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ABSTRACT: Classical forward genetics has been foundational to modern biology, and has been the paradigm for characterizing the role of genes in shaping phenotypes for decades. In recent years, reverse genetics has been used to identify the functions of genes, via the intentional introduction of variation and subsequent evaluation in physiological, molecular, and even population contexts. These approaches are complementary and whole genome analysis serves as a bridge between the two. We report in this article the whole genome sequencing of eighteen classical mutant strains of Neurospora crassa and the putative identification of the mutations associated with corresponding mutant phenotypes. Although some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype. For ten of the mutants, the mutant phenotype is recapitulated in classical or gene deletion mutants in Neurospora or other filamentous fungi. From thirteen to 137 nonsense mutations are present in each strain and indel sizes are shown to be highly skewed in gene coding sequence. Significant additional genetic variation was found in the eighteen mutant strains, and this variability defines multiple alleles of many genes. These alleles may be useful in further genetic and molecular analysis of known and yet-to-be-discovered functions and they invite new interpretations of molecular and genetic interactions in classical mutant strains.
G3 (Bethesda, Md.). 09/2011; 1(4):303-16.
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Viviana Cordeddu,
Elia Di Schiavi,
Len A Pennacchio,
Avi Ma'ayan,
Anna Sarkozy,
Valentina Fodale,
Serena Cecchetti,
Alessio Cardinale, Joel Martin,
Wendy Schackwitz, [......],
Angelo Selicorni,
Cesare Rossi,
Romano Tenconi,
Martin Zenker,
Daniela Merlo,
Bruno Dallapiccola,
Ravi Iyengar,
Paolo Bazzicalupo,
Bruce D Gelb,
Marco Tartaglia
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ABSTRACT: N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2(S2G) in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2(S2G) in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.
Nature Genetics 09/2009; 41(9):1022-6. · 35.53 Impact Factor
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Stéphane Le Crom,
Wendy Schackwitz,
Len Pennacchio,
Jon K Magnuson,
David E Culley,
James R Collett, Joel Martin,
Irina S Druzhinina,
Hugues Mathis,
Frédéric Monot,
Bernhard Seiboth,
Barbara Cherry,
Michael Rey,
Randy Berka,
Christian P Kubicek,
Scott E Baker,
Antoine Margeot
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ABSTRACT: Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.
Proceedings of the National Academy of Sciences 09/2009; 106(38):16151-6. · 9.68 Impact Factor
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Dennis R Harris,
Steve V Pollock,
Elizabeth A Wood,
Reece J Goiffon,
Audrey J Klingele,
Eric L Cabot,
Wendy Schackwitz, Joel Martin,
Julie Eggington,
Timothy J Durfee, [......],
Michael C Popelars,
Hao Li,
Sarit A Klugman,
Lindsay L Hamilton,
Lukas B Bane,
Len A Pennacchio,
Thomas J Albert,
Nicole T Perna,
Michael M Cox,
John R Battista
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ABSTRACT: We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D(37) values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.
Journal of bacteriology 07/2009; 191(16):5240-52. · 3.94 Impact Factor
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Zhen Zhang,
Deanne Alpert,
Richard Francis,
Bishwanath Chatterjee,
Qing Yu,
Terry Tansey,
Steven L Sabol,
Cheng Cui,
Yongli Bai,
Maxim Koriabine,
Yuko Yoshinaga,
Jan-Fang Cheng,
Feng Chen, Joel Martin,
Wendy Schackwitz,
Teresa M Gunn,
Kenneth L Kramer,
Pieter J De Jong,
Len A Pennacchio,
Cecilia W Lo
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ABSTRACT: Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENU-induced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8(C193R) mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.
