for carbohydrate-binding fragments of legume lectins

A new method of site-directed mutagenesis was developed to allow manipulation with extended plasmid-cloned gene fragments irrespective of their position and the presence of restriction sites. The method was used to obtain chimeric constructs encoding a Pisum sativum lectin with the native carbohydrate-binding region replaced by its counterpart from other legumes. The method can be used in plasmid construction to clone a coding gene fragment under the control of a promoter in a certain reading frame.