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A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops.

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species.

Base Sequence↗

A conserved upstream open reading frame mediates sucrose-induced repression of translation.

Sugars have been shown to regulate transcription of numerous genes in plants. Sucrose controls translation of the group S basic region leucine zipper (bZIP)-type transcription factor ATB2/AtbZIP11 (Rook et al., 1998a). This control requires the unusually long 5' untranslated region (UTR) of the gene. Point mutations and deletions of the 5'UTR have uncovered the sequences involved. A highly conserved upstream open reading frame (uORF) coding for 42 amino acids is essential for the repression mechanism. It is conserved in 5'UTRs of bZIP transcription factors from other Arabidopsis thaliana genes and many other plants. ATB2/AtbZIP11 is normally expressed in association with vascular tissues. Ectopic expression of a 5'UTR construct shows that the sucrose repression system is functional in all tissues. AtbZIP2 is another Arabidopsis bZIP transcription factor gene harboring the conserved uORF, which is regulated similarly via sucrose-induced repression of translation. This suggests a general function of the conserved uORF in sucrose-controlled regulation of expression. Our findings imply the operation of a sucrose-sensing pathway that controls translation of several plant bZIP transcription factor genes harboring the conserved uORF in their 5'UTRs. Target genes of such transcription factors will then be regulated in sucrose-dependent way.

5' Untranslated Regions↗

Sequence conserved for subcellular localization.

The more proteins diverged in sequence, the more difficult it becomes for bioinformatics to infer similarities of protein function and structure from sequence. The precise thresholds used in automated genome annotations depend on the particular aspect of protein function transferred by homology. Here, we presented the first large-scale analysis of the relation between sequence similarity and identity in subcellular localization. Three results stood out: (1) The subcellular compartment is generally more conserved than what might have been expected given that short sequence motifs like nuclear localization signals can alter the native compartment; (2) the sequence conservation of localization is similar between different compartments; and (3) it is similar to the conservation of structure and enzymatic activity. In particular, we found the transition between the regions of conserved and nonconserved localization to be very sharp, although the thresholds for conservation were less well defined than for structure and enzymatic activity. We found that a simple measure for sequence similarity accounting for pairwise sequence identity and alignment length, the HSSP distance, distinguished accurately between protein pairs of identical and different localizations. In fact, BLAST expectation values outperformed the HSSP distance only for alignments in the subtwilight zone. We succeeded in slightly improving the accuracy of inferring localization through homology by fine tuning the thresholds. Finally, we applied our results to the entire SWISS-PROT database and five entirely sequenced eukaryotes.

Amino Acid Sequence↗

Finding biologically relevant protein domain interactions: conserved binding mode analysis.

Proteins evolved through the shuffling of functional domains, and therefore, the same domain can be found in different proteins and species. Interactions between such conserved domains often involve specific, well-determined binding surfaces reflecting their important biological role in a cell. To find biologically relevant interactions we developed a method of systematically comparing and classifying protein domain interactions from the structural data. As a result, a set of conserved binding modes (CBMs) was created using the atomic detail of structure alignment data and the protein domain classification of the Conserved Domain Database. A conserved binding mode is inferred when different members of interacting domain families dock in the same way, such that their structural complexes superimpose well. Such domain interactions with recurring structural themes have greater significance to be biologically relevant, unlike spurious crystal packing interactions. Consequently, this study gives lower and upper bounds on the number of different types of interacting domain pairs in the structure database on the order of 1000-2000. We use CBMs to create domain interaction networks, which highlight functionally significant connections by avoiding many infrequent links between highly connected nodes. The CBMs also constitute a library of docking templates that may be used in molecular modeling to infer the characteristics of an unknown binding surface, just as conserved domains may be used to infer the structure of an unknown protein. The method's ability to sort through and classify large numbers of putative interacting domain pairs is demonstrated on the oligomeric interactions of globins.

Algorithms↗

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity.

During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.

Amino Acid Substitution↗

Objectives for multiple-species conservation planning.

The first step in conservation planning is to identify objectives. Most stated objectives for conservation, such as to maximize biodiversity outcomes, are too vague to be useful within a decision-making framework. One way to clarify the issue is to define objectives in terms of the risk of extinction for multiple species. Although the assessment of extinction risk for single species is common, few researchers have formulated an objective function that combines the extinction risks of multiple species. We sought to translate the broad goal of maximizing the viability of species into explicit objectives for use in a decision-theoretic approach to conservation planning. We formulated several objective functions based on extinction risk across many species and illustrated the differences between these objectives with simple examples. Each objective function was the mathematical representation of an approach to conservation and emphasized different levels of threat. Our objectives included minimizing the joint probability of one or more extinctions, minimizing the expected number of extinctions, and minimizing the increase in risk of extinction from the best-case scenario. With objective functions based on joint probabilities of extinction across species, any correlations in extinction probabilities had to be known or the resultant decisions were potentially misleading. Additive objectives, such as the expected number of extinctions, did not produce the same anomalies. We demonstrated that the choice of objective function is central to the decision-making process because alternative objective functions can lead to a different ranking of management options. Therefore, decision makers need to think carefully in selecting and defining their conservation goals.

