Journal: eLife

Article Title: A saturation-mutagenesis analysis of the interplay between stability and activation in Ras

doi: 10.7554/eLife.76595

Figure Lengend Snippet: ( A ) The three principal sites of cancer mutations in Ras, referred to as the three cancer hotspots – Gly 12, Gly 13, and Gln 61 – are shown in the K-Ras structure. The C-terminal helix extension is indicated. PDB ID: 2MSD . ( B ) Ras cycles between signaling-active GTP-bound and signaling-inactive GDP-bound states. ( C ) The bacterial-two-hybrid system couples the C-Raf-RBD•Ras-GTP interaction to the transcription of an antibiotic resistance gene . Ras is fused to the N-terminal domain of the α-subunit of the E. coli RNA polymerase. C-Raf-RBD is fused to the λ-cI protein. The GAP and the GEF can be co-expressed in the system. E. coli cells are transformed with a DNA library of ‘unselected’ variants. The bacteria are grown in the presence of an antibiotic for 9 hr, then the DNA library of ‘selected’ variants is isolated. Next-generation sequencing (NGS) is used to count the frequency of each variant in the unselected and selected samples. ( D ) Ba/F3 assay for Ras activity. Mutant H-Ras libraries are transfected into HEK 293T cells to generate a retroviral library of mutants. Ba/F3 cells are transduced with the retroviral library. After 24 hours, a fraction of the cells are used as the unselected population, and the remainder of the cells – the selected population – are cultured for 7 days in the absence of IL-3 before harvesting them by centrifugation. The genomic DNA of the selected and unselected populations is isolated and sequenced using NGS. The relative enrichment scores are calculated using the selected and unselected counts (see ).

Article Snippet: The extracted DNA was then amplified by PCR and attached to barcode oligonucleotides compatible with the MiSeq sequencing platform (Illumina, index1: i7 primers, and index: i5 primers).

Techniques: Transformation Assay, Bacteria, Isolation, Next-Generation Sequencing, Variant Assay, Activity Assay, Mutagenesis, Transfection, Retroviral, Transduction, Cell Culture, Centrifugation

Journal: eLife

Article Title: A saturation-mutagenesis analysis of the interplay between stability and activation in Ras

doi: 10.7554/eLife.76595

Figure Lengend Snippet: The pairwise difference (epistasis) is shown averaged over amino acids at each position ( ⟨ Δ Δ E x i ⟩ x ). compiles the activating sites where there is epistasis. ( B ) Comparison of enrichment values for substitutions at activating sites in the H-Ras+GAP and mammalian Ba/F3 cell experiments. Substitutions at the cancer hotspots are activating, whereas substitutions at other sites (e.g.,Val 14 or Asp 119) are gain-of-function only in the longer constructs. ( C ) The stability-dependent gain-of-function sites are mapped onto Ras structure. PDB ID: 2MSD . ( D ) Comparison of the pairwise difference ( Δ Δ E x i ) for activating mutations at the stability-dependent gain-of-function sites and at the cancer hotspots in long and short H-Ras+GAP datasets. Only mutations that are activating in the longer construct ( > 1.5 times the standard deviation) are considered in this analysis. ( E ) Sequence diversity analysis of the top-nine gain-of-function mutations found in the H-Ras 1-188 in mammalian Ba/F3 cells. Two separate multiple sequence alignments (MSAs) were used in the analysis: a MSA of the ~150 proteins in the human Ras superfamily , and a MSA of the Ras ortholog sequences found in an evolutionary analysis of metazoan Ras .

Article Snippet: The extracted DNA was then amplified by PCR and attached to barcode oligonucleotides compatible with the MiSeq sequencing platform (Illumina, index1: i7 primers, and index: i5 primers).

Techniques: Comparison, Construct, Standard Deviation, Sequencing

Journal: eLife

Article Title: A saturation-mutagenesis analysis of the interplay between stability and activation in Ras

doi: 10.7554/eLife.76595

Figure Lengend Snippet: ( A ) Structure of H-Ras with the top-nine sites of gain-of-function mutations in the H-Ras 1-188 in mammalian Ba/F3 cells. PDB ID: 5P21 . ( B ) The G-domain is conserved among the three proteins of the human Ras superfamily. Rab29 and Rap1B have sequence identities with H-Ras of ~27% and ~ 56%, respectively. Rab29 has alanine instead of glycine at positions corresponding to residues 12 and 13 of human Ras, and Rap1B, has a threonine instead of a glutamine at position 61. PDB IDs: 5P21, 3X1X, and 6HH2 ( ; ; ).

Article Snippet: The extracted DNA was then amplified by PCR and attached to barcode oligonucleotides compatible with the MiSeq sequencing platform (Illumina, index1: i7 primers, and index: i5 primers).

Techniques: Sequencing