Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays

Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis

Identification of differences in human and great ape phytanic acid metabolism that could influence gene expression profiles and physiological functions

<p>Abstract</p> <p>Background</p> <p>It has been proposed that anatomical differences in human and great ape guts arose in response to species-specific diets and energy demands. To investigate functional genomic consequences of these differences, we compared their physiological levels of phytanic acid, a branched chain fatty acid that can be derived from the microbial degradation of chlorophyll in ruminant guts. Humans who accumulate large stores of phytanic acid commonly develop cerebellar ataxia, peripheral polyneuropathy, and retinitis pigmentosa in addition to other medical conditions. Furthermore, phytanic acid is an activator of the PPAR-alpha transcription factor that influences the expression of genes relevant to lipid metabolism.</p> <p>Results</p> <p>Despite their trace dietary phytanic acid intake, all great ape species had elevated red blood cell (RBC) phytanic acid levels relative to humans on diverse diets. Unlike humans, chimpanzees showed sexual dimorphism in RBC phytanic acid levels, which were higher in males relative to females. Cultured skin fibroblasts from all species had a robust capacity to degrade phytanic acid. We provide indirect evidence that great apes, in contrast to humans, derive significant amounts of phytanic acid from the hindgut fermentation of plant materials. This would represent a novel reduction of metabolic activity in humans relative to the great apes.</p> <p>Conclusion</p> <p>We identified differences in the physiological levels of phytanic acid in humans and great apes and propose this is causally related to their gut anatomies and microbiomes. Phytanic acid levels could contribute to cross-species and sex-specific differences in human and great ape transcriptomes, especially those related to lipid metabolism. Based on the medical conditions caused by phytanic acid accumulation, we suggest that differences in phytanic acid metabolism could influence the functions of human and great ape nervous, cardiovascular, and skeletal systems.</p

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22 208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A−3 = 42%, G−3 = 36% and G+4 = 47%), only 37.4% of the genes adhere to the purine (R)−3/G+4 rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R−3 and G+4, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A+1T+2G+3 initiation codons adhered much more strongly to the R−3/G+4 rule. Among the intron-less histone genes, only the H3 genes adhere to the R−3/G+4 rule, while the H1, H2A, H2B and H4 genes usually lack either R−3 or G+4. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A+1→T+1 mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LR

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22 208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A(−3) = 42%, G(−3) = 36% and G(+4) = 47%), only 37.4% of the genes adhere to the purine (R)(−3)/G(+4) rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R(−3) and G(+4), potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A(+1)T(+2)G(+3) initiation codons adhered much more strongly to the R(−3)/G(+4) rule. Among the intron-less histone genes, only the H3 genes adhere to the R(−3)/G(+4) rule, while the H1, H2A, H2B and H4 genes usually lack either R(−3) or G(+4). To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A(+1)→T(+1) mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS

Synthesis and biologic properties of hydrophilic sapphyrins, a new class of tumor-selective inhibitors of gene expression

BACKGROUND: Sapphyrin analogues and related porphyrin-like species have attracted attention as anticancer agents due to their selective localization in various cancers, including hematologic malignancies, relative to surrounding tissues. Sapphyrins are electron affinic compounds that generate high yields of singlet oxygen formation. Although initially explored in the context of photodynamic therapy, sapphyrins have intrinsic anticancer activity that is independent of their photosensitizing properties. However, the mechanisms for their anticancer activity have not been fully elucidated. RESULTS: We have prepared a series of hydrophilic sapphyrins and evaluated their effect on proliferation, uptake, and cell death in adherent human lung (A549) and prostate (PC3) cancer cell lines and in an A549 xenograft tumor model. PCI-2050, the sapphyrin derivative with the highest in vitro growth inhibitory activity, significantly lowered 5-bromo-2'-deoxyuridine incorporation in S-phase A549 cells by 60% within eight hours and increased levels of reactive oxygen species within four hours. The growth inhibition pattern of PCI-2050 in the National Cancer Institute 60 cell line screen correlated most closely using the COMPARE algorithm with known transcriptional or translational inhibitors. Gene expression analyses conducted on A549 plateau phase cultures treated with PCI-2050 uncovered wide-spread decreases in mRNA levels, which especially affected short-lived transcripts. Intriguingly, PCI-2050 increased the levels of transcripts involved in RNA processing and trafficking, transcriptional regulation, and chromatin remodeling. We propose that these changes reflect the activation of cellular processes aimed at countering the observed wide-spread reductions in transcript levels. In our A549 xenograft model, the two lead compounds, PCI-2050 and PCI-2022, showed similar tumor distributions despite differences in plasma and kidney level profiles. This provides a possible explanation for the better tolerance of PCI-2022 relative to PCI-2050. CONCLUSION: Hydrophilic sapphyrins were found to display promise as novel agents that localize to tumors, generate oxidative stress, and inhibit gene expression

Improving the sensitivity and specificity of gene expression analysis in highly related organisms through the use of electronic masks

DNA microarrays are powerful tools for comparing gene expression profiles from closely related organisms. However, a single microarray design is frequently used in these studies. Therefore, the levels of certain transcripts can be grossly underestimated due to sequence differences between the transcripts and the arrayed DNA probes. Here, we seek to improve the sensitivity and specificity of oligonucleotide microarray-based gene expression analysis by using genomic sequence information to predict the hybridization efficiency of orthologous transcripts to a given microarray. To test our approach, we examine hybridization patterns from three Escherichia coli strains on E.coli K-12 MG1655 gene expression microarrays. We create electronic mask files to discard data from probes predicted to have poor hybridization sensitivity and specificity to cDNA targets from each strain. We increased the accuracy of gene expression analysis and identified genes that cannot be accurately interrogated in each strain using these microarrays. Overall, these studies provide guidelines for designing effective electronic masks for gene expression analysis in organisms where substantial genome sequence information is available

Diverse captive non-human primates with phytanic acid-deficient diets rich in plant products have substantial phytanic acid levels in their red blood cells

<p>Abstract</p> <p>Background</p> <p>Humans and rodents with impaired phytanic acid (PA) metabolism can accumulate toxic stores of PA that have deleterious effects on multiple organ systems. Ruminants and certain fish obtain PA from the microbial degradation of dietary chlorophyll and/or through chlorophyll-derived precursors. In contrast, humans cannot derive PA from chlorophyll and instead normally obtain it only from meat, dairy, and fish products.</p> <p>Results</p> <p>Captive apes and Old world monkeys had significantly higher red blood cell (RBC) PA levels relative to humans when all subjects were fed PA-deficient diets. Given the adverse health effects resulting from PA over accumulation, we investigated the molecular evolution of thirteen PA metabolism genes in apes, Old world monkeys, and New world monkeys. All non-human primate (NHP) orthologs are predicted to encode full-length proteins with the marmoset <it>Phyh</it> gene containing a rare, but functional, GA splice donor dinucleotide. <it>Acox2</it>, <it>Scp2</it>, and <it>Pecr</it> sequences had amino acid positions with accelerated substitution rates while <it>Amacr</it> had significant variation in evolutionary rates in apes relative to other primates.</p> <p>Conclusions</p> <p>Unlike humans, diverse captive NHPs with PA-deficient diets rich in plant products have substantial RBC PA levels. The favored hypothesis is that NHPs can derive significant amounts of PA from the degradation of ingested chlorophyll through gut fermentation. If correct, this raises the possibility that RBC PA levels could serve as a biomarker for evaluating the digestive health of captive NHPs. Furthermore, the evolutionary rates of the several genes relevant to PA metabolism provide candidate genetic adaptations to NHP diets.</p