A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.     

Life extension through neurofibromin mitochondrial regulation and antioxidant therapy for neurofibromatosis-1 in Drosophila melanogaster

We investigated the pathophysiology of neurofibromatosis-1 (NF1) in Drosophila melanogaster by inactivation or overexpression of the NF1 gene. NF1 gene mutants had shortened life spans and increased vulnerability to heat and oxidative stress in association with reduced mitochondrial respiration and elevated reactive oxygen species (ROS) production. Flies overexpressing NF1 had increased life spans, improved reproductive fitness, increased resistance to oxidative and heat stress in association with increased mitochondrial respiration and a 60% reduction in ROS production. These phenotypic effects proved to be modulated by the adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A pathway, not the Ras/Raf pathway. Treatment of wild-type D. melanogaster with cAMP analogs increased their life span, and treatment of NF1 mutants with metalloporphyrin catalytic antioxidant compounds restored their life span. Thus, neurofibromin regulates longevity and stress resistance through cAMP regulation of mitochondrial respiration and ROS production, and NF1 may be treatable using catalytic antioxidants.     

Genome-wide association study identifies a susceptibility locus for thoracic aortic aneurysms and aortic dissections spanning FBN1 at 15q21.1

Although thoracic aortic aneurysms and dissections (TAAD) can be inherited as a single-gene disorder, the genetic predisposition in the majority of affected people is poorly understood. In a multistage genome-wide association study (GWAS), we compared 765 individuals who had sporadic TAAD (STAAD) with 874 controls and identified common SNPs at a 15q21.1 locus that were associated with STAAD, with odds ratios of 1.6-1.8 that achieved genome-wide significance. We followed up 107 SNPs associated with STAAD with P < 1 × 10(-5) in the region, in two separate STAAD cohorts. The associated SNPs fall into a large region of linkage disequilibrium encompassing FBN1, which encodes fibrillin-1. FBN1 mutations cause Marfan syndrome, whose major cardiovascular complication is TAAD. This study shows that common genetic variants at 15q21.1 that probably act via FBN1 are associated with STAAD, suggesting a common pathogenesis of aortic disease in Marfan syndrome and STAAD.     

Widespread and nonrandom distribution of DNA palindromes in cancer cells provides a structural platform for subsequent gene amplification

Breakage-fusion-bridge cycles contribute to chromosome instability and generate large DNA palindromes that facilitate gene amplification in human cancers. The prevalence of large DNA palindromes in cancer is not known. Here, by using a new microarray-based approach called genome-wide analysis of palindrome formation, we show that palindromes occur frequently and are widespread in human cancers. Individual tumors seem to have a nonrandom distribution of palindromes in their genomes, and a subset of palindromic loci is associated with gene amplification. This indicates that the location of palindromes in the cancer genome can serve as a structural platform that supports subsequent gene amplification. Genome-wide analysis of palindrome formation is a new approach to identify structural chromosome aberrations associated with cancer.     

Evidence for a relationship between Ehlers-Danlos type VII C in humans and bovine dermatosparaxis.

Ehlers-Danlos (ED) syndrome type VII is characterized by the accumulation of collagen precursors in connective tissues. ED VII A and B are caused by mutations in the genes of alpha 1 and alpha 2 collagen I which result in the disruption of the cleavage site of procollagen I N-proteinase. The existence of ED VII C in humans has been hypothesized on the basis of a disorder in cattle and sheep related to the absence of the enzyme. We now present evidence for the existence of this disease in humans, characterized by skin fragility, altered polymers seen as hieroglyphic pictures with electron microscopy, accumulation of p-N-alpha 1 and p-N-alpha 2 collagen type I in the dermis and absence of processing of the p-N-I polypeptides in fibroblast cultures.     

Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm

The Drosophila melanogaster lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. Here we show that an essential genetic switch that differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. Our findings suggest that there is a close parallel between the molecular mechanisms functioning in the D. melanogaster cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm.     

Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection

Many human Y-chromosomal deletions are thought to severely impair reproductive fitness, which precludes their transmission to the next generation and thus ensures their rarity in the population. Here we report a 1.6-Mb deletion that persists over generations and is sufficiently common to be considered a polymorphism. We hypothesized that this deletion might affect spermatogenesis because it removes almost half of the Y chromosome's AZFc region, a gene-rich segment that is critical for sperm production. An association study established that this deletion, called gr/gr, is a significant risk factor for spermatogenic failure. The gr/gr deletion has far lower penetrance with respect to spermatogenic failure than previously characterized Y-chromosomal deletions; it is often transmitted from father to son. By studying the distribution of gr/gr-deleted chromosomes across the branches of the Y chromosome's genealogical tree, we determined that this deletion arose independently at least 14 times in human history. We suggest that the existence of this deletion as a polymorphism reflects a balance between haploid selection, which culls gr/gr-deleted Y chromosomes from the population, and homologous recombination, which continues to generate new gr/gr deletions.     

Hot-stop PCR: a simple and general assay for linear quantitation of allele ratios

We have developed a simple, quantitative assay for measurement of allele ratios that circumvents the problem of heteroduplex formation skewing the results of restriction endonuclease digestion of PCR products. This assay, 'hot-stop PCR', involves addition of a radiolabelled PCR primer at the final cycle. We applied the assay to analysis of loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) in tumours.