Computational protocol: The ubiquitin ligase CRL2ZYG11 targets cyclin B1 for degradation in a conserved pathway that facilitates mitotic slippage

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Protocol publication

[…] The zyg-11(ts) suppressor screen was performed as follows. zyg-11(ax491ts) hermaphrodites were synchronized by Na-hypochlorite isolation of eggs followed by L1 larval-stage arrest in M9 buffer with 5 µg/ml cholesterol (). L1-stage larvae were placed on agar plates with OP50 bacteria and grown to the L4 larval stage at 16°C. The L4 larvae were mutagenized with 0.3–1 mM N-ethyl-N-nitrosourea () and grown to gravid adults. F1 embryos arrested in the L1 stage were isolated from the mutagenized gravid adults and grown on OP50 bacteria at 16°C until they were gravid adults. F2 L1 larvae were isolated from F1 gravid adults, and 25 L1 larvae were placed on 6-cm nematode growth medium plates with OP50 bacteria and incubated at the nonpermissive temperature of 25°C for 3 wk. Plates in which the animals could reproduce and clear the plate of bacteria were considered to have potential suppressors. 19 suppressors were obtained from 100,000 screened F2 progeny. Each suppressor strain was outcrossed by mating with strain ET168, zyg-11(ax491ts) unc-4(e120). The unc-4 gene is tightly linked to the zyg-11 gene, with the two genes separated by only 1.1 genetic map units. The observation of suppression of Zyg-11 Unc animals indicated that the suppressors were extragenic.SNP mapping was performed as briefly described in this study. The suppressor strain (which has a Bristol N2 genetic background) was crossed with strain ET340, which contains the zyg-11(ax491ts) allele in a Hawaiianized genetic background (crossed six times into the Hawaiian strain CB4856), and grown at 16°C. 240 F2 progeny were cloned and grown at the nonpermissive temperature of 25°C to allow F3 progeny to develop. The plates of F3 progeny were segregated to identify the 30 most “suppressed” and 30 most “nonsuppressed” plates. Animals from these plates were pooled and used to isolate DNA for SNP analysis, generally by digesting the PCR-amplified product with the restriction enzyme DraI, which would distinguish N2 from the Hawaiian strain (). Subsequent SNP analysis was also performed with DNA from individual clones of suppressed animals to map recombination endpoints.Whole-genome sequencing of zyg-11(ax491ts); ek14 was performed by Cofactor Genomics using the Solexa genome analyzer (Illumina) 36-bp single-end reads were obtained at 13× coverage of the C. elegans genome. We used three programs to analyze the sequence reads. MAQGene, MAQ, and Galign programs were individually used to align the sequence reads to the C. elegans genome and annotate SNPs (; ; ). We also aligned the sequence reads to the genome with the program Bowtie, and the graphical results were visualized with the IGV genomics viewer (; ). All of these approaches identified the mutation of the cyb-2.1 gene in the ek14 strain (; ; ; ), which changes amino acid residue 120 from GAA (Glu, E) to AAA (Lys, K). […]

Pipeline specifications

Software tools MAQGene, galign, Bowtie, IGV
Application WGS analysis
Organisms Caenorhabditis elegans, Homo sapiens