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Choosing the right molecular genetic markers for studying biodiversity: from molecular evolution to practical aspects.

The use of molecular genetic markers (MGMs) has become widespread among evolutionary biologists, and the methods of analysis of genetic data improve rapidly, yet an organized framework in which scientists can work is lacking. Elements of molecular evolution are summarized to explain the origin of variation at the DNA level, its measures, and the relationships linking genetic variability to the biological parameters of the studied organisms. MGM are defined by two components: the DNA region(s) screened, and the technique used to reveal its variation. Criteria of choice belong to three categories: (1) the level of variability, (2) the nature of the information (e.g. dominance vs. codominance, ploidy, ... ) which must be determined according to the biological question and (3) some practical criteria which mainly depend on the equipment of the laboratory and experience of the scientist. A three-step procedure is proposed for drawing up MGMs suitable to answer given biological questions, and compiled data are organized to guide the choice at each step: (1) choice, determined by the biological question, of the level of variability and of the criteria of the nature of information, (2) choice of the DNA region and (3) choice of the technique.

Animals↗

Cloning and sequencing of the chaperonin-encoding Cctd gene from Fugu rubripes rubripes.

CCT, a chaperonin containing t-complex polypeptide 1 (TCP-1), is a cytosolic molecular chaperone involved in the folding of proteins. We have isolated the Cctd gene from a Fugu rubripes rubripes (Frr) genomic library using a rat Ccta cDNA as a probe, and cloned its cDNA by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of oligodeoxyribonucleotides corresponding to the 5' and 3' non-coding regions of Frr Cctd. Cctd spans a region of 4.7 kb and consists of at least 13 exons with small introns of about 144 bp on average. The Cctd cDNA sequence revealed a deduced polypeptide of 536 amino acids sharing a high degree of homology with that of the mouse Cctd cDNA (88%). Cctd is present as a single-copy gene, as shown by genomic Southern blot analysis, and can be used for evolutionary and classification analyses of Fugu species.

Amino Acid Sequence↗

The complete sequence of a Singapore isolate of odontoglossum ringspot virus and comparison with other tobamoviruses.

The complete sequence of a Singapore isolate of odontoglossum ringspot virus (ORSV-S1) comprises 6609 nucleotides (nt) and four open reading frames (ORFs 1 to 4). The 126/183-kDa RNA-dependent RNA polymerase (RdRp), 33-kDa movement protein (MP) and 18-kDa coat protein (CP) cistrons are located at nt 63-3401/4901, 4807-5718, and 5721-6197 on the genome, respectively. The 5' UTR contains three copies of an 8-base direct repeat and (CAA)n motifs. Characteristic tRNA-like structure and three consecutive homologous regions were present in the 3' UTR. The genomic RNA and MP of ORSV-S1 are one of the longest among all members of the TOV group. Phylogenetic analysis of all four genes indicates evolutionary divergence, but within each gene there are some degrees of evolutionary convergence. The conserved amino acid sequences in the MP can be used for the classification of tobamoviruses.

Amino Acid Sequence↗

Evolution: bacterial mutation in stationary phase.

A recent study indicates that the genomic mutation rate of the gut bacterium Escherichia coli is substantially higher in nongrowing than growing cultures. These findings are important in the light of the ongoing controversy over the generality and robustness of stationary phase mutagenesis and its evolutionary implications.

Analysis of Variance↗

Adaptive evolution and functional divergence of pepsin gene family.

In vertebrates, a large proportion of genes is organized in gene families. Paralogous gene groups generated by gene duplication are related by homology, high degree of sequence identity and similar structural architecture of their products. Aspartic proteinases form a widely distributed protein superfamily including cathepsins, pepsins, renin and napsin. In the present study, the nucleotide sequences coding for various pepsins in 30 vertebrate species have been used to derive a gene phylogeny. Gene duplication and losses have been inferred from a reconciled tree, reconstructed by combining information from gene tree and species tree. Our findings based on the results of the relative rate ratio test and maximum likelihood analysis suggest that each round of gene duplication is characterized by adaptive evolution, although instances of evolution under positive selection have been found also long after divergence of gene families. The results of functional divergence analysis provided statistical evidence for shifted evolutionary rate after gene duplication.

