Glucan synthase complex of Aspergillus fumigatus.

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.

Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase.

A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function.

Do products of the myc proto-oncogene play a role in transcriptional regulation of the prothymosin alpha gene?

The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the intact human prothymosin alpha gene or reporter constructs that mimic its structure. Rather, they suggest that the human prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes.

A GTP-binding protein regulates the activity of (1-->3)-beta-glucan synthase, an enzyme directly involved in yeast cell wall morphogenesis.

Synthesis of (1-->3)-beta-D-glucan, the major structural component of the yeast cell wall, is synchronized with the budding cycle. Membrane-bound, GTP-stimulated (1-->3)-beta-glucan synthase was dissociated by stepwise treatment with salt and detergents into two soluble fractions, A and B, both required for activity. Fraction A was purified about 800-fold by chromatography on Mono Q and Sephacryl S-300 columns. During purification, GTP binding to protein correlated with synthase complementing activity. A 20-kDa GTP-binding protein was identified by photolabeling in the purified preparation. This preparation no longer required GTP for activity, but incubation with another fraction from the Mono Q column (A1) led to hydrolysis of bound GTP to GDP with a concomitant return of the GTP requirement. Thus, fraction A1 appears to contain a GTPase-activating protein. These results show that the GTP-binding protein not only regulates glucan synthase activity but can be regulated in turn, constituting a potential link between cell cycle controls and wall morphogenesis.

Biosynthesis of cell wall and septum during yeast growth.

The primary septum that forms in yeast cells at cytokinesis consists of the polysaccharide chitin. Three chitin synthetases (Chs1, Chs2 and Chs3) have been identified in Saccharomyces cerevisiae. Cloning and disruption of the respective genes showed that Chs3 is responsible for the formation of a chitin ring at the base of an emerging bud and of chitin dispersed in the cell wall, whereas Chs2 catalyzes the synthesis of a chitin disc that completes the primary septum. Chs1 acts as a repair enzyme, replenishing chitin lost through excessive action of a chitinase that facilitates cell separation by degrading part of the septum. The major structural polysaccharide of the yeast cell wall is beta(1->3)glucan. The glucan synthetase complex is bound to the plasma membrane. By differential extraction of the membranes with salt and detergents two solubilized fractions have been obtained which are required, in addition to GTP, for glucan synthesis. Further purification of one of these fractions led to results that indicate a role for other proteins in the modulation of GTP stimulation. A G-protein system appears to function in the regulation of beta(1->3) glucan and cell wall formation in vivo.

CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae.

The CAL1 gene was cloned by complementation of the defect in Calcofluor-resistant calR1 mutants of Saccharomyces cerevisiae. Transformation of the mutants with a plasmid carrying the appropriate insert restored Calcofluor sensitivity, wild-type chitin levels and normal spore maturation. Southern blots using the DNA fragment as a probe showed hybridization to a single locus. Allelic tests indicated that the cloned gene corresponded to the calR1 locus. The DNA insert contains a single open-reading frame encoding a protein of 1,099 amino acids with a molecular mass of 124 kD. The predicted amino acid sequence shows several regions of homology with those of chitin synthases 1 and 2 from S. cerevisiae and chitin synthase 1 from Candida albicans. calR1 mutants have been found to be defective in chitin synthase 3, a trypsin-independent synthase. Transformation of the mutants with a plasmid carrying CAL1 restored chitin synthase 3 activity; however, overexpression of the enzyme was not achieved even with a high copy number plasmid. Since Calcofluor-resistance mutations different from calR1 also result in reduced levels of chitin synthase 3, it is postulated that the products of some of these CAL genes may be limiting for expression of the enzymatic activity. Disruption of the CAL1 gene was not lethal, indicating that chitin synthase 3 is not an essential enzyme for S. cerevisiae.

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle.

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.