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A novel anti-influenza virus compound, flutimide, was identified in extracts of a recently identified fungal species, Delitschia confertaspora (F. Pelaez, J.D. Polishook, M. Valldosera, and J.Guarro, Mycotaxon 50:115-122, 1994). The compound, a substituted 2,6-diketopiperazine, selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases. Similar to the 4-substituted 2,4-dioxobutanoic acids, a series of transcriptase inhibitors which we described previously (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J.Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994), this inhibitor, which is a natural product, affected neither the initiation nor the elongation of influenza virus mRNA synthesis, but it specifically targeted the cap-dependent endonuclease of the transcriptase. Additionally, the compound was inhibitory to the replication of influenza A and B viruses in cell culture. The selective antiviral properties of this compound further demonstrate the utility of influenza virus endonuclease as a target of antiviral agents.

Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus and is a member of a large structurally diverse group of secondary metabolites known as indole-diterpenes. By using degenerate primers for conserved domains of fungal geranylgeranyl diphosphate synthases, we cloned two genes, atmG and ggsA (an apparent pseudogene), from A. flavus. Adjacent to atmG are two other genes, atmC and atmM. These three genes have 64 to 70% amino acid sequence similarity and conserved synteny with a cluster of orthologous genes, paxG, paxC, and paxM, from Penicillium paxilli which are required for indole-diterpene biosynthesis. atmG, atmC, and atmM are coordinately expressed, with transcript levels dramatically increasing at the onset of aflatrem biosynthesis. A genomic copy of atmM can complement a paxM deletion mutant of P. paxilli, demonstrating that atmM is a functional homolog of paxM. Thus, atmG, atmC, and atmM are necessary, but not sufficient, for aflatrem biosynthesis by A. flavus. This provides the first genetic evidence for the biosynthetic pathway of aflatrem in A. flavus.Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus (17,18). This compound is a member of a large, structurally diverse group of secondary metabolites known as indole-diterpenes that includes paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines (34). These metabolites all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from either tryptophan or a tryptophan precursor. Different patterns of prenylation, hydroxylation, epoxidation, acetylation, and ring stereochemistry around the basic indole-diterpene ring structure define this structural diversity (34). The mechanism by which aflatrem and related indole-diterpenes cause tremorgenicity in mammals is not well defined, but biochemical and clinical studies indicate that these effects are due in part to effects on receptors and to interference with neurotransmitter release in the central and peripheral nervous systems (40).Very little is known about the pathways of indole-diterpene biosynthesis other than the origins of the indole and diterpene components (7, 10). Biosynthetic schemes based on the chemical identification of likely intermediates have been proposed, but until recently none of these steps had been validated by biochemical or genetic studies (25,30).Recently, a cluster of genes for paxilline biosynthesis was cloned from Penicillium paxilli (53). Key genes in this cluster include a GGPP synthase gene (paxG), a FAD-dependent monooxygenase gene (paxM), a prenyl transferase gene (paxC), and two cytochrome P450 monooxygenase genes, paxP and paxQ. The deletion of paxG, paxM, or paxC results in mutants that are defective in paxilline biosynthesis (53; B. Scott, L. McMillan, J. Astin, C. Young, A. Bryant, and E.Parker, unpublished results). PaxM and PaxC may catalyze the additi...

S U M M A R YThe wall mannan in dividing Saccharomyces cells was labelled by exposing the yeasts to fluorescein-conjugated concanavalin A. The yeasts were then allowed to grow in the absence of excess staining reagent and were examined by fluorescence microscopy at several intervals after the re-initiation of growth. The newly synthesized mannan was non-fluorescent and, thus, could be easily distinguished from the material present at the outset. Most new mannan was deposited in the wall surrounding the bud, and little if any of the mannan in the bud wall was derived from the surface of the mother cell. Deposition of new mannan in the mother cell was detected only in the bud scar. The distal tip of the growing bud was identified as the major site at which new mannan was inserted into the existing wall fabric. This area is also known to be the deposition site of newly synthesized glucan. Thus, with respect to these two major wall polysaccharides, wall replication in Saccharomyces cells resembles the apical mode exhibited by filamentous fungi.