A Survey of New Temperature-Sensitive, Embryonic-Lethal Mutations in C. elegans: 24 Alleles of Thirteen Genes

Abstract

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.

Structure of the C. elegans ZYG-1 Cryptic Polo Box Suggests a Conserved Mechanism for Centriolar Docking of Plk4 Kinases

“…In contrast, very few conserved hydrophilic residues are present on the reverse side of the dimer (Figure 1G, H). Nine of thirteen temperature sensitive ( ts) zyg-1 mutations (O’Connell et al, 2001; Kemp et al, 2007; O’Rourke et al, 2011) are in the CPB (Figure S1A, B). In contrast to the conserved basic residues, which cluster near the dimer midline, the majority of ts CPB mutations (8/9) are in non-conserved residues in PB1 near the dimer periphery (Figure S1C, D).…”

The million mutation project: A new approach to genetics in Caenorhabditis elegans

“…Strains would be stable and genes across the genome would be targets. Because of their potential value we have acquired temperature-sensitive embryonic lethal strains from several investigators and begun sequence analysis (O'Rourke et al 2011). How many different mutated genes are represented in these collections is unclear, and more fundamentally the fraction of essential genes that can yield conditional alleles remains unknown.…”

Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

“…To test whether the activity of MEI-1/MEI-2 is also required continuously to maintain structure after spindle assembly, we did temperature-shift experiments with a fast-acting temperature-sensitive (ts) mutant, mei-1(or642) (O'Rourke et al ., 2011). Embryos within the uterus of mei-1(or642) worms grown at 16°C were arrested after spindle assembly at metaphase I by depletion of a component of the anaphase-promoting complex mat-1 (Davis et al ., 2002) by RNA interference (RNAi).…”