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PubMed2001

Structure, sequence and function of a marsupial LIF gene: conservation of IL-6 family cytokines.

Leukaemia Inhibitory Factor (LIF) is a multifunctional cytokine with an obligate role in the mouse in embryonic implantation. In this paper we demonstrate the existence of a functional LIF gene in the marsupial Sminthopsis crassicaudata, and the presence of LIF-related sequences in the monotreme Tachyglossus aculeatus (Australian echidna). Isolation of genomic and cDNA clones from S. crassicaudata, indicated that the LIF gene is highly conserved between marsupials and monotremes in terms of sequence and genomic organisation. Critical functional residues within the LIF sequence were also conserved including residues implicated in intracellular LIF activity, and in interaction with the receptor subunits LIFR and gp130. These findings suggest that the structure and biochemical function of the protein is likely to be conserved. Consistent with this, purified recombinant S. crassicaudata LIF interacted functionally with mouse receptor components and was sufficient for maintenance of mouse embryonic stem (ES) cells in the undifferentiated state. Conservation of LIF outside eutherians is intriguing given the markedly divergent reproductive strategies which include, for some marsupial species, embryonic diapause, and in monotremes, the absence of implantation. The availability of marsupial LIF probes provides an opportunity to investigate conservation of expression and function in these mammals.

Amino Acid Sequence↗

PubMed2003

Conservation of regulatory elements between two species of Drosophila.

BACKGROUND: One of the important goals in the post-genomic era is to determine the regulatory elements within the non-coding DNA of a given organism's genome. The identification of functional cis-regulatory modules has proven difficult since the component factor binding sites are small and the rules governing their arrangement are poorly understood. However, the genomes of suitably diverged species help to predict regulatory elements based on the generally accepted assumption that conserved blocks of genomic sequence are likely to be functional. To judge the efficacy of strategies that prefilter by sequence conservation it is important to know to what extent the converse assumption holds, namely that functional elements common to both species will fall within these conserved blocks. The recently completed sequence of a second Drosophila species provides an opportunity to test this assumption for one of the experimentally best studied regulatory networks in multicellular organisms, the body patterning of the fly embryo. RESULTS: We find that 50%-70% of known binding sites reside in conserved sequence blocks, but these percentages are not greatly enriched over what is expected by chance. Finally, a computational genome-wide search in both species for regulatory modules based on clusters of binding sites suggests that genes central to the regulatory network are consistently recovered. CONCLUSIONS: Our results indicate that binding sites remain clustered for these "core modules" while not necessarily residing in conserved blocks. This is an important clue as to how regulatory information is encoded in the genome and how modules evolve.

Algorithms↗

PubMed2005

NemaFootPrinter: a web based software for the identification of conserved non-coding genome sequence regions between C. elegans and C. briggsae.

BACKGROUND: NemaFootPrinter (Nematode Transcription Factor Scan Through Philogenetic Footprinting) is a web-based software for interactive identification of conserved, non-exonic DNA segments in the genomes of C. elegans and C. briggsae. It has been implemented according to the following project specifications:a) Automated identification of orthologous gene pairs. b) Interactive selection of the boundaries of the genes to be compared. c) Pairwise sequence comparison with a range of different methods. d) Identification of putative transcription factor binding sites on conserved, non-exonic DNA segments. RESULTS: Starting from a C. elegans or C. briggsae gene name or identifier, the software identifies the putative ortholog (if any), based on information derived from public nematode genome annotation databases. The investigator can then retrieve the genome DNA sequences of the two orthologous genes; visualize graphically the genes' intron/exon structure and the surrounding DNA regions; select, through an interactive graphical user interface, subsequences of the two gene regions. Using a bioinformatics toolbox (Blast2seq, Dotmatcher, Ssearch and connection to the rVista database) the investigator is able at the end of the procedure to identify and analyze significant sequences similarities, detecting the presence of transcription factor binding sites corresponding to the conserved segments. The software automatically masks exons. DISCUSSION: This software is intended as a practical and intuitive tool for the researchers interested in the identification of non-exonic conserved sequence segments between C. elegans and C. briggsae. These sequences may contain regulatory transcriptional elements since they are conserved between two related, but rapidly evolving genomes. This software also highlights the power of genome annotation databases when they are conceived as an open resource and the possibilities offered by seamless integration of different web services via the http protocol. AVAILABILITY: The program is freely available at http://bio.ifom-firc.it/NTFootPrinter.

Animals↗

PubMed2001

Conservation of the binding site for the arginine repressor in all bacterial lineages.

BACKGROUND: The arginine repressor ArgR/AhrC is a transcription factor universally conserved in bacterial genomes. Its recognition signal (the ARG box), a weak palindrome, is also conserved between genomes, despite a very low degree of similarity between individual sites within a genome. Thus, the arginine repressor is different from two other universal transcription factors - HrcA, whose recognition signal is very strongly conserved both within and between genomes, and LexA/DinR, whose signal is strongly conserved within, but not between, genomes. The arginine regulon is well studied in Escherichia coli and to some extent in Bacillus subtilis and some other genomes. Here, we apply the comparative genomic approach to the prediction of the ArgR-binding sites in all completely sequenced bacterial genomes. RESULTS: Orthologs of ArgR/AhrC were identified in the complete genomes of E. coli, Haemophilus influenzae, Vibrio choleras, B. subtilis, Mycobacterium tuberculosis, Thermotoga maritima, Chlamydia pneumoniae and Deinococcus radiodurans. Candidate arginine repressor binding sites were identified upstream of arginine transport and metabolism genes. CONCLUSIONS: We found that the ArgR/AhrC recognition signal is conserved in all genomes that contain genes encoding orthologous transcription factors of this family. All genomes studied except M. tuberculosis contain ABC transport cassettes (related to the Art system of E. coli) belonging to the candidate arginine regulons.

Bacteria↗

PubMed2003

Phylogenetically and spatially conserved word pairs associated with gene-expression changes in yeasts.

BACKGROUND: Transcriptional regulation in eukaryotes often involves multiple transcription factors binding to the same transcription control region, and to understand the regulatory content of eukaryotic genomes it is necessary to consider the co-occurrence and spatial relationships of individual binding sites. The determination of conserved sequences (often known as phylogenetic footprinting) has identified individual transcription factor binding sites. We extend this concept of functional conservation to higher-order features of transcription control regions. RESULTS: We used the genome sequences of four yeast species of the genus Saccharomyces to identify sequences potentially involved in multifactorial control of gene expression. We found 989 potential regulatory 'templates': pairs of hexameric sequences that are jointly conserved in transcription regulatory regions and also exhibit non-random relative spacing. Many of the individual sequences in these templates correspond to known transcription factor binding sites, and the sets of genes containing a particular template in their transcription control regions tend to be differentially expressed in conditions where the corresponding transcription factors are known to be active. The incorporation of word pairs to define sequence features yields more specific predictions of average expression profiles and more informative regression models for genome-wide expression data than considering sequence conservation alone. CONCLUSIONS: The incorporation of both joint conservation and spacing constraints of sequence pairs predicts groups of target genes that are specific for common patterns of gene expression. Our work suggests that positional information, especially the relative spacing between transcription factor binding sites, may represent a common organizing principle of transcription control regions.

Base Sequence↗

PubMed2004

The structure of a rigorously conserved RNA element within the SARS virus genome.

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-A resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3' end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90 degrees kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.

Binding Sites↗

PubMed2004

Highly conserved non-coding sequences are associated with vertebrate development.

In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH), in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.

Animals↗

PubMed2005

Genome trees from conservation profiles.

The concept of the genome tree depends on the potential evolutionary significance in the clustering of species according to similarities in the gene content of their genomes. In this respect, genome trees have often been identified with species trees. With the rapid expansion of genome sequence data it becomes of increasing importance to develop accurate methods for grasping global trends for the phylogenetic signals that mutually link the various genomes. We therefore derive here the methodological concept of genome trees based on protein conservation profiles in multiple species. The basic idea in this derivation is that the multi-component "presence-absence" protein conservation profiles permit tracking of common evolutionary histories of genes across multiple genomes. We show that a significant reduction in informational redundancy is achieved by considering only the subset of distinct conservation profiles. Beyond these basic ideas, we point out various pitfalls and limitations associated with the data handling, paving the way for further improvements. As an illustration for the methods, we analyze a genome tree based on the above principles, along with a series of other trees derived from the same data and based on pair-wise comparisons (ancestral duplication-conservation and shared orthologs). In all trees we observe a sharp discrimination between the three primary domains of life: Bacteria, Archaea, and Eukarya. The new genome tree, based on conservation profiles, displays a significant correspondence with classically recognized taxonomical groupings, along with a series of departures from such conventional clusterings.

