Mortierella alpina was transformed successfully to hygromycin B resistance by using a homologous histone H4 promoter to drive gene expression and a homologous ribosomal DNA region to promote chromosomal integration. This is the first description of transformation in this commercially important oleaginous organism. Two pairs of histone H3 and H4 genes were isolated from this fungus. Each pair consisted of one histone H3 gene and one histone H4 gene, transcribed divergently from an intergenic promoter region. The pairs of encoded histone H3 or H4 proteins were identical in amino acid sequence. At the DNA level, each histone H3 or H4 open reading frame showed 97 to 99% identity to its counterpart but the noncoding regions had little sequence identity. Unlike the histone genes from other filamentous fungi, all four M. alpina genes lacked introns. During normal vegetative growth, transcripts from the two histone H4 genes were produced at approximately the same level, indicating that either histone H4 promoter could be used in transformation vectors. The generation of stable, hygromycin B-resistant transformants required the incorporation of a homologous ribosomal DNA region into the transformation vector to promote chromosomal integration.The filamentous fungus Mortierella alpina produces up to 50% of its biomass as triacyglycerol, which is rich in long-chain polyunsaturated fatty acids (7, 26). These fatty acids are important both nutritionally and pharmacologically, and there is much interest in developing microbial processes for their production (19,26). To date, manipulation of M. alpina to produce oils with different fatty acid contents has been carried out by strain mutagenesis (7). The recent isolation of several fatty acid desaturase genes from this fungus has presented the opportunity of using recombinant methods to modify the fatty acid composition of its oil (14,22,27,38). To achieve this goal, a DNA transformation system must be developed for M. alpina, because there are no reports of transformation in this organism.Efficient transformation vectors usually contain a homologous promoter to drive expression of the selection marker. In the case of the fungus Phanerochaete chrysosporium (12) and in Tetrahymena thermophila (17), dominant antibiotic resistance markers have been expressed using a strong, homologous histone H4 promoter. Histone H3 and H4 promoters have also been used to express reporter genes in yeast and plants (3,11). Additionally, a number of fungal histone genes have been characterized (10,21,28,39). Most histone genes are highly expressed, and their regulation is tightly coupled to DNA synthesis during the cell cycle (3, 32). The use of a histone promoter to express selection markers should, however, present no problems in fungal cultures which normally grow asynchronously.In the present paper, we describe the isolation and characterization of two pairs of histone H3 and H4 genes from M. alpina and the use of one of the histone H4 promoters in a transformation vector to drive expression of t...

cerevisiae. This indicated that the M. alpina ∆9-desaturase had a substrate preference for stearic acid (18 :0) rather than palmitic acid (16 :0). Genomic clone ∆9-2 (ole2) also encoded a protein of 445 aa which had 86 % identity to the ∆9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strainspecific or induced under certain physiological conditions.

A subtraction library was prepared from cultures of Aspergillus niger that had or had not been exposed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn2+-containing superoxide dismutase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control conditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stresses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined.