High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in <em>Chlamydomonas reinhardtii</em>

Abstract

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii 1 . These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a widetarget screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

Interregulation of CDKA/CDK1 and the Plant-Specific Cyclin-Dependent Kinase CDKB in Control of the Chlamydomonas Cell Cycle

“…Its cell division cycle is rapid, continuous, and cultures can be synchronized. These features allowed isolation of temperature-sensitive lethal mutations inactivating diverse components in cell cycle control and execution (Tulin and Cross 2014;Breker et al 2016). Among the mutated genes were CDKA1 and CDKB1.…”

Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii

“…We began the study by isolating thousands of mutants harboring Ts-lethal mutations in essential genes using a highly efficient robotically assisted pipeline (Tulin and Cross, 2014;Breker et al, 2016). In order to enrich for mutagenized genes with potential cell cycle involvement, we used time-lapse microscopy to focus on mutants with little detectable problems with cell growth, but which fail to successfully complete a division cycle (Figure 1; Supplemental Figure 1) (assays and logic previously described in detail in Tulin and Cross, 2014;Breker et al, 2016). This quality control step reduces the number of mutants to be analyzed by a factor of ∼10 to 100, but still leaves many more mutants than could practically be sequence identified by the rather laborious bulked segregant sequence analysis strategy of Tulin and Cross (2014).…”

“…Second, some candidate causative lesions are in genes previously identified as bearing independent causative mutations. In such cases, we test the new mutant for failure to complement the previous mutation in heterozygous diploids and for failure to recombine to produce Ts+ progeny in meiosis (Breker et al, 2016). Third, we can select for reversion of temperature sensitivity and determine whether the revertants alter the coding sequence at or near the site of the candidate mutation, implying that this mutation was causative.…”

“…Medium was prepared as described (Dutcher, 1995;Harris, 2008). Mutant isolation, complementation, linkage, and reversion analysis was as described (Breker et al, 2016).…”

“…In one experiment, we detected ∼5700 genetic lesions in 28 sequenced specimens and uniquely assigned an average of ∼200 to each specimen ( Figure 2C). The number of lesions assigned per mutant varied from 87 to 386, due in part to diverse UV dosages during mutagenesis (Breker et al, 2016).…”