Animals↗

PubMed2002

Methodologies for conservation assessments of the genetic biodiversity of aquatic macro-organisms.

International organizations and biodiversity scientists recognize three levels of biodiversity: genetic, species, and ecosystem. However, most studies with the goal of assessing biodiversity collect data at only a single level--that of the species. Even when multiple levels of biodiversity are considered, usually only ecosystem diversity is also evaluated. Genetic diversity is virtually never considered. Yet, genetic diversity is essential for the maintenance of populations and species over ecological and evolutionary time periods. Moreover, because components of genetic diversity are independent of either species or ecosystem diversity, genetic diversity can provide a unique measure by which to assess the value of regions for conservation. Regions can be valuable for conservation of their genetic resources regardless of their levels of species or ecosystem uniqueness or diversity. In general, the same methods and statistical programs that are used to answer questions about population genetics and phylogenetics are applicable to conservation genetics. Thus, numerous genetic techniques, laboratory methods, and statistical programs are available for assessing regional levels of genetic diversity for conservation considerations. Here, we provide the rationale, techniques available, field and laboratory protocols, and statistical programs that can be used to estimate the magnitude and type of genetic diversity in regions. We also provide information on how to obtain commonly utilized statistical programs and the type of analyses that they include. The guide that we present here can be used to conduct investigations of the genetic diversity of regions under consideration for conservation of their natural resources.

Animals↗

PubMed2006

Floristic conservation value, nested understory floras, and the development of second-growth forest.

Nestedness analysis can reveal patterns of plant composition and diversity among forest patches. For nested floral assemblages, the plants occupying any one patch are a nested subset of the plants present in successively more speciose patches. Elimination of sensitive understory plants with human disturbance is one of several mechanisms hypothesized to generate nonrandom, nested floral distributions. Hypotheses explaining distributions of understory plants remain unsubstantiated across broad landscapes of varying forest types and disturbance histories. We sampled the vegetation of 51 floodplain and 55 upland forests across Illinois (USA) to examine how the diversity, composition, and nestedness of understory floras related to their overstory growth and structure (basal area), and their overall floristic conservation value (mean C). We found that plant assemblages were nested with respect to site species richness, such that rare plants indicated diverse forests. Floras were also nested with respect to site mean C and basal area (BA). However, in an opposite pattern from what we had expected, floras of high-BA stands were nested subsets of those of low-BA stands. A set of early-successional plants restricted to low-BA stands, and more importantly, the absence of a set of true forest plants in high-BA stands, accounted for this pattern. Additionally, we observed a decrease in species richness with increasing BA. These results are consistent with the hypothesis that recovery of true forest plants does not occur concurrently with overstory regeneration following massive anthropogenic disturbance. Nestedness by site mean C indicates that high conservation value (conservative) plants co-occur in highly diverse stands; these forests are assumed to be less disturbed historically. Because site mean C was uncorrelated with BA, BA-neutral disturbances such as livestock usage are suggested as accounting for between-site differences in mean C. When considered individually, conservative plants were actually more likely to be found in low-BA stands (uplands only). This suggests that floras of historically more open-canopied oak-hickory uplands are being degraded by canopy closure from increasing density of "mesophytic, nonpyrogenic" trees. It also indirectly suggests that recent moderate logging is uncorrelated with floristic conservation values.

Conservation of Natural Resources↗

PubMed1999

A conserved sequence block in the murine and human TCR J alpha region: assessment of regulatory function in vivo.

Temporal control of rearrangement at the TCR alpha/delta locus is crucial for development of the gamma delta and alpha beta T cell lineages. Because the TCR delta locus is embedded within the alpha locus, rearrangement of any V alpha-J alpha excises the delta locus, precluding expression of a functional gamma delta TCR. Approximately 100 kb spanning the C delta-C alpha region has been sequenced from both human and mouse, and comparison has revealed an unexpectedly high degree of conservation between the two. Of interest in terms of regulation, several highly conserved sequence blocks (> 90% over > 50 bp) were identified that did not correspond to known regulatory elements such as the TCR alpha and delta enhancers or to coding regions. One of these blocks lying between J alpha 4 and J alpha 3, which appears to be conserved in other vertebrates, has been shown to augment TCR alpha enhancer function in vitro and differentially bind factors from nuclear extracts. To further assess a plausible regulatory role for this element, we have created mice in which this conserved sequence block is either deleted or replaced with a neomycin resistance gene driven by the phosphoglycerate kinase promoter (pgk-neor). Deletion of this conserved sequence block in vivo did have a local effect on J alpha usage, echoing the in vitro data. However, its replacement with pgk-neor had a much more dramatic, long range effect, perhaps underscoring the importance of maintaining overall structure at this locus.

