No abstract

Arabidopsis thaliana is a well-established model organism for plant genetics, and its recently sequenced genome reveals a wealth of enzymes similar to known examples that biosynthesize secondary metabolites. We describe experiments that exploit this genomic information to identify novel terpenoids. A predicted open reading frame with high similarity to known oxidosqualene cyclases was shown to convert 3(S)-oxidosqualene to the previously unknown triterpene alcohol (3S,13S,14R)-malabarica-8,17,21-trien-3-ol, which we named thalianol. Genome mining offers a systematic approach to exhaustively characterize the biosynthetic potential of an organism, and is considerably more sensitive than classical approaches. Because even rare transcripts can be heterologously expressed at high levels, genome mining coupled to heterologous expression may be more sensitive than classical extraction approaches for isolating and characterizing trace metabolites.

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Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA. In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper. We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper. DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains. These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.

Drosophila peripheral nerves contain motor and sensory axons surrounded by two layers of glia: an inner peripheral glia and an outer perineurial glia. Although it is known that intercellular signalling occurs among cells of the peripheral nerve, the effects of this signalling on nerve structure is incompletely understood. It was previously shown that growth of the perineurial glia is negatively regulated by five genes: ine, which encodes a putative neurotransmitter transporter, eag, which encodes a potassium channel, push, which encodes a large, Zn2+ finger‐containing protein, amn, which encodes a putative neuropeptide with similarity to pituitary adenylate cyclase activator peptide (PACAP), and NF1, the Drosophila orthologue of human NF1. Single or various double mutants in any of these genes exhibit perineurial glial hypertrophy. We found that the Ras12A/Rase2f heteroallelic combination, which decreases Ras activity, rescues the thick perineurial glia phenotype exhibited in ine; NF1 double mutants. This result demonstrates that Ras activity is required for the effect of NF1 mutations on perineurial glial growth. We also expressed a constitutively active Ras transgene in an ine– background specifically in peripheral glia using a Gal4‐UAS system. These larvae exhibited perineurial glia thickness comparable to that of ine; NF1 double mutants. Thus, expression of constitutively active Ras in peripheral glia mimics the effect of NF1 mutations on perineurial glial growth. These results demonstrate that Ras activity is both necessary and sufficient to activate perineurial glial growth in peripheral nerves and that Ras activity controls perineurial glial growth in a cell nonautonomous manner.