We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells 1 . Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P 2 ), a phospholipid present in small quantities that regulates membrane trafficking in the endosome-lysosome axis in yeast 2 . Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2β (early transposon 2β) 3 into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC (suppressor of actin) domain PtdIns(3,5)P 2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns(3,5)P 2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosomelysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated

Membrane fusion and fi ssion events in intracellular traffi cking are controlled by both intraluminal Ca 2 + release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca 2 + release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P 2 , an endolysosome-specifi c PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specifi city and potency. Both PI(3,5)P 2 -defi cient cells and cells that lack TRPML1 exhibited enlarged endolysosomes / vacuoles and traffi cking defects in the late endocytic pathway. We fi nd that the enlarged vacuole phenotype observed in PI(3,5)P 2 -defi cient mouse fi broblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P 2 -dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca 2 + release from the vacuole. In this study, we show that this release requires both PI(3,5)P 2 production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane traffi cking by transducing information regarding PI(3,5)P 2 levels into changes in juxtaorganellar Ca 2 + , thereby triggering membrane fusion / fi ssion events.

Protein localization to membrane-enclosed organelles is a central feature of cellular organization. Using protein correlation profiling, we have mapped 1,404 proteins to ten subcellular locations in mouse liver, and these correspond with enzymatic assays, marker protein profiles, and confocal microscopy. These localizations allowed assessment of the specificity in published organellar proteomic inventories and demonstrate multiple locations for 39% of all organellar proteins. Integration of proteomic and genomic data enabled us to identify networks of coexpressed genes, cis-regulatory motifs, and putative transcriptional regulators involved in organelle biogenesis. Our analysis ties biochemistry, cell biology, and genomics into a common framework for organelle analysis.