Adaptation, Physiological↗

Stochastic modeling of T cell receptor gamma gene rearrangement.

The mechanisms controlling the recombination process of the gamma genes that encode the gamma chain of the antigen receptor of the gammadelta T lymphocytes are unclear. Based on experimental data on the recombination status of the two major TCR gamma genes expressed in V(gamma)4+ and V(gamma)1+ thymocytes, we tested the plausibility of three possible rearrangement mechanisms: (1) a time window mechanism according to which the two chromosomes are accessible to the recombination machinery during a defined period of time; (2) a feedback mechanism in which recombination stops shortly after the first in-frame rearrangement event anywhere in both chromosomes; and (3) a feedback mechanism with asynchronous chromosome accessibility, in which there is a first period when only one chromosome is accessible for recombination, followed by a second period when both chromosomes are accessible; shortly after the first in-frame rearrangement event, during any of these two periods, recombination will definitely stop. We model the time window mechanism using a pure probabilistic approach and the two feedback mechanisms using a continuous-time Markov chain formalism. We used maximum likelihood methodology to infer the goodness-of-fit of the models showing evidence for the last model, which best fits the data. Further analysis of this model suggests an evolutionary tradeoff between allelic and isotypic exclusion and the probability that a precursor differentiates into a mature gammadelta T lymphocyte.

Alleles↗

Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens.

Since the early evolution of land plants from primitive green algae, flavonoids have played an important role as UV protective pigments in plants. Flavonoids occur in liverworts and mosses, and the first committed step in the flavonoid biosynthesis is catalyzed by chalcone synthase (CHS). Although higher plant CHSs have been extensively studied, little information is available on the enzymes from bryophytes. Here we report the cloning and characterization of CHS from the moss, Physcomitrella patens. Taking advantage of the available P. patens EST sequences, a CHS (PpCHS) was cloned from the gametophores of P. patens, and heterologously expressed in Escherichia coli. PpCHS exhibited similar kinetic properties and substrate preference profile to those of higher plant CHS. p-Coumaroyl-CoA was the most preferred substrate, suggesting that PpCHS is a naringenin chalcone producing CHS. Consistent with the evolutionary position of the moss, phylogenetic analysis placed PpCHS at the base of the plant CHS clade, next to the microorganism CHS-like gene products. Therefore, PpCHS likely represents a modern day version of one of the oldest CHSs that appeared on earth. Further, sequence analysis of the P. patens EST and genome databases revealed the presence of a CHS multigene family in the moss as well as the 3'-end heterogeneity of a CHS gene. Of the 19 putative CHS genes, 10 genes are expressed and have corresponding ESTs in the databases. A possibility of the functional divergence of the multiple CHS genes in the moss is discussed.

Acyltransferases↗

Evo-devo and constraints on selection.

Developmental bias, or genetic channeling, can influence the tempo and direction of evolution and, thus, become reflected in patterns of biodiversity. Twenty years ago, this notion rested on armchair descriptions of potential constraints on evolution. Now, a broad evo-devo approach involving both evolutionary and developmental genetics provides experimental analysis of such bias, revealing how shared genetic or developmental pathways among morphological traits contribute to the evolution of complexity and diversity, and that developmental bias itself evolves, generating variation in evolvability. As I discuss here, it is now possible to determine the relative importance of natural selection and of genetical and developmental architecture as determinants of organic diversity, particularly for examples of adaptive radiations and parallel evolution.

Animals↗

The future of human spaceflight.

After the Apollo Moon program, the international space station represents a further milestone of humankind in space, International follow-on programs like a manned return to the Moon and a first manned Mars Mission can be considered as the next logical step. More and more attention is also paid to the topic of future space tourism in Earth orbit, which is currently under investigation in the USA, Japan and Europe due to its multibillion dollar market potential and high acceptance in society. The wide variety of experience, gained within the space station program, should be used in order to achieve time and cost savings for future manned programs. Different strategies and roadmaps are investigated for space tourism and human missions to the Moon and Mars, based on a comprehensive systems analysis approach. By using DLR's software tool FAST (Fast Assessment of Space Technologies), different scenarios will be defined, optimised and finally evaluated with respect to mission architecture, required technologies, total costs and program duration. This includes trajectory analysis, spacecraft design on subsystem level, operations and life cycle cost analysis. For space tourism, an expected evolutionary roadmap will be described which is initiated by short suborbital tourism and ends with visionary designs like the Space Hotel Berlin and the Space Hotel Europe concept. Furthermore the potential space tourism market, its economic meaning as well as the expected range of the costs of a space ticket (e.g. $50,000 for a suborbital flight) will be analysed and quantified. For human missions to the Moon and Mars, an international 20 year program for the first decades of the next millennium is proposed, which requires about $2.5 Billion per year for a manned return to the Moon program and about $2.6 Billion per year for the first 3 manned Mars missions. This is about the annual budget, which is currently spend by the USA only for the operations of its Space Shuttle fleet which generally proofs the affordability of such ambitious programs after the build-up of the International Space Station, when corresponding budget might become again available.

Equipment Design↗

Complement systems in invertebrates. The ancient alternative and lectin pathways.

The complement system in higher vertebrates is composed of about thirty proteins that function in three activation cascades and converge in a single terminal pathway. It is believed that these cascades, as they function in the higher vertebrates, evolved from a few ancestral genes through a combination of gene duplications and divergences plus pathway duplication (perhaps as a result of genome duplication). Evidence of this evolutionary history is based on sequence analysis of complement components from animals in the vertebrate lineage. There are fewer components and reduced or absent pathways in lower vertebrates compared to mammals. Modern examples of the putatively ancestral complement system have been identified in sea urchins and tunicates, members of the echinoderm phylum and the protochordate subphylum, which are sister groups to the vertebrates. Thus far, this simpler system is composed of homologues of C3, factor B, and mannose binding protein associated serine protease suggesting the presence of simpler alternative and lectin pathways. Additional components are predicted to be present. A complete analysis of this invertebrate defense system, which evolved before the invention of rearranging genes, will provide keys to the primitive beginnings of innate immunity in the deuterostome lineage of animals.

Amino Acid Sequence↗

The cloning and sequencing of a ribosomal L18 protein from an evolutionary divergent eukaryote, Trypanosoma brucei.

Eukaryotic ribosomal proteins are highly conserved across widely divergent species, suggesting that strong functional constraints prevent divergence of important amino acid motifs. Using this as a basis, an evolutionary approach could be used to identify putative functional motifs. We obtained the DNA sequence of the ribosomal protein L18 from the evolutionary divergent protozoan parasite, Trypanosoma brucei. Analysis of this sequence showed that it had 46% and 43% identity with the human and yeast sequences, respectively, and 30% of amino acid residues were identical across all the species analysed. Using these data, amino acids essential to the structure and function of ribosomal protein L18 can be inferred and could provide valuable information for molecular modelling and mutational studies.

Amino Acid Sequence↗

Sequence heterogeneity within the 5'-terminal region of the hepatitis GB virus C genome and evidence for genotypes.

BACKGROUND: GB virus C is a positive-strand RNA virus that is associated with hepatitis in humans. GB virus C bears some resemblance to hepatitis C virus in its genomic sequence and organization. However, unlike hepatitis C virus, an open reading frame possessing a complete core protein was not identified in the original isolate. METHODS: To verify the sequence at the 5'-end of the GB virus C genome, we amplified approximately 600 nucleotides from this region from 35 globally distributed individuals. The nucleotide sequences were translated in all possible reading frames and then examined for conserved motifs indicative of nucleocapsid or core-like peptides. RESULTS: Forty-two unique GB virus C sequences were obtained from the 35 individuals. The deduced amino acid sequences upstream of the putative E1 gene from each isolate varied in length and composition, such that a conserved core-like sequence was not apparent. No core-like sequences were evident in the other reading frames. There was, however, a single methionine codon held in common among all isolates, although it was located very near the presumed amino-terminus of the putative E1 protein. Further analysis of the sequences for their evolutionary relatedness demonstrated the existence of five GB virus C subtypes that demonstrated a significant correlation with geographic distribution. CONCLUSIONS: GB virus C differs from hepatitis C virus and GB virus B in that it does not encode a nucleocapsid or core protein. The existence of GB virus C subtypes emphasizes the importance of investigating the correlation between infecting subtype and the severity of liver disease and/or responsiveness to treatment of GB virus C-associated hepatitis.

