Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries. DNA engineering is conducted routinely in Escherichia coli, not only for genetic studies in bacteria, but also for constructing DNA molecules to be used in studies of other organisms. Most cloning methods use restriction endonuclease cleavage followed by DNA joining with DNA ligase. These in vitro cleavage and joining reactions are the basis for creating DNA recombinants and for cloning of DNA segments on plasmid vectors in which additional modification and amplification can take place. In the yeast Saccharomyces cerevisiae, strategies that generate novel DNA junctions exploit homologous recombination (1). The DNA double-strand break and repair recombination pathway is very efficient in yeast. Functions in this pathway recombine transformed linear DNA with homologous DNA in the yeast cell. Moreover, this recombination is proficient with short synthetic oligonucleotides, thereby permitting recombinant DNA to be generated in vivo without using DNA restriction and ligation (2-4). However, subsequent manipulation of recombinant DNA molecules in yeast, as opposed to E. coli, is laborious, especially if it is to be used for recombinant DNA work in other organisms.Unlike yeast, E. coli is not readily transformed by linear DNA fragments due in part to the rapid degradation of the DNA by the intracellular RecBCD exonuclease (5). Mutant recBCD strains lacking the exonuclease do not rapidly degrade linear DNA; however, such strains are extremely poor growing, are defective for recombination, and do not support efficient replication of the many plasmids used in recombinant DNA work. In certain recA ϩ backgrounds, the recBCD defect for recombination is suppressed, allowing linear DNA to be taken up by the cell and to be recombined (6). Other homologous recombination strategies in E. coli recBC derivatives hav...

Fas antigen is a cell-surface protein that mediates apoptosis. It is expressed in various tissues including the thymus and has structural homology with a number of cell-surface receptors, including tumour necrosis factor receptor and nerve growth factor receptor. Mice carrying the lymphoproliferation (lpr) mutation have defects in the Fas antigen gene. The lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus-like autoimmune disease, indicating an important role for Fas antigen in the negative selection of autoreactive T cells in the thymus.

Amyloid precursor protein (APP) is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides (Abeta40 and 42) that aggregate to form senile plaques in the brains of patients with Alzheimer's disease (AD). The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin (PS 1 or 2). Missense mutations in PS1 (and PS2) occur in patients with early-onset familial AD (FAD), and previous studies in transgenic mice and cultured cell models demonstrated that FAD-PS1 variants shift the ratio of Abeta40 : 42 to favor Abeta42. One hypothesis to explain this outcome is that mutant PS alters the specificity of gamma-secretase to favor production of Abeta42 at the expense of Abeta40. To test this hypothesis in vivo, we studied Abeta40 and 42 levels in a series of transgenic mice that co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain. We demonstrate a direct correlation between the concentration of Abeta42 and the rate of amyloid deposition. We further show that the shift in Abeta42 : 40 ratios associated with the expression of FAD-PS1 variants is due to a specific elevation in the steady-state levels of Abeta42, while maintaining a constant level of Abeta40. These data suggest that PS1 variants do not simply alter the preferred cleavage site for gamma-secretase, but rather that they have more complex effects on the regulation of gamma-secretase and its access to substrates.