Jeffrey C. Edman

The Cryptococcus neoformans STE12α gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific

Cryptococcus neoformans possesses two mating types, MATα and MATa. α‐Cells are more virulent than a‐cells and are also, unlike a‐cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFα, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12α gene, however, has one striking difference from other fungal STE12 genes; it is found only in α‐cells. The existence of STE12α in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter

A Cryptococcus neoformans galactose auxotroph was created by ultraviolet light mutagenesis and complemented with a C. neoformans genomic library. The translated sequence of the complementing DNA revealed a high degree of simlarity to a number of UDP glucose‐D‐gatactose‐1‐phosphate uridylyitransferases. Expression of C. neoformans GAL7 mRNA followed a pattern similar to Saccharomyces cerevisiae expression; it was first observed within 2.5 min of induction and fully induced by 30 min. The gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two genes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MFα, which encodes a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, β‐glucuronidase (gusA), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7 p::GUS fusion was used to quantify inducibitity of the GAL7 promoter, the level of enzyme activity was at least 500‐fold greater for cells grown in galactose than for cells grown in glucose. The GAL7 promoter is the first inducible promoter characterized in C neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.

Cloning, sequence analysis and expression of the gene encoding imidazole glycerol phosphate dehydratase in Cryptococcus neoformans

A cDNA from Cryptococcus neoformans, encoding imidazole glycerol phosphate dehydratase (IGPD), was isolated by complementation of a his3 mutant strain of Saccharomyces cerevisiae. The C. neoformans HIS3 cDNA encodes an approx. 22-kDa protein with a high degree of amino-acid sequence similarity to IGPDs from ten other microorganisms, as well as Arabidopsis thaliana. Most striking are two conserved HHXXE regions and several conserved His, Asp and Glu residues. The cDNA was engineered for expression in Escherichia coli and an approx. 26-kDa protein was identified by SDS-PAGE. DNA and N-terminal sequence analyses confirmed that this protein was C. neoformans IGPD. Furthermore, IGPD assays of crude extracts from IGPD-producing E. coli cells demonstrated that the C. neoformans protein was catalytically active.

Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.

A HUMAN HEPATOMA CELL LINE CONTAINS HEPATITIS B DNA AND RNA SEQUENCES

ABSTRACT The genome of a human hepatoma cell line, the Alexander cell, contains at least six integrated copies of Hepatitis B viral DNA. Four of these integrated sequences appear to be full length viral genomes. Integration occurs at a specific region of the viral DNA, which is near the nick present in the native Hepatitis B genome. Alexander cells produce a major transcript of 2000 bases which hybridizes specifically to the viral surface antigen gene.