Base Sequence↗

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Devescovinidae and Calonymphidae (Trichomonadea).

Small subunit rRNA sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the polymastigont Calonymphidae, including Snyderella tabogae, Calonympha grassii, and Metacoronympha senta. The yet-unidentified sequence Nk9 previously obtained from the termite Neotermes koshunensis, has also been shown to derive from the Devescovinidae Devescovina sp. by in situ hybridization. These new sequences were analyzed by distance, parsimony, and likelihood methods in a broad phylogeny including all identified parabasalid sequences available in databases. All analyses revealed the emergence of a very well supported Devescovinidae/Calonymphidae group but showed an unexpected dichotomy of the Calonymphidae represented by the "Coronympha" and "Calonympha" groups. It strongly suggests that the polymastigont state observed in the Calonymphidae might be explained by at least two independent evolutionary events. In a second phylogenetic analysis, some yet-unidentified parabasalid sequences likely deriving from the Devescovinidae/Calonymphidae taxa, were added to our data set. This analysis confirmed the polyphyly of the Calonymphidae. A tentative identification is proposed for each of these sequences, and hypotheses on the origin of the Devescovinidae and Calonymphidae are discussed. Tritrichomonas foetus or a close relative might be the best candidate for the ancestor of the Devescovinidae, fairly consistent with morphology-based hypotheses. Regarding the Calonymphidae, the origin of the "Coronympha" group might be found within the Devescovinidae, related to Foaina, whereas the "Calonympha" group may directly descend from Tritrichomonas or related species.

Animals↗

Molecular evolution of CXC chemokines: extant CXC chemokines originate from the CNS.

The mammalian CXC chemokine system comprises 16 ligands and six receptors, and its actions stretch well beyond the immune system. Recent elucidation of the pufferfish genome, a representative of an evolutionary ancient vertebrate class, has enabled analysis of the mammalian CXC chemokine system in a phylogenetic context. Comparison of the phylogenies of vertebrate CXC chemokines revealed that fish and mammals have found different solutions to similar problems, grafted on the same basic structural motif. Phylogenetic analyses showed that the large, highly redundant CXC chemokine family is a very recent phenomenon that is exclusive to higher vertebrates. Moreover, its ancestral role is found within the central nervous system and not within the immune system.

Algorithms↗

Transition metal-catalyzed nonoxidative decarboxylation reactions.

Decarboxylases typically utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates. The recent characterization of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) has revealed that this enzyme adopts a TIM-barrel (beta/alpha)(8) fold and employs a mononuclear transition metal center to decarboxylate the substrate in an oxidant-independent fashion. Thus, ACMSD represents a type of decarboxylation reaction that has been so far uncharacterized in biological systems. Several close homologues of ACMSD were analyzed, including isoorotate decarboxylase (IDCase), 5-carboxyvanillic acid decarboxylase (5-CVD), gamma-resorcylate decarboxylase (gamma-RSD), and 4-oxalomesaconate hydratase (OMAH). These enzymes are involved in the catabolism of tryptophan and vanillate, the biodegradation of hydroxylbenzoates, and the thymidine salvage pathways in certain organisms. They possess the signature sequence motifs of the amidohydrolase superfamily and likely share the same structural and mechanistic characteristics as that of ACMSD. Analysis of the sequence conservation and evolutionary relationship of ACMSD-related proteins suggests an emerging ACMSD protein family that includes ACMSD and ACMSD-like decarboxylases and hydratases with diverse substrate specificities, many of which are poorly understood in regard to their functions and mechanisms. Progress in the biochemical and structural characterization of ACMSD not only sheds light on the active site of this protein family but also promises the elucidation of the detailed catalytic mechanism of these novel transition metal-dependent nonoxidative decarboxylation reactions.

Binding Sites↗

Protein structures of common bean (Phaseolus vulgaris) alpha-amylase inhibitors.

