Browsing by Author "Weller, Jennifer W."

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    Comparative Genome Analysis of Three Brucella spp. and a Data Model for Automated Multiple Genome Comparison
    Sturgill, David Matthew (Virginia Tech, 2003-07-30)
    Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented.
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    Incorporation of Physico-Chemical Parameters Into Design of Microarray Experiments
    Ratushna, Vladyslava G. (Virginia Tech, 2005-05-06)
    Microarrays containing long oligonucleotides provide sensitive and specific detection of gene expression and are becoming a popular experimental platform. In the process of designing an oligonucleotide microarray for Brucella, we optimized the overall design of the array and created probes to distinguish among the known Brucella species. A 3-way genome comparison identified a set of genes which occur uniquely in only one or two of the sequenced Brucella genomes. Reverse transcriptase PCR assays of over one hundred unique and pairwise-differential regions identified in Brucella revealed several groups of genes that are transcribed in vivo with potential significance for virulence. The structural and thermodynamic properties of a set of 70mer oligonucleotide probes for a combined B. abortus, B. melitensis and B. suis microarray were modeled to help perform quantitative interpretation of the microarray data. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrated that properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and an oligonucleotide probe in a microarray experiment. Despite relatively high hybridization temperatures used in the modeling process, parts of the target molecules are predicted to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. Features in the Brucella genomes with potential diagnostic use were identified, and the extent to which target secondary structure, a molecular property which is not considered in the array design process, may influence the quality of results was characterized.
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    Secondary structure in the target as a confounding factor in synthetic oligomer microarray design
    Ratushna, Vladyslava G.; Weller, Jennifer W.; Gibas, Cynthia J. (2005-03-08)
    Background Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment. Results Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65°C, 28 ± 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Brucella using a freely available oligo design software package. 21 ± 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65°C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure. Conclusion Secondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly.