Figure 1
Dideoxysequence traces of the L19F and T20A double mutation in K-RAS. (A) Identification of double peaks at the third base of codon 19 and first base of codon 20 in a colorectal cancer. This sequence trace did not determine whether both base changes were on the same or two independent alleles. Harvesting of tumour genomic DNA and sequencing methodology has been described in detail elsewhere [16]. (B) Sequence trace (upper panel), demonstrating the normal leucine and threonine codons at positions 19 and 20 of the K-RAS sequence. Sequence trace from a bacterial clone (lower panel) showing the appearance of the double mutation L19F/T20A in the same allele. For cloning of tumour derived PCR amplicons, products were ligated into TOPOBlunt plasmids using ZeroBlunt TOPO cloning kits (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Bacterial DNA was then isolated using Miniprep kits (Qiagen, Crawley, UK) according to the manufacturer's instructions and sequenced using M13 universal primers on a ABI3730xl Platform sequencer (Applied Biosystems, Warrington, UK).
Figure 2
Focus formation by mutated K-RAS cDNA. Methylene blue stained transformed foci following transfection of K-RAS cDNA expression vectors containing: (A) G12D (B) G12V (C) L19F alone (D) T20A alone (E) L19F/T20A co-mutation and (F) wildtype sequences. "No DNA" controls describe cells treated with transfection reagents but no DNA. Colonies formed following transfection with the L19F/T20A double mutant K-RAS cDNA were consistently smaller than those formed following transfection with the codon 12 mutants. K-RAS cDNA containing plasmids were lipofected with Lipofectamine (Invitrogen, Paisley, UK) into NIH3T3 mouse fibroblast cells, and seeded onto 60 mm plates at a density of 100 000 cells per plate. The cells were grown for 14 days, with the media replaced every 72 hours. After 14 days plates were fixed in 10% formalin then stained with 1% methylene blue (Sigma-Aldrich, Gillingham, UK), in 70% ethanol solution. Colonies above 3 mm in diameter were counted and were statistically analysed using Mann-Whitney U tests with P values of less than or equal to 0.05 considered statistically significant.
Figure 3
Quantification of focus formation by different K-RAS mutations. Histogram of mean focus numbers obtained from focus formation assays performed in 4 separate experiments after NIH3T3 cells were transfected with K-RAS cDNA expression vectors containing G12V, G12D, L19F, T20A, L19F/T20A or wildtype K-RAS sequences. Error bars represent ±SD. Full focus counts for all transfections and all experiments and the sequences of the cDNA constructs used for NIH3T3 cell transfection are described in Additional File section (Additional File 1).
Figure 4
Modelling of the mutant codon 19 phenylalanine and codon 20 alanine side chains into the K-RAS protein. (A) The GDP/GTP binding pocket of wildtype K-RAS. The white residues are codons 19 and 20. The loop on the left of the helix forms the GDP/GTP binding pocket of K-RAS. Residues coloured blue represent the mutational hotspot codons 12 and 13. The shortened K-RAS protein structure used for modelling was obtained from the Protein Data Bank (PDB ID:3gft) and visualised using PyMOL (http://www.pymol.org). (B) The GDP/GTP binding pocket of wildtype K-RAS with wildtype side chains of codons 19 and 20 and a non-hydrolysable GTP analogue (used during crystallisation) adjacent to codons 12 and 13. (C and D) Modelling of the mutant phenylalanine (codon 19) and alanine (codon 20) side chains into the K-RAS protein, predicts projection of the bulky phenylalanine side chain into the main body of the protein and replacement of the OH containing side chain of threonine with the small aliphatic side chain of alanine. The mutant side chains were modelled into positions of the protein which produced the fewest conflicting Van der Waals radii and the configuration with the least steric interference with other amino acid side chains.