A glucoamylase ::green fluorescent protein fusion (GLA ::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA ::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA ::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA ::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA ::sGFP by Western analysis. A strain containing the GLA : :sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA ::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA ::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

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The utility of the Aspergillus fumigatus cellobiohydrolase cbhB promoter for controlled gene expression has been investigated. cbhB message was present at high levels in the presence of carboxymethylcellulose and undetected in the presence of glucose. A reporter construct using the cbhB promoter showed similar behaviour and gave lower message levels than the Aspergillus nidulans alcA promoter under repressing conditions. An RNAi construct driven by the cbhB promoter was used to down-regulate the alb1 gene; transformants showed low alb1 message levels and a loss-of-function phenotype with carboxymethylcellulose, while both wild-type message levels and phenotype were seen with glucose. The cbhB promoter is therefore tightly controlled and can be exploited for the study of A. fumigatus.

The John Day Formation of central and eastern Oregon, contains a widespread assemblage of both ash-flow and airfall tuffs, yet only a few corresponding caldera sources have been identified in the region. Investigators have long speculated on the sources of tuffs in the John Day Formation and have suggested that these pyroclastic rocks were vented from now buried eruptive centers in or marginal to a nascent Cascade Range. Recent detailed geologic mapping in the John Day and Clarno Formations, however, indicates the presence of at least three large-scale rhyolite caldera complexes centered along the northeast-trending axis of the Blue Mountains. This field guide describes a three-day geologic transect, from the scenic high desert of central Oregon eastward across the axis of the Blue Mountains, that will examine the physical volcanology and geologic setting of the 41.50-39.35 Ma Wildcat Mountain caldera exposed along the crest of the Ochoco Mountains, the 29.56 Ma Crooked River caldera at Prineville, and the 29.8 to 28.1 Ma Tower Mountain caldera near Ukiah.

Fusarium venenatum JeRS 325, a strain which produces recombinant glucoamylase under control of a growth rate independent promoter was transformed with a plasmid carrying the Aspergillus niger glucoamylase gene under control of its own growth rate correlated promoter. Some disruption of the original recombinant genes occurred and at pH 5.8 the double transformant did not produce as much glucoamylase as JeRS 325 in batch culture. However, the double transformant still produced as much glucoamylase as JeRS 325 in fed-batch cultures, illustrating the potential for the combined use of growth rate independent and dependent promoters to improve production of recombinant proteins in fed-batch culture systems.