Two nucleotide sequences for genes that encode alpha-amylase inhibitor 4 (alphaAI-4) from white kidney bean (WKB) cv. 858, designated gene alphaAI-4 (Accession No. ), and alpha-amylase inhibitor 5 (alphaAI-5) from black bean (BB), designated gene alphaAI-5 (Accession No. ), were determined. Genes alphaAI-4 and alphaAI-5 encode 244 amino acid prepro-alphaAI-4 and prepro-alphaAI-5 polypeptides that are 93 and 95% identical with alpha-amylase inhibitor l (alphaAI-l; Hoffman, L. M.; Ma, Y.; Barker, R. F. Nucleic Acids Res. 1982, 10, 7819-7828), 40 and 43% identical with red kidney bean lectin, and 52 and 55% identical with arcelin l of wild-type bean. The high degree of sequence similarity indicates the evolutionary relationship among these genes. PCR analysis of genomic DNA purified from six genotypes of Phaseolus vulgaris showed very similar band patterns in 2% agarose gel, another indication of the conserved size homology among these genes. Proteolytic processing sites were located between Asn77 and Ser78 for pro-alphaAI-4 and pro-alphaAI-5. A bend next to Asn77 in three-dimensional model structures of alphaAI-4 and alphaAI-5 proinhibitors indicates that the proteolytic cleavage is necessary to remove the conformational constraint for activation to the mature protein. Mature WKB alphaAI-4 was composed of four subunits (2alpha2beta) and had a molecular weight of 50000 determined by multiangle laser light scattering and 56714 determined by laser-assisted time-of-flight mass spectrometry.

Amino Acid Sequence↗

The identification of inducible cytoplasmic/nuclear carbohydrate-binding proteins urges to develop novel concepts about the role of plant lectins.

During the last few years compelling evidence has been presented for the occurrence of cytoplasmic/nuclear plant lectins that are not detectable in normal plants but are only induced upon application of well-defined stress conditions. Since both the regulation of the expression and the subcellular location indicate that these 'non-classical lectins' are good candidates to play a physiologically important role as mediators of specific protein-carbohydrate-interactions within the plant cell, a critical assessment is made of the impact of these findings on the development of novel concepts about the role of plant lectins. Based on an analysis of the biochemical, molecular and evolutionary data of a jasmonate-induced chitin-binding lectin from tobacco leaves and a salt/jasmonate-induced leaf lectin from rice it is concluded that these lectins most probably interact with endogenous glycans located within the cytoplasmic/nuclear compartment of the plant cell. Several working mechanisms are proposed to explain how these inducible lectins may fulfill an important regulatory or structural role in stressed cells. In addition, the question of the evolutionary relationship(s) between the newly discovered inducible lectins and their 'classical' constitutively expressed homologs is addressed. Evidence is presented that the 'non-classical lectins' represent the main evolutionary line and that some of their corresponding genes were used as templates for genes encoding storage protein-like 'classical' homologs.

Cell Nucleus↗

Substitution of murine for human CD4 residues identifies amino acids critical for HIV-gp120 binding.

Human CD4 is the receptor for the gp120 envelope glycoprotein of human immunodeficiency virus and is essential for virus entry into the host cell. Sequence analysis of CD4 has suggested an evolutionary origin from a structure with four immunoglobulin-related domains. Only the two NH2-terminal domains are required to mediate gp120 binding. The extracellular segment of murine CD4 has an overall 50% identity with its human counterpart at the amino-acid level, but fails to bind gp120. To define those residues of human CD4 critical for gp120 binding, we have taken advantage of this species difference and substituted all non-conserved murine for human CD4 residues between amino-acid positions 27-167. We used oligonucleotide-directed mutagenesis to create each of 16 individual mutant human CD4 molecules containing from 1-4 amino-acid substitutions. Introduction of as few as three amino acids into corresponding positions of human CD4 abrogates gp120 binding. Furthermore, these critical residues are located in domain I with a contribution from domain II. Modelling studies using the three-dimensional coordinates of the V kappa Bence-Jones REI homodimer localize the site in domain I to the C" beta strand within CDR2 but projecting away from the homologues of principle antigen-binding regions CDR 1 and 3.

Amino Acid Sequence↗