Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, we propose the use of NotI clone microarrays and genomic subtraction, because NotI recognition sites are closely associated with CpG islands and genes. We show here that the CODE (Cloning Of DEleted sequences) genomic subtraction procedure can be adapted to NotI flanking sequences and to CpG islands. Because the sequence complexity of this procedure is greatly reduced, only two cycles of subtraction are required. A NotI-CODE procedure can be used to prepare NotI representations (NRs) containing 0.1-0.5% of the total DNA. The NRs contain, on average, 10-fold less repetitive sequences than the whole human genome and can be used as probes for hybridization to NotI microarrays. These microarrays, when probed with NRs, can simultaneously detect copy number changes and methylation. NotI microarrays offer a powerful tool with which to study carcinogenesis. R epresentational difference analysis (RDA) (1) and restriction fragment length polymorphism subtraction (2) were reproducibly successful in cloning deleted sequences. However, these methods are sensitive to minor impurities, are laborious, and suffer from a number of limitations (e.g., the inability to detect differences due to point mutations, small deletions, or insertions). Furthermore, the PCR amplification after the first hybridization step and before the nuclease treatment may give rise to artifacts. Excess driver DNA can result in reduced efficiency in amplification of the tester-tester duplexes because of the potential formation of residual driver-driver and drivertester duplexes that act as competitors. As RDA is based mainly on the specific amplification of the desired products and requires 95-110 PCR cycles, it suffers from a ''plateau effect'' that is characterized by a decline in the exponential rate of accumulation of amplification products. However, the major problem results from the inefficiency of the multiple restriction digestion and ligation reactions that are used in this method, and which lead to the generation of false positives. Furthermore, these experiments result in the cloning of products that usually do not represent functional genes. Similarly, the methylation-sensitive representational difference analysis (3), aimed at CpG-rich sequences, suffers from the same limitations as the original RDA.Recently, we developed a procedure for cloning deleted sequences (Cloning Of DEleted sequences, CODE) (4) that is free from some of the limitations inherent in the RDA and restriction fragment length polymorphism subtraction protocols. Our major objective was to improve the subtractive enrichment, thereby avoiding excessive PCR kinetic enrichment steps that often generate small DNA products.

The mechanism(s) by which DNA vaccines trigger the activation of Ag-specific T cells is incompletely understood. A series of in vivo and in vitro experiments indicates plasmid transfection stimulates muscle cells to up-regulate expression of MHC class I and costimulatory molecules and to produce multiple cytokines and chemokines. Transfected muscle cells gain the ability to directly present Ag to CD8 T cells through an IFN-regulatory factor 3-dependent process. These findings suggest that transfected muscle cells at the site of DNA vaccination may contribute to the magnitude and/or duration of the immune response initiated by professional APCs.

The innate immune system is triggered when pathogen associated molecular patterns (PAMPs) expressed by infectious microorganisms interact with toll-like receptors (TLR) present on immune cells. Individual TLRs signal through distinct molecular pathways. For example, TLR9 interacts with unmethylated CpG motifs expressed by bacterial DNA and triggers via a MyD88 dependent pathway whereas TLR3 recognizes viral RNA through a MyD88-independent pathway. Bioinformatic analysis of microarray data was used to identify the regulatory patterns underlying changes in gene expression induced when RAW 264.7 macrophages were stimulated via TLR9 by CpG oligonucleotides (ODN) and/or via TLR3 by poly (I:C). While the genes activated by each ligand mediated similar functions, poly (I:C) elicited a larger and more diverse change in gene expression. Co-stimulation with both ligands accelerated gene expression and synergistically activated genes primarily associated with immune function. This is the first work to compare global changes in gene regulation triggered by distinct TLR pathways and clarify their impact on gene expression.

Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.

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