The genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or N-RAS.

The RAS/mitogen-activated protein kinase pathway sends external growth-promoting signals to the nucleus. BRAF, a critical serine/threonine kinase in this pathway, is frequently activated by somatic mutation in melanoma. Using a cohort of 115 patients with primary invasive melanomas, we show that BRAF mutations are statistically significantly more common in melanomas occurring on skin subject to intermittent sun exposure than elsewhere (23 of 43 patients; P<.001, two-sided Fisher's exact test). By contrast, BRAF mutations in melanomas on chronically sun-damaged skin (1 of 12 patients) and melanomas on skin relatively or completely unexposed to sun, such as palms, soles, subungual sites (6 of 39 patients), and mucosal membranes (2 of 21 patients) are rare. We found no association of mutation status with clinical outcome or with the presence of an associated melanocytic nevus. The mutated BRAF allele was frequently found at an elevated copy number, implicating BRAF as one of the factors driving selection for the frequent copy number increases of chromosome 7q in melanoma. In summary, the uneven distribution of BRAF mutations strongly suggests distinct genetic pathways leading to melanoma. The high mutation frequency in melanomas arising on intermittently sun-exposed skin suggests a complex causative role of such exposure that mandates further evaluation.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.

In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly 67 amide nitrogen to the Ser 65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg 96 and Glu 222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant ‫؍‬ 7.5 ؋ 10 3 h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9 -10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7-10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu 222 carboxylate titrates with an apparent pK a of 6.5 in R96M and that the Gly 67 amide nitrogen titrates with an apparent pK a of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu 222 plays the role of a general base, facilitating proton abstraction from the Gly 67 amide nitrogen or the Tyr 66 ␣-carbon. Arg 96 fulfills the role of an electrophile by lowering the respective pK a values and stabilizing the ␣-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.