Conservation of Natural Resources↗

Conserving old-growth forest diversity in disturbance-prone landscapes.

A decade after its creation, the Northwest Forest Plan is contributing to the conservation of old-growth forests on federal land. However the success and outlook for the plan are questionable in the dry provinces, where losses of old growth to wildfire have been relatively high and risks of further loss remain. We summarize the state of knowledge of old-growth forests in the plan area, identify challenges to conserve them, and suggest some conservation approaches that might better meet the goals of the plan. Historically, old-growth forests in these provinces ranged from open, patchy stands, maintained by frequent low-severity fire, to a mosaic of dense and open stands maintained by mixed-severity fires. Old-growth structure and composition were spatially heterogeneous, varied strongly with topography and elevation, and were shaped by a complex disturbance regime of fire, insects, and disease. With fire suppression and cutting of large pines (Pinus spp.) and Douglas-firs (Pseudotsuga menziesii [Mirbel] Franco), old-growth diversity has declined and dense understories have developed across large areas. Challenges to conserving these forests include a lack of definitions needed for planning of fire-dependent old-growth stands and landscapes, and conflicts in conservation goals that can be resolved only at the landscape level. Fire suppression has increased the area of the dense, older forest favored by Northern Spotted Owls (Strix occidentalis caurina) but increased the probability of high-severity fire. The plan allows for fuel reduction in late-successional reserves; fuel treatments, however apparently have not happened at a high enough rate or been applied in a landscape-level approach. Landscape-level strategies are needed that prioritize fuel treatments by vegetation zones, develop shaded fuel breaks in strategic positions, and thin and apply prescribed fire to reduce ladder fuels around remaining old trees. Evaluations of the current and alternative strategies are needed to determine whether the current reserve-matrix approach is the best strategy to meet plan goals in these dynamic landscapes.

Biodiversity↗

Conservation value of clustered housing developments.

Traditionally, exurban lands in Colorado have been subdivided into a grid of parcels ranging from 2 to 16 ha. From an ecological perspective, this dispersed pattern of development effectively maximizes the individual influence of each home on the land. Clustered housing developments, designed to maximize open space, are assumed to benefit plant and wildlife communities of conservation interest. They have become a popular alternative for rural development despite the lack of empirical evidence demonstrating their conservation benefits. To better inform rural land-use planning, we evaluated clustered housing developments by comparing their spatial pattern with that of dispersed housing developments and by comparing their conservation value with that of both dispersed housing developments and undeveloped areas in Boulder County, Colorado. We used four indicators to assess conservation value: (1) densities of songbirds, (2) nest density and survival of ground-nesting birds, (3) presence of mammals, and (4) percent cover and proportion of native and non-native plant species. Clustered and dispersed housing developments did not differ on the majority of variables we examined. Both types of housing development had significantly higher densities of non-native and human-commensal species and significantly lower densities of native and human-sensitive species than undeveloped areas. More rigorous ecological guidelines and planning on a regional scale may help create clustered developments with higher conservation value.

Animals↗

Testing hypotheses for the success of different conservation strategies.

Evaluations of the success of different conservation strategies are still in their infancy. We used four different measures of project outcomes--ecological, economic, attitudinal, and behavioral--to test hypotheses derived from the assumptions that underlie contemporary conservation solutions. Our hypotheses concerned the effects of natural resource utilization, market integration, decentralization, and community homogeneity on project success. We reviewed the conservation and development literature and used a specific protocol to extract and code the information in a sample of papers. Although our results are by no means conclusive and suffer from the paucity of high-quality data and independent monitoring (80% of the original sample of 124 projects provided inadequate information for use in this study), they show that permitted use of natural resources, market access, and greater community involvement in the conservation project are all important factors for a successful outcome. Without better monitoring schemes in place, it is still impossible to provide a systematic evaluation of how different strategies are best suited to different conservation challenges.

Bias↗

Using ecological-niche modeling as a conservation tool for freshwater species: live-bearing fishes in central Mexico.

