Discovery of a novel insecticidal protein from Chromobacterium piscinae, with activity against Western Corn Rootworm, Diabrotica virgifera virgifera.

Western corn rootworm (WCR), Diabrotica virgifera virgifera, is one of the most significant pests of corn in the United States. Although transgenic solutions exist, increasing resistance concerns make the discovery of novel solutions essential. In order to find a novel protein with high activity and a new mode of action, a large microbial collection was surveyed for toxicity to WCR using in vitro bioassays. Cultures of strain ATX2024, identified as Chromobacterium piscinae, had very high activity against WCR larvae. The biological activity from the strain was purified using chromatographic techniques and fractions were tested against WCR larvae. Proteins in the final active fraction were identified by mass spectrometry and N-terminal sequencing and matched to the genome of ATX2024. A novel 58.9kDa protein, identified by this approach, was expressed in a recombinant expression system and found to have specific activity against WCR. Transgenic corn events containing this gene showed good protection against root damage by WCR, with average scores ranging between 0.01 and 0.04 on the Iowa State node injury scale. Sequence analysis did not reveal homology to any known insecticidal toxin, suggesting that this protein may act in a novel way to control WCR. The new WCR active protein is named GNIP1Aa, for Gram Negative Insecticidal Protein.

Identification of polymorphic antioxidant response elements in the human genome.

Single nucleotide polymorphisms (SNPs) in transcription factor binding sites (TFBSs) may affect the binding of transcription factors, lead to differences in gene expression and phenotypes and therefore affect susceptibility to environmental exposure. We developed an integrated computational system for discovering functional SNPs in TFBSs in the human genome and predicting their impact on the expression of target genes. In this system, we (i) construct a position weight matrix (PWM) from a collection of experimentally discovered TFBSs; (ii) predict TFBSs in SNP sequences using the PWM and map SNPs to the upstream regions of genes; (iii) examine the evolutionary conservation of putative TFBSs by phylogenetic footprinting; (iv) prioritize candidate SNPs based on microarray expression profiles from tissues in which the transcription factor of interest is either deleted or over-expressed and (v) finally, analyze association of SNP genotypes with gene expression phenotypes. The application of our system has been tested to identify functional polymorphisms in the antioxidant response element (ARE), a cis-acting enhancer sequence found in the promoter region of many genes that encode antioxidant and Phase II detoxification enzymes/proteins. In response to oxidative stress, the transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) binds to AREs, mediating transcriptional activation of its responsive genes and modulating in vivo defense mechanisms against oxidative damage. Using our novel computational tools, we have identified a set of polymorphic AREs with functional evidence, showing the utility of our system to direct further experimental validation of genomic sequence variations that could be useful for identifying high-risk individuals.

Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes.

Single nucleotide polymorphisms (SNPs) in the human genome are DNA sequence variations that can alter an individual's response to environmental exposure. SNPs in gene coding regions can lead to changes in the biological properties of the encoded protein. In contrast, SNPs in non-coding gene regulatory regions may affect gene expression levels in an allele-specific manner, and these functional polymorphisms represent an important but relatively unexplored class of genetic variation. The main challenge in analyzing these SNPs is a lack of robust computational and experimental methods. Here, we first outline mechanisms by which genetic variation can impact gene regulation, and review recent findings in this area; then, we describe a methodology for bioinformatic discovery and functional analysis of regulatory SNPs in cis-regulatory regions using the assembled human genome sequence and databases on sequence polymorphism and gene expression. Our method integrates SNP and gene databases and uses a set of computer programs that allow us to: (1) select SNPs, from among the >9 million human SNPs in the NCBI dbSNP database, that are similar to cis-regulatory element (RE) consensus sequences; (2) map the selected dbSNP entries to the human genome assembly in order to identify polymorphic REs near gene start sites; (3) prioritize the candidate polymorphic RE containing genes by searching the existing genotype and gene expression data sets. The applicability of this system has been demonstrated through studies on p53 responsive elements and is being extended to additional pathways and environmentally responsive genes.

Functionally distinct polymorphic sequences in the human genome that are targets for p53 transactivation.

The p53 tumor suppressor protein is a master regulatory transcription factor that coordinates cellular responses to DNA damage and cellular stress. Besides mutations in p53, or in proteins involved in the p53 response pathway, genetic variation in promoter response elements (REs) of p53 target genes is expected to alter biological responses to stress. To identify SNPs in p53 REs that may modify p53-controlled gene expression, we developed an approach that combines a custom bioinformatics search to identify candidate SNPs with functional yeast and mammalian cell assays to assess their effect on p53 transactivation. Among approximately 2 million human SNPs, we identified >200 that seem to disrupt functional p53 REs. Eight of these SNPs were evaluated in functional assays to determine both the activity of the putative RE and the impact of the candidate SNPs on transactivation. All eight candidate REs were functional, and in every case the SNP pair exhibited differential transactivation capacities. Additionally, six of the eight genes adjacent to these SNPs are induced by genotoxic stress or are activated directly by transfection with p53 cDNA. Thus, this strategy efficiently identifies SNPs that may differentially affect gene expression responses in the p53 regulatory pathway.

Sequence context at human single nucleotide polymorphisms: overrepresentation of CpG dinucleotide at polymorphic sites and suppression of variation in CpG islands.

Human polymorphisms originate as mutations, and the influence of context on mutagenesis should be reflected in the distribution of sequences surrounding single nucleotide polymorphisms (SNPs). We have performed a computational survey of nearly two million human SNPs to determine if sequence-dependent hotspots for polymorphism exist in the human genome. Here we show that sequences containing CpG dinucleotides, which occur at low frequencies in the human genome, are 6.7-fold more abundant at polymorphic sites than expected. In contrast, polymorphisms in CpG sequences located within CpG islands, important regulatory regions that modulate gene expression, are 6.8-fold less prevalent than expected. The distribution of polymorphic alleles at CpGs in CpG islands is also significantly different from that in non-island regions. These data strongly support a role for 5-methylcytosine deamination in the generation of human variation, and suggest that variation at CpGs in islands is suppressed.