Animals↗

PubMed1999

Invariant or highly conserved TCR alpha are expressed on double-negative (CD3+CD4-CD8-) and CD8+ T cells.

TCR alpha (TCRA) expression was examined in RNA samples from PBMC and isolated populations of CD4+, CD8+, and DN T cells from 15 healthy individuals. The expressed TCR repertoire was surveyed using spectratype analysis, a technique that displays the distribution of complementarity determining region 3 (CDR3) lengths for each TCRAV gene family. The results revealed the presence of unusual populations of double-negative (DN; CD4-CD8-CD3+) T cells that express invariant or conserved TCRAV4A, AV7, AV19, and AV24 chains. Each of the conserved TCRA families was over-represented in >70% of the individuals studied, and all individuals expressed at least one of the over-represented TCRAV families. Over-represented conserved AV4A or AV7 sequences were also present in CD8+ T cells from most donors. The extent of TCRA sequence conservation is unparalleled. TCRAV4A, AV19, and AV24 sequences were invariant, although AV4A and AV19 transcripts contained N region additions. TCRAV24 transcripts derived from the direct juxtaposition of V and J gene segments. TCRAV7 sequences showed some diversity in two amino acids encoded at junctions of V and J gene segments. Although derivation of DN T cells with conserved TCRA chains is puzzling, the wide-spread expression of these unusual cells suggests an important function.

Adult↗

PubMed1999

Phylogenetic inference from conserved sites alignments.

Molecular sequences provide a rich source of data for inferring the phylogenetic relationships among species. However, recent work indicates that even an accurate multiple alignment of a large sequence set may yield an incorrect phylogeny and that the quality of the phylogenetic tree improves when the input consists only of the highly conserved, motif regions of the alignment. This work introduces two methods of producing multiple alignments that include only the conserved regions of the initial alignment. The first method retains conserved motifs, whereas the second retains individual conserved sites in the initial alignment. Using parsimony analysis on a mitochondrial data set containing 19 species among which the phylogenetic relationships are widely accepted, both conserved alignment methods produce better phylogenetic trees than the complete alignment. Unlike any of the 19 inference methods used before to analyze this data, both methods produce trees that are completely consistent with the known phylogeny. The motif-based method employs far fewer alignment sites for comparable error rates. For a larger data set containing mitochondrial sequences from 39 species, the site-based method produces a phylogenetic tree that is largely consistent with known phylogenetic relationships and suggests several novel placements. J. Exp. Zool. ( Mol. Dev. Evol.) 285:128-139, 1999.

Amino Acid Sequence↗

PubMedDate not supplied

Assessing conservation biology priorities through the development of biodiversity indicators.

Biodiversity erosion is now a recognized phenomenon within the scientific community, and it is understood that there is an urgent need for action to relieve this loss. However, no consensus exists on how to go about achieving this goal. This is due to two kinds of problems: namely, that there are many different reasons which can motivate conservation attempts and hence they can lead to different priorities, sometimes in direct contrast with one another; and that of the complexity of the natural world which we have to operate in. Regarding the latter, several authors have lamented a severe lack of theoretical support for many conservation decisions. We briefly review some of the criteria that are commonly adopted in conservation, and indicate some of their problems; we then introduce a new theoretical framework based on the use of a series of biological indicators, many of which have already been proposed in the past but never fully explained and/or adopted. In addition, we propose two indices for one of these potential indicators, i.e. evolutionary potential. We conclude with an appeal for a greater collaboration between conservation scientists and evolutionary biologists, in order to encompass evolutionary evidence in the decision-making process. In doing so, we hope to increase the chances of successful conservation efforts.

Animals↗

PubMed2002

Conservative region of the genes encoding four adhesins of Helicobacter pylori: cloning, sequence analysis and biological information analysis.

