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.     

Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration

Calorie restriction (CR) extends lifespan in a wide spectrum of organisms and is the only regimen known to lengthen the lifespan of mammals. We established a model of CR in budding yeast Saccharomyces cerevisiae. In this system, lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-dependent kinase (PKA). Lifespan extension in a mutant with reduced PKA activity requires Sir2 and NAD (nicotinamide adenine dinucleotide). In this study we explore how CR activates Sir2 to extend lifespan. Here we show that the shunting of carbon metabolism toward the mitochondrial tricarboxylic acid cycle and the concomitant increase in respiration play a central part in this process. We discuss how this metabolic strategy may apply to CR in animals.     

Semaphorin 7A promotes axon outgrowth through integrins and MAPKs

Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.     

The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.     

The genome sequence of the filamentous fungus Neurospora crassa

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes--more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca2+ signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.     

Genome sequence and analysis of the tuber crop potato

Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop.     

Human trampling effects on regeneration and age structures of Pinus edulis and Juniperus monosperma

We examined effects of human foot traffic on age structures and densities of seedlings and saplings of Pinus edulis (Colorado pinyon) and Junisperus monosperma (one-seed juniper) in a heavily used urban park, Garden of the Gods, Colorado. Age structures show no stand-destroying disturbances, but they do contain small peaks 85-95 yr ago (minimum age), which have been interpreted as responses to heavy grazing. For Pinus edulis reverse J-shaped age structures indicate a strongly reproducing population, while flat age structures of J. monosperma show low present reproduction. Young trees showed strong preferences for establishing under existing trees and shrubs and not among herbs or on bare soil. Pinus edulis seedling density was reduced by 73% in heavily trampled areas compared to lightly trampled areas. However, there were no differences in density when only the area protected by rocks, shrubs, or trees was considered. This indicates that direct effects such as physical damage and soil erosion kill young trees, and indirect effects, such as lower seed production, do not cause the lower densities in heavily trampled areas. Pinus edulis saplings and J. monosperma seedlings and saplings showed no differences in density across trampling intensities. In heavily trampled areas of Garden of the Gods Park, recent increases in use have apparently reduced Pinus edulis seedling establishment enough that long-term regeneration is threatened. Managers of all pinyon-juniper woodlands must recognize that in areas strongly impacted by foot traffic, and also presumably by similar disturbances such as vehicle traffic, sufficient regeneration likely does not occur to replace trees. The areal extent of severely disturbed areas should be limited, and managers should seek to avoid further degradation of less damaged areas.     

Quantum phase transition in a resonant level coupled to interacting leads

A Luttinger liquid is an interacting one-dimensional electronic system, quite distinct from the 'conventional' Fermi liquids formed by interacting electrons in two and three dimensions. Some of the most striking properties of Luttinger liquids are revealed in the process of electron tunnelling. For example, as a function of the applied bias voltage or temperature, the tunnelling current exhibits a non-trivial power-law suppression. (There is no such suppression in a conventional Fermi liquid.) Here, using a carbon nanotube connected to resistive leads, we create a system that emulates tunnelling in a Luttinger liquid, by controlling the interaction of the tunnelling electron with its environment. We further replace a single tunnelling barrier with a double-barrier, resonant-level structure and investigate resonant tunnelling between Luttinger liquids. At low temperatures, we observe perfect transparency of the resonant level embedded in the interacting environment, and the width of the resonance tends to zero. We argue that this behaviour results from many-body physics of interacting electrons, and signals the presence of a quantum phase transition. Given that many parameters, including the interaction strength, can be precisely controlled in our samples, this is an attractive model system for studying quantum critical phenomena in general, with wide-reaching implications for understanding quantum phase transitions in more complex systems, such as cold atoms and strongly correlated bulk materials.