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PubMed1995

Polymerase chain reaction-based assays for the characterisation of plant genetic resources.

Plant genetic resources are an important component of biodiversity and provide the basic genetic variability that allow new and improved cultivars to be developed. Numerous germplasm collections have been established and it is important to established that such collections are representative and accessible to breeders and biotechnologists. Molecular markers provide the best estimate of genetic diversity since they are independent of the confounding effects of environmental factors. Assays based on the polymerase chain reaction (PCR) are considered to meet both the technical and genetical requirements for the characterisation of plant and animal genetic resources. Two main approaches are described, based on anonymous and defined primers. The use of both randomly amplified polymorphic DNA (RAPD) and microsatellites or simple sequence repeats (SSR) for the characterisation of perennial tree species, and distribution of variability within gene pools is reported. The detection of interspecific gene introgression between coffee species with RAPD markers is described together with the use of microsatellites to genotype potato. The use of PCR-based assays will facilitate the evaluation and utilisation of plant genetic resources.

Base Sequence↗
PubMed2006

Patterns of sequence conservation in presynaptic neural genes.

BACKGROUND: The neuronal synapse is a fundamental functional unit in the central nervous system of animals. Because synaptic function is evolutionarily conserved, we reasoned that functional sequences of genes and related genomic elements known to play important roles in neurotransmitter release would also be conserved. RESULTS: Evolutionary rate analysis revealed that presynaptic proteins evolve slowly, although some members of large gene families exhibit accelerated evolutionary rates relative to other family members. Comparative sequence analysis of 46 megabases spanning 150 presynaptic genes identified more than 26,000 elements that are highly conserved in eight vertebrate species, as well as a small subset of sequences (6%) that are shared among unrelated presynaptic genes. Analysis of large gene families revealed that upstream and intronic regions of closely related family members are extremely divergent. We also identified 504 exceptionally long conserved elements (> or =360 base pairs, > or =80% pair-wise identity between human and other mammals) in intergenic and intronic regions of presynaptic genes. Many of these elements form a highly stable stem-loop RNA structure and consequently are candidates for novel regulatory elements, whereas some conserved noncoding elements are shown to correlate with specific gene expression profiles. The SynapseDB online database integrates these findings and other functional genomic resources for synaptic genes. CONCLUSION: Highly conserved elements in nonprotein coding regions of 150 presynaptic genes represent sequences that may be involved in the transcriptional or post-transcriptional regulation of these genes. Furthermore, comparative sequence analysis will facilitate selection of genes and noncoding sequences for future functional studies and analysis of variation studies in neurodevelopmental and psychiatric disorders.

Animals↗
PubMed2006

Utility of mitochondrial DNA barcodes in species conservation.

Molecular tools are a standard part of many conservation studies and can be informative at many different levels of analysis, although there are inherent limitations and strengths of different genes or parts of genes to inform specific questions. Animal DNA barcodes, 600- to 800-base-pair segments of the mitochondrial gene cytochrome oxidase I, have been proposed as a means to quantify global biodiversity. Although mitochondrial (mt) DNA has a long history of use at the species level, recent analyses suggest that the use of a single gene, particularly mitochondrial, is unlikely to yield data that are balanced, universally acceptable, or sufficient in taxonomic scope to recognize many species lineages. Mitochondrial and nuclear genomes have different patterns of evolution and modes of inheritance, which can result in very different assessments of biodiversity. The ramifications of choosing a particular definition of species (species concept) need to be carefully considered because current efforts have designated DNA barcodes as the universal species concept without demonstrating its superiority over preexisting concepts. The results of such a barcoding paradigm may include a failure to recognize significant portions of biodiversity or nuclear/mitochondrial mixed lineages and could spuriously focus conservation resources on populations with relatively minor mtDNA divergence. DNA barcodes are most likely to provide potentially useful information for groups that are already well studied, and such taxa do not constitute the majority of biodiversity or those in most need of research attention. DNA barcode-length sequences are an important source of data but, when used alone or out of context, may offer only a fraction of the information needed to characterize species while taking resources from broader studies that could produce information essential to robust and informed conservation decisions.

Biodiversity↗
PubMed2006

VNTRDB: a bacterial variable number tandem repeat locus database.

Variable number tandem repeat-PCR (VNTR-PCR) is a novel method developed for molecular typing of microorganisms. This method has proven useful in epidemiological studies in medical microbiology. Although hundreds of bacterial genomes have been sequenced, variable number tandem repeats (TRs) derived from comparative genome analyses are scarce. This may hamper their application to the surveillance of bacteria in molecular epidemiology. Here, we present a freely accessible variable number tandem repeat database (VNTRDB) that is intended to be a resource for helping in the discovery of putatively polymorphic tandem repeat loci and to aid with assay design by providing the flanking sequences that can be used in subsequent PCR primer design. In order to reveal possible polymorphism, each TR locus was obtained by comparing the sequences between different sets of bacterial genera, species or strains. Through this comparison, TRs which are unique to a genus can also be identified. Moreover, a visualization tool is provided to ensure that the copy number and locus length of repeats are correct. The VNTRDB is available at http://vntr.csie.ntu.edu.tw/.

Bacteria↗
PubMed2005

Impact of natural variation in bacterial F17G adhesins on crystallization behaviour.

Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies.

Adhesins, Bacterial↗
PubMed2026

Comparative Analysis of Chloroplast Genomes Reveals Molecular Evolution and Phylogenetic Relationships in Fraxinus (Fraxinus mandshurica).

Fraxinus mandshurica (Manchurian ash) is an ecologically and economically valuable hardwood tree native to Northeast Asia, yet its genomic resources remain limited. We assembled its complete chloroplast (cp) genome (155,559 bp) using hybrid PacBio and Illumina sequencing and performed comparative, phylogenetic, and evolutionary analyses. The cp genome exhibits a typical quadripartite structure encoding 132 gene copies, comprising 114 unique genes (80 protein-coding, 30 tRNA, and 4 rRNA genes), with 18 genes duplicated in the inverted repeat (IR) regions. Simple sequence repeat analysis revealed dominance of mononucleotide A/T repeats. Phylogenetic analysis of 53 complete cp genomes strongly supported the monophyly of Oleaceae and resolved F. mandshurica as sister to the North American F. nigra, consistent with previously proposed Miocene intercontinental dispersal scenarios between East Asia and North America. Most protein-coding genes were under strong purifying selection (Ka/Ks << 1), whereas petB, rpl2, and several ndh genes showed elevated Ka/Ks values that are suggestive of altered selective constraint but are based on very few substitutions and are therefore not, on their own, evidence of positive selection. Nucleotide diversity (Pi) analysis identified 15 hypervariable intergenic spacers (mean Pi = 0.067), among which trnM-CAU-rps14, ndhJ-ndhK, and petL-petG represent promising candidate barcode regions requiring further validation. This study provides a high-quality, fully annotated cp genome of F. mandshurica and a valuable genomic resource for future phylogenetic, population genetic, and conservation studies of this important genus.

Fraxinus↗
PubMed2005

Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies.

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.

Animals↗
PubMed2002

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis.

As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.

Cloning, Molecular↗
PubMed1980

Evaluation of the resources required for the implementation of a new therapeutic protocol: the Institut Gustave Roussy method.

All treatments give rise to a sequence of events which represent the work-load. Before implementing a new treatment, it is essential to identify the sequence of the induced events. Their cost must be evaluated to ensure that it can be undertaken, and if so, for the necessary logistics to be set up. It must be stressed that the means required are not solely financial, but also involve the availability of an appropriate infrastructure where required, which can include: an increase in staff, additional equipment or premises, a broadening of working hours such as, for example, extending the activity of some laboratories over the weekend. At the Gustave-Roussy Institute, we are now developing the method set out in this paper, which should enable us to rapidly forecast the consequences of any modification in the medical approach to our patients. It is based on the evaluation of "consumption" of the staff, premises, equipment and drugs required, in terms of time.

Drug Therapy↗
PubMed2003

The apoptosis database.

