David R. Rank

Sequence variation and phylogenetic history of the mouse Ahr gene

The Ahr locus encodes for the aryl hydrocarbon receptor (AHR), which plays an important toxicological and developmental role. Sequence variation in this gene was studied in 13 different mouse lines that included eight laboratory strains, two Mus musculus subspecies and three additional Mus species. The data presented represent the largest study of sequence variation across multiple mouse lines in a single gene (approximately equal to 15.9 kb/mouse line). Among all mice, the average frequency of all polymorphisms in the intronic regions was 20.3 variants/kb and the average exonic frequency was 14.1 variants/kb. For substitutions alone, the average frequencies in the intronic and exonic regions for all mice were 13.3 and 8.9 substitutions/kb, respectively. Between laboratory strains, the average intronic and exonic frequencies for all polymorphisms dropped to 5.4 and 2.9 variants/kb, respectively. There were 111 non-synonymous polymorphisms that resulted in 42 different amino acid changes, of which only 10 amino acid changes had been previously identified. Based on the nucleotide sequence, the phylogenetic history of the gene showed mice from the Ahr(b2) and Ahr(d) alleles in separate branches while mice from the Ahr(b1) and Ahr(b3) alleles exhibited a more complex history. Evolutionarily, the AHR protein as a whole appears to be under purifying selective pressure (K(a) : K(s) ratio = 0.237). Despite significant functional constraint in the basic helix-loop-helix and PAS domains, ligand binding is not constrained to the high-affinity allele, which supports further the role of the AHR in development and its importance beyond the adaptive response to environmental toxicants.

Mining the human genome using microarrays of open reading frames.

To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.

Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry.

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length. Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.

Sub-microliter DNA sequencing for capillary array electrophoresis

DNA sequencing from sub-microliter samples was demonstrated for capillary array electrophoresis by optimizing the analysis of 500 nl reaction aliquots of full-volume reactions and by preparing 500 nl reactions within fused-silica capillaries. Sub-microliter aliquots were removed from the pooled reaction products of 10 microl dye-primer cycle-sequencing reactions and analyzed without modifying either the reagent concentrations or instrument workflow. The impact of precipitation methods, resuspension buffers, and injection times on electrokinetic injection efficiency for 500 nl aliquots were determined by peak heights, signal-to-noise ratios, and changes in base-called readlengths. For 500 nl aliquots diluted to 5 microl in 60% formamide-1 mM EDTA and directly injected, a five-fold increase in signal-to-noise ratios was obtained by increasing injection times from 10 to 80 s without a corresponding increase in peak widths or reduction in readlengths. For 500 nl aliquots precipitated in alcohol, 80 +/- 5% template recovery and a two-fold decrease in conductivity was obtained, resulting in a two-fold increase in peak heights and 50 to 100 bases increase in readlengths. In a comparison of aliquot volumes and precipitation methods, equivalent readlengths were obtained for 500 nl, 4 microl, and 8 microl aliquots by simply adjusting the electrokinetic injection conditions. To ascertain the robustness of this methodology for genomic sequencing, 96 Arabidopsis thaliana subclones were sequenced, with a yield of 38 624 bases obtained from 500 nl aliquots versus 30 764 bases from standard scale reactions. To demonstrate 500 nl sample preparation, reactions were performed in fused-silica capillary reaction chambers using air-based thermal cycling. A readlength of 690 bases was obtained for the polymerase chain reaction product of an Arabidopsis subclone without modifying the reagent concentrations, post-reaction processing or electrokinetic injection workflow. These results demonstrated the fundamental feasibility of small-volume DNA sequencing for high-throughput capillary electrophoresis.

A conditional density error model for the statistical analysis of microarray data

MOTIVATION In many microarray experiments, relatively few intra- and inter-array replicate measurements are made due to significant cost limitations and sample availability. Compounding this problem is a lack of robust statistical methods for analyzing gene expression data with limited experimental replicates. As a result, the interpretation of the results of these experiments are difficult with little understanding of the probability of type I and type II errors. RESULTS The variability in a series of replicate microarray measurements was modelled using a combination of parametric and non-parametric methods. A 3-dimensional surface was created for the conditional distribution of the variability given the mean signal intensity in both the Cy3 and Cy5 channels. The results were used as the basis for developing statistical methods for analyzing gene expression data with limited experimental replicates. AVAILABILITY The statistical analysis scripts are available upon request.

Developing toxicologically predictive gene sets using cDNA microarrays and bayesian classification

Publisher Summary The potential applications of predictive statistical models in toxicology based on gene expression measurements are numerous. For example, short-term studies measuring gene expression could be used to predict long-term toxicity studies like those still performed by the National Toxicology Program (NTP). Other short-term gene expression studies could be used to predict which chemicals would be teratogenic or cause more subtle developmental changes after human prenatal exposure. In either case, the application of microarray analysis and predictive statistical models has the potential to be extremely useful from both economic and human health perspectives. The application of predictive statistical models to chemically induced gene expression is the next logical step in the developing field of toxicogenomics. The development of these models may eventually open the door to a new era of toxicological testing where relatively short and inexpensive microarray studies may allow the assessment of the human health risks associated with a previously untested chemical. However, the accuracy and applicability of these models are highly dependent of the quality of the training sets used in their development. As the public gene expression database grows, more chemicals may be added to training the models and those models will become more predictive.

M13-102 : A VECTOR FOR FACILITATING CONSTRUCTION AND IMPROVING QUALITY OF M13 SHOTGUN LIBRARIES

A modified vector, M13-102, is described which utilizes the previously reported M13-100 direct selection strategy for shotgun cloning [Guilfoyle and Smith, Nucleic Acids Res. 22 (1994) 100-107]. In these vectors, direct selection replaces the need for phosphatase treatment of vector DNA and is achieved by insertional inactivation of M13 gene X. When not inactivated, the engineered overproduction of the M13 gene X product mediates phage replication repression. M13-102 contains two new additions: (1) a sequence enabling triple-helix-mediated affinity capture (TAC) for purification of linearized vector DNA, and (2) universal primer sequences for wider compatibility with commercial instruments that support fluorescence-based sequencing. Using a biotinylated homopyrimidine oligodeoxyribonucleotide as third-strand probe, TAC is performed on streptavidin-coated magnetic beads [Ji et al., Genetics Analysis: Techniques and Applications 11 (1994) 43-47], and serves as a rapid and efficient alternative to gel purification. To reduce tandem insertions, phosphatase treatment of insert DNA was easily invoked without sacrificing cloning efficiency. The combined capabilities of direct selection, TAC purification and phosphatase treatment of inserts should facilitate library construction and improve overall library quality.

AUTOMATED DNA SEQUENCERS: THE NEXT GENERATION

Optically detected DNA sequencing technologies facilitate sequencing the 3 billion base pairs of the human genome. Faster, higher throughput optically detected electrophoresis instrumentation will be necessary to bring the cost of sequencing the entire genome down and to complete the project by 2005.