Proceedings of the National Academy of Sciences 03/2009; 106(9):3219-24. · 9.68 Impact Factor
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Nadav Ahituv,
Nihan Kavaslar,
Wendy Schackwitz,
Anna Ustaszewska, Joel Martin,
Sybil Hebert,
Heather Doelle,
Baran Ersoy,
Gregory Kryukov,
Steffen Schmidt,
Nir Yosef,
Eytan Ruppin,
Roded Sharan,
Christian Vaisse,
Shamil Sunyaev,
Robert Dent,
Jonathan Cohen,
Ruth McPherson,
Len A Pennacchio
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ABSTRACT: Body weight is a quantitative trait with significant heritability in humans. To identify potential genetic contributors to this phenotype, we resequenced the coding exons and splice junctions of 58 genes in 379 obese and 378 lean individuals. Our 96-Mb survey included 21 genes associated with monogenic forms of obesity in humans or mice, as well as 37 genes that function in body weight-related pathways. We found that the monogenic obesity-associated gene group was enriched for rare nonsynonymous variants unique to the obese population compared with the lean population. In addition, computational analysis predicted a greater fraction of deleterious variants within the obese cohort. Together, these data suggest that multiple rare alleles contribute to obesity in the population and provide a medical sequencing-based approach to detect them.
The American Journal of Human Genetics 05/2007; 80(4):779-91. · 10.60 Impact Factor
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Marco Tartaglia,
Len A Pennacchio,
Chen Zhao,
Kamlesh K Yadav,
Valentina Fodale,
Anna Sarkozy,
Bhaswati Pandit,
Kimihiko Oishi,
Simone Martinelli,
Wendy Schackwitz, [......],
Cinzia Neri,
Isabella Vasta,
Kate Gibson,
Cynthia J Curry,
Juan Pedro López Siguero,
Maria Cristina Digilio,
Giuseppe Zampino,
Bruno Dallapiccola,
Dafna Bar-Sagi,
Bruce D Gelb
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ABSTRACT: Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.
Nature Genetics 02/2007; 39(1):75-9. · 35.53 Impact Factor
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Joel Martin,
Cliff Han,
Laurie A Gordon,
Astrid Terry,
Shyam Prabhakar,
Xinwei She,
Gary Xie,
Uffe Hellsten,
Yee Man Chan,
Michael Altherr, [......],
Jeremy Schmutz,
Jane Grimwood,
Paul Richardson,
Daniel S Rokhsar,
Evan E Eichler,
Paul Gilna,
Susan M Lucas,
Richard M Myers,
Edward M Rubin,
Len A Pennacchio
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ABSTRACT: Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.
Nature 01/2005; 432(7020):988-94. · 36.28 Impact Factor
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Joel Martin,
Cliff Han,
Laurie A. Gordon,
Astrid Terry,
Shyam Prabhakar,
Xinwei She,
Gary Xie,
Uffe Hellsten,
Yee Man Chan,
Michael Altherr, [......],
Jeremy Schmutz,
Jane Grimwood,
Paul Richardson,
Daniel S. Rokhsar,
Evan E. Eichler,
Paul Gilna,
Susan M. Lucas,
Richard M. Myers,
Edward M. Rubin,
Len A. Pennacchio
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ABSTRACT: Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.
Nature 12/2004; 432(7020):988-994. · 36.28 Impact Factor
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Jeremy Schmutz, Joel Martin,
Astrid Terry,
Olivier Couronne,
Jane Grimwood,
Steve Lowry,
Laurie A. Gordon,
Duncan Scott,
Gary Xie,
Wayne Huang, [......],
Mark Dickson,
Jan-Fang Cheng,
Evan E. Eichler,
Anne Olsen,
Len A. Pennacchio,
Daniel S. Rokhsar,
Paul Richardson,
Susan M. Lucas,
Richard M. Myers,
Edward M. Rubin
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ABSTRACT: Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.
Nature 09/2004; 431(7006):268-274. · 36.28 Impact Factor
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Marco Tartaglia,
Len A Pennacchio,
Chen Zhao,
Kamlesh K Yadav,
Valentina Fodale,
Anna Sarkozy,
Bhaswati Pandit,
Kimihiko Oishi,
Simone Martinelli,
Wendy Schackwitz, [......],
Cinzia Neri,
Isabella Vasta,
Kate Gibson,
Cynthia J Curry,
Juan Pedro López Siguero,
Maria Cristina Digilio,
Giuseppe Zampino,
Bruno Dallapiccola,
Dafna Bar-Sagi,
Bruce D Gelb
