scholarly journals Contextual diversity of the human cell-essential proteome

2017 ◽  
Author(s):  
Thierry Bertomeu ◽  
Jasmin Coulombe-Huntington ◽  
Andrew Chatr-Aryamontri ◽  
Karine Bourdages ◽  
Yu Xia ◽  
...  
Keyword(s):  
Human Cell ◽  
Essential Gene ◽  
B Cell Lymphoma ◽  
Cell Types ◽  
Bimodal Distribution ◽  
Essential Genes ◽  
Analysis Tool ◽  
Contextual Diversity ◽  
Alternatively Spliced ◽  
Alternatively Spliced Exons

AbstractEssential genes define central biological functions required for cell growth, proliferation and survival, but the nature of gene essentiality across human cell types is not well understood. We assessed essential gene function in a Cas9-inducible human B-cell lymphoma cell line using an extended knockout (EKO) library of 278,754 sgRNAs that targeted 19,084 RefSeq genes, 20,852 alternatively-spliced exons and 3,872 hypothetical genes. A new statistical analysis tool called RANKS identified 2,280 essential genes, 234 of which had not been reported previously. Essential genes exhibited a bimodal distribution across 10 cell lines screened in different studies, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines were associated with more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes. Genes essential in fewer lines tended to form context-specific modules and encode subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively-spliced exons in essential genes were dispensable and tended to encode disordered regions. We also detected a significant fitness defect for 44 newly evolved hypothetical reading frames. These results illuminate the nature and evolution of essential gene functions in human cells.

scholarly journals A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome

2017 ◽  
Vol 38 (1) ◽  
Author(s):  
Thierry Bertomeu ◽  
Jasmin Coulombe-Huntington ◽  
Andrew Chatr-aryamontri ◽  
Karine G. Bourdages ◽  
Etienne Coyaud ◽  
...  
Keyword(s):  
Stress Responses ◽  
B Cell Lymphoma ◽  
Human Cells ◽  
Essential Genes ◽  
Analysis Tool ◽  
Contextual Diversity ◽  
Genome Wide ◽  
A Genome ◽  
Alternatively Spliced ◽  
Alternatively Spliced Exons

ABSTRACT To interrogate genes essential for cell growth, proliferation and survival in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribosome biogenesis and stress responses. Essential genes exhibited a bimodal distribution across 10 different cell lines, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively spliced exons in essential genes were dispensable and were enriched for disordered regions. Fitness defects were observed for 44 newly evolved hypothetical reading frames. These results illuminate the contextual nature and evolution of essential gene functions in human cells.

A novel way to purify recombinant baculoviruses by using bacmid

2009 ◽  
Vol 29 (2) ◽  
pp. 71-75 ◽  
Author(s):  
Wu-Jie Su ◽  
Wei-De Shen ◽  
Bing Li ◽  
Yan Wu ◽  
Guang Gao ◽  
...  
Keyword(s):  
Insect Cells ◽  
Nuclear Polyhedrosis Virus ◽  
Essential Gene ◽  
Essential Genes ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Recombinant Bacmid ◽  
Bmnpv Bacmid ◽  
Nuclear Polyhedrosis ◽  
Recombinant Bacmids

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

scholarly journals Profiling Bioactivity of the ToxCast Chemical Library Using BioMAP Primary Human Cell Systems

2009 ◽  
Vol 14 (9) ◽  
pp. 1054-1066 ◽  
Author(s):  
Keith A. Houck ◽  
David J. Dix ◽  
Richard S. Judson ◽  
Robert J. Kavlock ◽  
Jian Yang ◽  
...  
Keyword(s):  
Human Cell ◽  
Regulatory Networks ◽  
Cell Types ◽  
Environmental Chemicals ◽  
Data Sets ◽  
Chemical Library ◽  
Data Set ◽  
Cell Systems ◽  
Disease Biology ◽  
Nfκb Pathway