OBJECTIVE: To clone the conserved regions of the genes encoding the 4 adhesins (BabA, AlpA, AlpB and HopZ) of Helicobacter pylori (H. pylori) and analyze their sequences and biological information, thus facilitating further research in the molecular mechanism and immunogenicity of H. pylori adhesins. METHODS: Common conserved region (designated as CB) was identified from the confirmed sequences (by ANTHEPROT V4.3c software package) of the 4 adhesin proteins. Their DNA sequences were deduced, according to which primers specific to CB were designed for subsequent PCR, and the products were inserted directionally into pET-22b(+) vector to construct recombinant clones of the conserved region. The DNA sequences were determined with the basic local alignment sequence tool (BLAST) and the biological properties analyzed with ANTHEPROT V4.3c software package. RESULTS: The recombinant plasmid containing the CB sequence was constructed. DNA sequencing showed an open reading frame of 588 bp in length, encoding 195 amino acids. The homogencity of conservative region of the 4 adhesion genes was above 50%. The corresponding protein possessed a relative molecular mass (Mr) of 22 500 as predicted by ANTHEPROT V4.3c software prediction, with excellent antigenicity and hydrophobicity. There were 836 767 sequences analyzed with BLAST, in which those with homogencity of 40% with the identified CB sequence were categorized into H. pylori sequences. CONCLUSION: There are conservative regions in the 4 adhesin genes with similar homogencity, suggesting similar molecular basis for adhesion of the adhesins. Biological information analysis indicates that CB has excellent immunogenicity and strict species specificity.

Adhesins, Bacterial↗

PubMed1992

Conserved T and B cell epitopes on the M protein of group A streptococci. Induction of bactericidal antibodies.

To identify conserved T and B cell epitopes on the M protein of group A beta-hemolytic streptococci, overlapping synthetic peptides that span the conserved carboxyl-terminal segment of the M-5 protein were constructed and used to immunize a panel of H-2 congenic mice. Proliferative T cell epitopes were identified and, in many cases, mice immunized with these peptides produced high titer antibodies to the same peptides indicating that these proliferative epitopes could also stimulate Th cells. Peptide-specific T cells and antisera were tested for their reactivity with porcine myosin, tropomyosin, human heart myosin synthetic peptides, and extracts of human pericardial and atrial heart tissue. Although there was minimal response of M peptide-specific T cells to any of these Ag, certain M peptide-specific antisera reacted to immunoblotted porcine myosin and to an immunoblotted extract of human atrial heart tissue. However, two conserved peptides, LRRDLDASREAKKQVEKALE and KLTEKEKAELQAKLEAEAKA, stimulated peptide-specific antibodies in B10.BR and B10.D2 mice respectively, which reacted minimally if at all with human atrial heart tissue extract. Furthermore, antisera to the former peptide, in a bactericidal assay involving human monocytes, could mediate killing of streptococci (82% of bacteria). Although this level of killing is less than that produced by antisera to the highly polymorphic type-specific aminoterminus (up to 100% killing), it provides evidence that conserved epitopes can be the targets of bactericidal antibodies. These conserved epitopes may be useful in a vaccine because they also stimulate T cells, thus allowing development of immunologic memory and natural boosting of an immune response after natural exposure.

Amino Acid Sequence↗

PubMed2005

[Genetic risks in plant ex situ conservation].

Conserving genetic diversity of rare and endangered species and their evolutionary potential is one of the long-term goals of ex-situ conservation. Some potential genetic risks in ex-situ conservation in botanical gardens are presented. The preserved species may lack genetic representativity because of poor sampling. Inappropriate plantations, inadequate records and unclear kinships jeopardize endangered species to genetic confusion, inbreeding depression or outbreeding depression. Artificial selection and habitat conversion also potentially result endangered plants in adapting to ex-situ conservation, which had been usually overlooked. All the genetic risks can decrease the success of reintroduction and recovery. Therefore, appropriate genetic management should be carried out in botanical gardens to decrease or avoid genetic risks in ex-situ conservation.

Adaptation, Biological↗

PubMed2005

[The application of DNA molecular markers in conservation of the rare and endangered medicinal plants].

In this paper, the advance in DNA molecular markers techniques in recent years was reviewed. The application of DNA markers in conservation of the rare and endangered medicinal plants was explicated, of which included identification of germ-plasm resource, determination of the habitats unite which should be protected in situ, sampling strategies of ex-situ conservation, evaluation of the conservation effects of the rare and endangered medicinal plants, as well as elucidation of their endangered mechanism etc. The information could help drawing up conservation strategies and conservation measures for references.

Conservation of Natural Resources↗