Ecological-niche modeling is an important tool for conservation assessment of terrestrial species; however, its applicability has been poorly explored in the aquatic realm. Goodeines are a monophyletic group of viviparous freshwater fishes that are well known in central Mexico, with 41 species in 19 genera. Given the number of threats to biodiversity in the region, goodeines represent an excellent model with which to test novel conservation approaches. We assessed the conservation status of the goodeines (37 species), based on their potential distributions predicted by ecological-niche models generated with the genetic algorithm for rule-set prediction (GARP). Predictions of species' distributions performed well in six out of eight species for which sufficient information was available to perform estimations of the area under the curve (AUC) in receiver operating characteristic plots. Extensive field surveys conducted in recent years in most cases confirm the models' predictions. Species richness exhibited a nested pattern, in which the number of species increased toward the center of the distribution of the group. At the basin level, the Río Ameca Basin had the highest number of species (11), chiefly because of the high number of microendemic species (6). Human activities within water bodies (e.g., extensive aquaculture) and drainages (e.g., agriculture, ranching, industrial activities) have affected most goodeines severely, given the deleterious effects of pollution and introductions of exotic species, such as carp (Cyprinus carpio, Ctenopharingodon idella) and tilapia (Oreochromis spp.). Our results paint a pessimistic picture for the long-term survival of many goodeines in their natural environment, and realistic conservation measures are complex and would require immediate protection of specific areas that we have identified. Ecological-niche modeling is a suitable tool for conservation assessment of freshwater species, but availability of environmental information on aquatic systems (e.g., temperature, water speed, pH, oxygen concentration) would improve distributional predictions.

Algorithms↗

Immunogenic B-cell epitopes of Babesia bovis rhoptry-associated protein 1 are distinct from sequences conserved between species.

Babesia bovis merozoite apical membrane polypeptide Bv60 was found to be rhoptry associated by immuno-electron microscopy and was redesignated rhoptry-associated protein 1 (RAP-1). The N-terminal 300 amino acids of RAP-1 have a high level of sequence similarity to the same N-terminal region of p58, its homolog from Babesia bigemina. However, the interspecies conserved sequences did not include RAP-1 surface-exposed B-cell epitopes as defined by monoclonal antibodies. Furthermore, neither heterologous B. bigemina immune nor monospecific anti-p58 bovine serum binds to whole RAP-1, indicating that the major B-cell epitopes recognized by these sera are also not encoded by the conserved sequences. Truncated RAP-1, expressed by a subclone encoding the N-terminal 235 amino acids, is weakly bound by antibodies in heterologous sera. A peptide representing the longest conserved amino acid sequence (amino acids 121 to 134) in this region is also weakly bound by antibodies in immune bovine sera, and rabbit antibodies induced by and strongly reactive with the peptide alone fail to bind native or denatured RAP-1. Thus, although the conserved region may contain one or more poorly immunogenic B-cell epitopes, these epitopes are inaccessible to antibody in whole RAP-1. The results indicate that the major immunogenic B-cell epitopes of RAP-1, including surface-accessible epitopes bound by monoclonal antibodies, are distinct from the conserved sequences representing putative functional domains.

Amino Acid Sequence↗

Characterization of conserved T- and B-cell epitopes in Plasmodium falciparum major merozoite surface protein 1.

Vaccines for P. falciparum will need to contain both T- and B-cell epitopes. Conserved epitopes are the most desirable, but they are often poorly immunogenic. The major merozoite surface protein 1 (MSP-1) is currently a leading vaccine candidate antigen. In this study, six peptides from conserved or partly conserved regions of MSP-1 were evaluated for immunogenicity in B10 congenic mice. Following immunization with the peptides, murine T cells were tested for the ability to proliferate in vitro and antibody responses to MSP-1 were evaluated in vivo. The results showed that one highly conserved sequence (MSP-1#1, VTHESYQELVKKLEALEDAV; located at amino acid positions 20 to 39) and one partly conserved sequence (MSP-1#23, GLFHKEKMILNEEEITTKGA; located at positions 44 to 63) contained both T- and B-cell epitopes. Immunization of mice with these peptides resulted in T-cell proliferation and enhanced production of antibody to MSP-1 upon exposure to merozoites. MSP-1#1 stimulated T-cell responses in three of the six strains of mice evaluated, whereas MSP-1#23 was immunogenic in only one strain. Immunization with the other four peptides resulted in T-cell responses to the peptides, but none of the resulting peptide-specific T cells recognized native MSP-1. These results demonstrate that two sequences located in the N terminus of MSP-1 can induce T- and B-cell responses following immunization in a murine model. Clearly, these sequences merit further consideration for inclusion in a vaccine for malaria.

Amino Acid Sequence↗

PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus.

Abortion, Veterinary↗

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus.

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.

Amino Acid Sequence↗

A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation.