The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency’s (EPA’s) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems, with a total of 87 assay endpoints resulting in more than 100,000 data points. Within the context of the BioMAP database, ToxCast compounds could be classified based on their ability to cause overt cytotoxicity in primary human cell types or according to toxicity mechanism class derived from comparisons to activity profiles of BioMAP reference compounds. ToxCast chemicals with similarity to inducers of mitochondrial dysfunction, cAMP elevators, inhibitors of tubulin function, inducers of endoplasmic reticulum stress, or NFκB pathway inhibitors were identified based on this BioMAP analysis. This data set is being combined with additional ToxCast data sets for development of predictive toxicity models at the EPA. ( Journal of Biomolecular Screening 2009:1054-1066)

scholarly journals Positive Selection in Alternatively Spliced Exons of Human Genes

2008 ◽  
Vol 83 (1) ◽  
pp. 94-98 ◽  
Author(s):  
Vasily E. Ramensky ◽  
Ramil N. Nurtdinov ◽  
Alexei D. Neverov ◽  
Andrei A. Mironov ◽  
Mikhail S. Gelfand
Keyword(s):  
Positive Selection ◽  
Human Genes ◽  
Alternatively Spliced ◽  
Alternatively Spliced Exons

Elements regulating an alternatively spliced exon of the rat fibronectin gene

1993 ◽  
Vol 13 (9) ◽  
pp. 5301-5314 ◽  
Author(s):  
G S Huh ◽  
R O Hynes
Keyword(s):  
Splice Sites ◽  
Cell Type ◽  
Long Distance ◽  
Sequence Elements ◽  
Alternatively Spliced ◽  
A Cell ◽  
Cell Type Specific ◽  
Alternatively Spliced Exons

We have investigated the regulation of splicing of one of the alternatively spliced exons in the rat fibronectin gene, the EIIIB exon. This 273-nucleotide exon is excluded by some cells and included to various degrees by others. We find that EIIIB is intrinsically poorly spliced and that both its exon sequences and its splice sites contribute to its poor recognition. Therefore, cells which recognize the EIIIB exon must have mechanisms for improving its splicing. Furthermore, in order for EIIB to be regulated, a balance must exist between the EIIIB splice sites and those of its flanking exons. Although the intron upstream of EIIIB does not appear to play a role in the recognition of EIIIB for splicing, the intron downstream contains sequence elements which can promote EIIIB recognition in a cell-type-specific fashion. These elements are located an unusually long distance from the exon that they regulate, more than 518 nucleotides downstream from EIIIB, and may represent a novel mode of exon regulation.

Label-free RF biosensors for human cell dielectric spectroscopy

2009 ◽  
Vol 1 (6) ◽  
pp. 497-504 ◽  
Author(s):  
Claire Dalmay ◽  
Arnaud Pothier ◽  
Mathilde Cheray ◽  
Fabrice Lalloue ◽  
Marie-Odile Jauberteau ◽  
...  
Keyword(s):  
Human Cell ◽  
Cell Types ◽  
Label Free ◽  
Microwave Frequencies ◽  
Non Invasive ◽  
System Cell ◽  
Electromagnetic Characteristics ◽  
Biosensor Chip ◽  
Rf Signal

This paper presents an original biosensor chip allowing determination of intrinsic relative permittivity of biological cells at microwave frequencies. This sensor permits non-invasive cell identification and discrimination using an RF signal to probe intracellular medium of biological samples. Indeed, these sensors use an RF planar resonator that allows detection capabilities on less than 10 cells, thanks to the microscopic size of its sensitive area. Especially, measurements between 15 and 35 GHz show the ability label-free biosensors to differentiate two human cell types using their own electromagnetic characteristics. The real part of permittivity of cells changes from 20 to 48 for the nervous system cell types studied. The proposed biodetection method is detailed and we show how the accuracy and the repeatability of measurements have been improved to reach reproducible measurements.

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