Carette_Science_2009 - Haploid Genetic Screens in Human...

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Unformatted text preview: Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens Jan E. Carette, 1 Carla P. Guimaraes, 1 Malini Varadarajan, 1 Annie S. Park, 1 Irene Wuethrich, 1 Alzbeta Godarova, 1 Maciej Kotecki, 2 Brent H. Cochran, 2 Eric Spooner, 1 Hidde L. Ploegh, 1,3 Thijn R. Brummelkamp 1 * Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast. I dentification of gene products that play a role in human disease drives much of today ’ s biomedical research. Classical genetics with induced mutations, as pioneered by Muller in 1927 ( 1 ), is the most powerful unbiased approach to elucidate the genetic components that underlie biological processes. The study of cultured human cells allows the recapitulation of many essential elements of human disease. However, the inability to efficiently generate and recover bi-allelic mutants in human diploid cells limits the con- tribution of mutagenesis-based genetics to the understanding of human disease. The identification of cellular genes exploited by bacteria and viruses is essential to elucidate the mechanisms by which these pathogens cause disease ( 2 – 4 ). Bacterial toxins contribute greatly to pathogenicity of the microbes that produce them. Identification of host proteins involved in toxin cytotoxicity should help to identify targets for therapeutic intervention in diseases caused by bacteria, many of which now show increased resistance to conventional antibiotics. Likewise, an understanding of how viruses depend on host proteins to enter the cell, replicate their genome, and spread may accelerate the development of antiviral drugs. Influenza virus remains a threat to human health, causing several hundred thou- sands of deaths annually and many more in the course of a pandemic ( 5 ). The rapid spread of new strains of influenza A [for instance, avian (H5N1) and swine (H1N1) influenza] and the emergence of drug-resistant influenza strains ( 6 ) limit the effectiveness of vaccines and current antiviral therapeutics. Thus, we developed a method for genetic screens in human cells and isolated genes required for the action of several bacterial toxins and influenza viruses....
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This note was uploaded on 11/01/2010 for the course A B taught by Professor C during the Spring '10 term at HKU.

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Carette_Science_2009 - Haploid Genetic Screens in Human...

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