Hepatitis C virus (HCV) initiates translation of its polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of the 341-nucleotide (nt) 5' nontranslated RNA (5'NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which secondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5' border of the HCV IRES and thus may function in viral translation. This prediction was tested by a mutational analysis of putative helical structures that examined the impact of both covariant and noncovariant nucleotide substitutions on IRES activity in vivo and in vitro. Results of these experiments provide support for predicted base pair interactions between nt 44 to 52 and 111 to 118 and between nt 65 to 70 and 97 to 102 of the HCV 5'NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted in reciprocal changes in V1 nuclease cleavage patterns within the opposing strand of the putative helix, consistent with the predicted base pair interactions. IRES activity was highly dependent on maintenance of the stem-loop II structure but relatively tolerant of covariant nucleotide substitutions within predicted helical segments. Sequence alignments suggested that the deduced domain II structure is conserved within the IRESs of pestiviruses as well as the novel flavivirus GB virus B. Despite marked differences in primary nucleotide sequence within conserved helical segments, the sequences of the intervening single-stranded loop segments are highly conserved in these different viruses. This suggests that these segments of the viral RNA may interact with elements of the host translational machinery that are broadly conserved among different mammalian species.

5' Untranslated Regions↗

The central conserved cystine noose of the attachment G protein of human respiratory syncytial virus is not required for efficient viral infection in vitro or in vivo.

The G glycoprotein of human respiratory syncytial virus (RSV) was identified previously as the viral attachment protein. Although we and others recently showed that G is not essential for replication in vitro, it does affect the efficiency of replication in a cell type-dependent fashion and is required for efficient replication in vivo. The ectodomain of G is composed of two heavily glycosylated domains with mucin-like characteristics that are separated by a short central region that is relatively devoid of glycosylation sites. This central region contains a 13-amino acid segment that is conserved in the same form among RSV isolates and is overlapped by a second segment containing four cysteine residues whose spacings are conserved in the same form and which create a cystine noose. The conserved nature of the cystine noose and flanking 13-amino acid segment suggested that this region likely was important for attachment activity. To test this hypothesis, we constructed recombinant RSVs from which the region containing the cysteine residues was deleted together with part or all of the conserved 13-amino acid segment. Surprisingly, each deletion had little or no effect on the intracellular synthesis and processing of the G protein, the kinetics or efficiency of virus replication in vitro, or sensitivity to neutralization by soluble heparin in vitro. In addition, neither deletion had any discernible effect on the ability of RSV to infect the upper respiratory tract of mice and both resulted in a 3- to 10-fold reduction in the lower respiratory tract. Thus, although the G protein is necessary for efficient virus replication in vivo, this activity does not require the central conserved cystine noose region.

Amino Acid Sequence↗

Identification of a conserved RNA motif essential for She2p recognition and mRNA localization to the yeast bud.

In Saccharomyces cerevisiae, over twenty mRNAs localize to the bud tip of daughter cells, playing roles in processes as different as mating type switching and plasma membrane targeting. The localization of these transcripts depends on interactions between a cis-acting localization element(s) or zipcodes and the RNA-binding protein She2p. While previous studies identified four different localization elements in the bud-localized ASH1 mRNA, the main determinants for She2p recognition are still unknown. To investigate the RNA-binding specificity of She2p, we isolated She2p-binding RNAs by in vivo selection from libraries of partially randomized ASH1 localization elements. The RNAs isolated contained a similar loop-stem-loop structure with a highly conserved CGA triplet in one loop and a single conserved cytosine in the other loop. Mutating these conserved nucleotides or the stem separating them resulted in the loss of She2p binding and in the delocalization of a reporter mRNA. Using this information, we identified the same RNA motif in two other known bud-localized transcripts, suggesting that this motif is conserved among bud-localized mRNAs. These results show that mRNAs with zipcodes lacking primary sequence similarity can rely on a few conserved nucleotides properly oriented in their three-dimensional structure in order to be recognized by the same localization machinery.

Base Sequence↗

The sex-determining gene doublesex in the fly Megaselia scalaris: conserved structure and sex-specific splicing.

The well-known sex-determining cascade of Drosophila melanogaster serves as a paradigm for the pathway to sexual development in insects. But the primary sex-determining signal and the subsequent step, Sex-lethal (Sxl), have been shown not to be functionally conserved in non-Drosophila flies. We isolated doublesex (dsx), which is a downstream step in the cascade, from the phorid fly Megaselia scalaris, which is a distant relative of D. melanogaster. Conserved properties, e.g., sex-specific splicing, structure of the female-specific 3' splice site, a splicing enhancer region with binding motifs for the TRA2/RBP1/TRA complex that activates female-specific splicing in Drosophila, and conserved domains for DNA-binding and oligomerization in the putative DSX protein, indicate functional conservation of dsx in M. scalaris. Hence, the dsx step of the sex-determining pathway appears to be conserved among flies and probably in an even wider group of insects, as the analysis of a published cDNA from the silkmoth indicates.

Amino Acid Sequence↗