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In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA-) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis.

Farnesylpyrophosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis. Several classes of essential metabolites, including sterols, quinones, carotenoids and gibberellins, are terpenoids with high biological activity. The structural gene for FPP synthase was isolated from two ascomycete fungi, Neurospora crassa and Gibberella fujikuroi. A comparative analysis of the nucleotide sequences of both FPPS genes revealed the presence of introns at the same positions at the 5' end of the coding regions. Furthermore, the most conserved region of the gene was isolated from two other plant pathogenic fungi, Sphaceloma manihoticola and Claviceps purpurea, by PCR. Sequence analysis showed a high degree of similarity between the deduced proteins of all known FPP synthase genes. In contrast to animals, all analyzed fungi contain a single copy of the gene, although FPP is the precursor for essential sterol and quinone biosynthesis and secondary metabolites, such as gibberellins, as well. Transcription analysis in different light regimes has shown that the FPPS genes in G. fujikuroi and N. crassa are not regulated by light induction.

The GPD 1 gene of Claviceps purpurea coding for glyceraldehyde-3-phosphate dehydrogenase was cloned and sequenced, including 1,800 bp of its 5' upstream region. This gene shows an identical structure to the gpd gene of Podospora anserina and Cryphonectria parasitica (one intron at an identical position) with high homology at both the DNA and amino-acid levels. Two fragments of the promoter spanning from the ATG to -500 bp and to -1,400 bp were fused to the phleomycin-resistance gene. Both constructs transformed C. purpurea at a high rate. The enhanced expression of the long vector construct indicates the presence of additional elements between -500 bp and -1,400 bp upstream of the initiation codon.