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Title: An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

Abstract

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1];  [1];  [1];  [1];  [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3];  [1]; ORCiD logo [1]
  1. Carnegie Inst. for Science, Stanford, CA (United States). Dept. of Plant Biology
  2. Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Plant Biology
  3. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
Publication Date:
Research Org.:
Univ. of California, Los Angeles, CA (United States); Carnegie Inst. for Science, Stanford, CA (United States); Univ. of Minnesota, Minneapolis, MN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Science Foundation (NSF); National Inst. of Health (NIH) (United States)
OSTI Identifier:
1418625
Grant/Contract Number:  
FC02-02ER63421; MCB-1146621; R24 GM092473
Resource Type:
Accepted Manuscript
Journal Name:
Plant Cell
Additional Journal Information:
Journal Volume: 28; Journal Issue: 2; Journal ID: ISSN 1040-4651
Publisher:
American Society of Plant Biologists
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Li, Xiaobo, Zhang, Ru, Patena, Weronika, Gang, Spencer S., Blum, Sean R., Ivanova, Nina, Yue, Rebecca, Robertson, Jacob M., Lefebvre, Paul A., Fitz-Gibbon, Sorel T., Grossman, Arthur R., and Jonikas, Martin C. An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii. United States: N. p., 2016. Web. doi:10.1105/tpc.15.00465.
Li, Xiaobo, Zhang, Ru, Patena, Weronika, Gang, Spencer S., Blum, Sean R., Ivanova, Nina, Yue, Rebecca, Robertson, Jacob M., Lefebvre, Paul A., Fitz-Gibbon, Sorel T., Grossman, Arthur R., & Jonikas, Martin C. An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii. United States. doi:10.1105/tpc.15.00465.
Li, Xiaobo, Zhang, Ru, Patena, Weronika, Gang, Spencer S., Blum, Sean R., Ivanova, Nina, Yue, Rebecca, Robertson, Jacob M., Lefebvre, Paul A., Fitz-Gibbon, Sorel T., Grossman, Arthur R., and Jonikas, Martin C. Wed . "An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii". United States. doi:10.1105/tpc.15.00465. https://www.osti.gov/servlets/purl/1418625.
@article{osti_1418625,
title = {An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii},
author = {Li, Xiaobo and Zhang, Ru and Patena, Weronika and Gang, Spencer S. and Blum, Sean R. and Ivanova, Nina and Yue, Rebecca and Robertson, Jacob M. and Lefebvre, Paul A. and Fitz-Gibbon, Sorel T. and Grossman, Arthur R. and Jonikas, Martin C.},
abstractNote = {The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.},
doi = {10.1105/tpc.15.00465},
journal = {Plant Cell},
number = 2,
volume = 28,
place = {United States},
year = {2016},
month = {1}
}

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