Biomedical subjects

Thomas J Albert

Publications and source records attributed to Thomas J Albert.

9 recordsLinked to original sources

PubMed2006

The UBX-regulated network in the haltere imaginal disc of D. melanogaster.

Hox proteins have been proposed to act at multiple levels within regulatory hierarchies and to directly control the expression of a plethora of target genes. However, for any specific Hox protein or tissue, very few direct in vivo-regulated target genes have been identified. Here, we have identified target genes of the Hox protein Ultrabithorax (UBX), which modifies the genetic regulatory network of the wing to generate the haltere, a modified hindwing. We used whole-genome microarrays and custom arrays including all predicted transcription factors and signaling molecules in the Drosophila melanogaster genome to identify differentially expressed genes in wing and haltere imaginal discs. To elucidate the regulation of selected genes in more detail, we isolated cis-regulatory elements (CREs) for genes that were specifically expressed in either the wing disc or haltere disc. We demonstrate that UBX binds directly to sites in one element, and these sites are critical for activation in the haltere disc. These results indicate that haltere and metathoracic segment morphology is not achieved merely by turning off the wing and mesothoracic development programs, but rather specific genes must also be activated to form these structures. The evolution of haltere morphology involved changes in UBX-regulated target genes, both positive and negative, throughout the wing genetic regulatory network.

Animals↗

PubMed2006

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale.

We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.

Adaptation, Physiological↗

PubMed2006

Interrogating genomic diversity of E. coli O157:H7 using DNA tiling arrays.

Here, we describe a novel microarray-based approach for investigating the genomic diversity of Escherichia coli O157:H7 in a semi-high throughput manner using a high density, oligonucleotide-based microarray. This microarray, designed to detect polymorphisms at each of 60,000 base-pair (bp) positions within an E. coli genome, is composed of overlapping 29-mer oligonucleotides specific for 60 equally spaced, 1000-bp loci of the E. coli O157:H7 strain EDL933 chromosome. By use of a novel 12-well microarray that permitted the simultaneous investigation of 12 strains, the genomes of 44 individual isolates of E. coli O157:H7 were interrogated. These analyses revealed more than 150 single nucleotide polymorphisms (SNPs) and several deletions and amplifications in the test strains. Pyrosequencing was used to confirm the usefulness of the novel SNPs by determining their allelic frequency among a collection of diverse isolates of E. coli O157:H7. The tiling DNA microarray system would be useful for the tracking and identification of individual strains of E. coli O157:H7 needed for forensic investigations.

Amino Acid Substitution↗

PubMed2006

Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms.

Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7x10(-9) per site per year), we estimate that the most recent common ancestor of the contemporary beta-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens.

Chromosome Mapping↗

PubMed2005

Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis.

PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.

Bacterial Proteins↗

PubMed2005

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori.

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.

Anti-Infective Agents↗

PubMed2004

Tracking the evolution of the SARS coronavirus using high-throughput, high-density resequencing arrays.

Mutations in the SARS-Coronavirus (SARS-CoV) can alter its clinical presentation, and the study of its mutation patterns in human populations can facilitate contact tracing. Here, we describe the development and validation of an oligonucleotide resequencing array for interrogating the entire 30-kb SARS-CoV genome in a rapid, cost-effective fashion. Using this platform, we sequenced SARS-CoV genomes from Vero cell culture isolates of 12 patients and directly from four patient tissues. The sequence obtained from the array is highly reproducible, accurate (>99.99% accuracy) and capable of identifying known and novel variants of SARS-CoV. Notably, we applied this technology to a field specimen of probable SARS and rapidly deduced its infectious source. We demonstrate that array-based resequencing-by-hybridization is a fast, reliable, and economical alternative to capillary sequencing for obtaining SARS-CoV genomic sequence on a population scale, making this an ideal platform for the global monitoring of SARS-CoV and other small-genome pathogens.

Animals↗

PubMed2003

Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays.

Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.

Molecular Structure↗

PubMed2002

Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized to Drosophila melanogaster and mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.

Animals↗