The apoptosis database is a public resource for researchers and students interested in the molecular biology of apoptosis. The resource provides functional annotation, literature references, diagrams/images, and alternative nomenclatures on a set of proteins having 'apoptotic domains'. These are the distinctive domains that are often, if not exclusively, found in proteins involved in apoptosis. The initial choice of proteins to be included is defined by apoptosis experts and bioinformatics tools. Users can browse through the web accessible lists of domains, proteins containing these domains and their associated homologs. The database can also be searched by sequence homology using basic local alignment search tool, text word matches of the annotation, and identifiers for specific records. The resource is available at http://www.apoptosis-db.org and is updated on a regular basis.

Animals↗
PubMed1999

A comparison of sequence and structure protein domain families as a basis for structural genomics.

MOTIVATION: Protein families can be defined based on structure or sequence similarity. We wanted to compare two protein family databases, one based on structural and one on sequence similarity, to investigate to what extent they overlap, the similarity in definition of corresponding families, and to create a list of large protein families with unknown structure as a resource for structural genomics. We also wanted to increase the sensitivity of fold assignment by exploiting protein family HMMs. RESULTS: We compared Pfam, a protein family database based on sequence similarity, to Scop, which is based on structural similarity. We found that 70% of the Scop families exist in Pfam while 57% of the Pfam families exist in Scop. Most families that occur in both databases correspond well to each other, but in some cases they are different. Such cases highlight situations in which structure and sequence approaches differ significantly. The comparison enabled us to compile a list of the largest families that do not occur in Scop; these are suitable targets for structure prediction and determination, and may be useful to guide projects in structural genomics. It can be noted that 13 out of the 20 largest protein families without a known structure are likely transmembrane proteins. We also exploited Pfam to increase the sensitivity of detecting homologs of proteins with known structure, by comparing query sequences to Pfam HMMs that correspond to Scop families. For SWISSPROT+TREMBL, this yielded an increase in fold assignment from 31% to 42% compared to using FASTA only. This method assigned a structure to 22% of the proteins in Saccharomyces cerevisiae, 24% in Escherichia coli, and 16% in Methanococcus jannaschii.

Computational Biology↗
PubMed1999

A maize map standard with sequenced core markers, grass genome reference points and 932 expressed sequence tagged sites (ESTs) in a 1736-locus map.

We have constructed a 1736-locus maize genome map containing1156 loci probed by cDNAs, 545 probed by random genomic clones, 16 by simple sequence repeats (SSRs), 14 by isozymes, and 5 by anonymous clones. Sequence information is available for 56% of the loci with 66% of the sequenced loci assigned functions. A total of 596 new ESTs were mapped from a B73 library of 5-wk-old shoots. The map contains 237 loci probed by barley, oat, wheat, rice, or tripsacum clones, which serve as grass genome reference points in comparisons between maize and other grass maps. Ninety core markers selected for low copy number, high polymorphism, and even spacing along the chromosome delineate the 100 bins on the map. The average bin size is 17 cM. Use of bin assignments enables comparison among different maize mapping populations and experiments including those involving cytogenetic stocks, mutants, or quantitative trait loci. Integration of nonmaize markers in the map extends the resources available for gene discovery beyond the boundaries of maize mapping information into the expanse of map, sequence, and phenotype information from other grass species. This map provides a foundation for numerous basic and applied investigations including studies of gene organization, gene and genome evolution, targeted cloning, and dissection of complex traits.

Chromosome Mapping↗
PubMed2001

Of mice and genome sequence.

Availability of the mouse genome sequence will have a major impact on the study of vertebrate evolution, mammalian biology, and animal models of human disease. Resources to explore genome biology in mice will maximize the effect of this watershed event.

Animals↗
PubMed1998

Expanding yeast knowledge online.

The completion of the Saccharomyces cerevisiae genome sequencing project and the continued development of improved technology for large-scale genome analysis have led to tremendous growth in the amount of new yeast genetics and molecular biology data. Efficient organization, presentation, and dissemination of this information are essential if researchers are to exploit this knowledge. In addition, the development of tools that provide efficient analysis of this information and link it with pertinent information from other systems is becoming increasingly important at a time when the complete genome sequences of other organisms are becoming available. The aim of this review is to familiarize biologists with the type of data resources currently available on the World Wide Web (WWW).

Amino Acid Sequence↗
PubMed2002

Pescador: the PEptides in Solution ConformAtion Database: Online Resource.

In recent years a large body of data has been obtained from Nuclear Magnetic Resonance and Circular Dichroism experiments on the influence of the amino acid sequence and various other parameters on the conformational state of peptides in solution. Interpreting the experimental data in terms of the conformational populations of the peptides remains a key problem, for which current solutions leave appreciable room for improvement. Considering that making this body of data available for surveys and analysis should be instrumental in tackling the problem, we undertook the development of Pescador: The 'PEptides in Solution ConformAtion Database: Online Resource'. Pescador contains data from NMR and CD spectroscopy on peptides in solution as well as information on the structural parameters derived from these data. It also features specialized Web-based tools for data deposition, and means for readily accessing the stored information for analysis purposes. To illustrate the use of the database in deriving information for the conformational analysis of peptides, we show how the alpha proton delta-values stored in Pescador and measured by NMR for different peptides in different laboratories can be used to derive a new set of 'random coil' chemical shift values. Firstly, we show these values to be very similar to those obtained experimentally for model peptides in water, and their variation with increasing Tri-Fluoro-Ethanol (TFE) concentration is similar to that reported for model peptides. We show, furthermore, that the chemical shift data in Pescador can be used to derive correction factors that take into account effects of neighboring residues. These correction factors compare favorably with those recently derived from a series of model GGXGG peptides (Schwarzinger et al., 2001). These encouraging results suggest that, as the quantity of NMR data on peptide deposited in Pescador increases, surveys of these data should be a valuable means of deriving key parameters for the analysis of peptide conformation.

Circular Dichroism↗
PubMed1997

Development of a Haemophilus two-dimensional protein database.

Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strain of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A "knock-out" mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to protein synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.

Bacterial Proteins↗
PubMed2000

Is judgment of random time intervals biased and capacity-limited?

Recently, random generation of time intervals has been proposed as a procedure to impair executive processing in a dual-task paradigm without substantial interference with phonological and visuo-spatial working memory resources. A fundamental assumption of this procedure is that humans are able to distinguish time sequences on their degree of randomness. The present study tests this assumption. To this end, non-biased, repetition-biased, alternation-biased sequences and repetitive rhythms were judged under conditions of higher or lower executive load. In Exp. 1, load depended on the presentation speed, while in Exp. 2, a dual-task condition was used with either a concurrent number-copying task or an arithmetic task. It was found that the participants could distinguish repetitive rhythms from more or less random sequences, and that both accuracy of this judgment and latency were affected by the concurrent load. The findings are taken as a first indication that random time judgment is capacity-limited.

Decision Making↗
PubMed2004

Gene expression profiling of systemically wound-induced defenses in hybrid poplar.

As part of an ongoing effort to identify genes involved in poplar defense responses, and to provide a resource for comparative analysis of woody and non-woody plant defense, we generated expressed sequence tags (ESTs) from a library constructed from systemically wounded leaves of hybrid poplar (Populus trichocarpa x P. deltoides). Partial sequences were obtained from the 5' ends of 928 individual cDNAs, which could be grouped into 565 non-overlapping sequences. Of these, 447 sequences were singletons, while the remainder fell into 118 clusters containing up to 17 partially overlapping ESTs. Approximately 81% of the EST sequences showed similarity to previously described sequences in public databases. Of these, the distribution of gene functions within the EST set indicated that approximately 11% of the ESTs encode proteins potentially involved in defense or secondary metabolism, while photosynthesis and primary metabolism accounted for 45% of the expressed genes. Two types of defense proteins, Kunitz trypsin inhibitors and chitinases, were found among the ten most abundant ESTs, indicating the significant impact of wounding on the leaf transcriptome and suggesting that these functions are important for hybrid poplar defense. In the course of this work, three new wound-inducible Kunitz trypsin inhibitor-like genes and two new chitinase-like genes were characterized. A suite of other systemically wound-induced genes were identified using northern and macroarray analysis, indicating diversity and multiplicity in the induced defense response. Overall, we demonstrate that defense-related genes of hybrid poplar have a variety of functions, and show remarkably diverse expression patterns upon wounding.

Crosses, Genetic↗