The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300 -400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.hole-genome sequences provide the foundation for the creation of relatively complete collections of strains carrying defined mutations in individual genes. Such libraries should facilitate the comprehensive identification of genes required for a wide range of biological processes. A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach (1). Other projects, also following a strategy of gene-by-gene disruption, are underway for Escherichia coli (E. coli genome project, www. genome.wisc.edu͞functional͞tnmutagenesis.htm), and have recently been completed for Bacillus subtilis (2).An alternative strategy for generating mutant libraries consists of ''random'' whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites. The approach is cost-effective and applicable to a wide variety of microbes (3, 4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5). Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth, although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined (6)(7)(8). In this report, we describe the generation and initial phenotypic analysis of a near-saturation library of transposon insertion mutants of the opportunistic pathogen Pseudomonas aeruginos...

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H+-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.

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