Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma.

UNLABELLED: NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. SIGNIFICANCE: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation.

An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein.

BACKGROUND AND AIMS: Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants. METHODOLOGY: Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically. PRINCIPAL FINDINGS: Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H(0): ß (slope) = 0 mean counts per pixel (ng s mm(-2))(-1), r(2) > 0.994, n = 31, P < 1.75 × 10(-29)). CONCLUSIONS: Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with small budgets.