exceeded 30. However, Northern analysis showed that mRNA was present in the CaADH2 stationary phase cells grown in YPD. This may reflect the greater sensitivity of Northern analysis. Having established that and mRNAs are expressed in cells grown CaADH1 CaADH2 C. albicans under a variety of conditions, both genes were cloned and expressed in (Fig. 2). S. cerevisiae Assays of enzyme activity in affinity-purified extracts from Adh1p- or Adh2p-expressing cells demonstrated only the CaAdh1p-expressing strain exhibited greater activity than the empty cassette vector pABC3-his control (Fig. 3). Although the presence of CaAdh2p in the purified cell extract from the AD/CaADH2-his strain was confirmed by Western blot using an anti-His antibody (Fig. 2), extracts from this strain showed enzyme activities similar to those in extracts from the control empty vector strain (Fig. 3). This would suggest that CaAdh2p is inactive under the conditions tested, and that the activity observed for the AD/CaADH2-his strain represents the background Adh activities of the host AD strain. In order to confirm that the ethanol-utilising activity in extracts from the AD/CaADH1-his recombinant strain was mediated by the cloned CaAdh1p rather than contaminating ScAdh2p, CaAdh1p was expressed in a strain in which was disrupted. was successfully ScADH2 ScADH2 disrupted in the parental AD strain. In this recombinant strain (AD/ScADH2) no significant NAD reduction was detected in extracts when ethanol was given as the substrate (Fig. 4) confirming that ScAdh2p catalyses the ethanol utilising activity in (Ciriacy 1975a). When S. cerevisiae ScADH2 was disrupted in a recombinant strain expressing CaAdh1p, ethanol utilising activity was present (Fig. 4) confirming that CaAdh1p catalyses the utilisation of ethanol which, under the conditions tested, would result in production of acetaldehyde. in vitro
exceeded 30. However, Northern analysis showed that CaADH2 mRNA was present in the stationary phase cells grown in YPD. This may reflect the greater sensitivity of Northern analysis. Having established that CaADH1 and CaADH2 mRNAs are expressed in C. albicans cells grown under a variety of conditions, both genes were cloned and expressed in S. cerevisiae (Fig. 2). Assays of enzyme activity in affinity-purified extracts from Adh1p- or Adh2p-expressing cells demonstrated only the CaAdh1p-expressing strain exhibited greater activity than the empty cassette vector pABC3-his control (Fig. 3). Although the presence of CaAdh2p in the purified cell extract from the AD/CaADH2-his strain was confirmed by Western blot using an anti-His antibody (Fig. 2), extracts from this strain showed enzyme activities similar to those in extracts from the control empty vector strain (Fig. 3). This would suggest that CaAdh2p is inactive under the conditions tested, and that the activity observed for the AD/CaADH2-his strain represents the background Adh activities of the host AD strain. In order to confirm that the ethanol-utilising activity in extracts from the AD/CaADH1-his recombinant strain was mediated by the cloned CaAdh1p rather than contaminating ScAdh2p, CaAdh1p was expressed in a strain in which ScADH2 was disrupted. ScADH2 was successfully disrupted in the parental AD strain. In this recombinant strain (AD/ScADH2) no significant NAD reduction was detected in extracts when ethanol was given as the substrate (Fig. 4) confirming that ScAdh2p catalyses the ethanol utilising activity in S. cerevisiae (Ciriacy 1975a). When ScADH2 was disrupted in a recombinant strain expressing CaAdh1p, ethanol utilising activity was present (Fig. 4) confirming that CaAdh1p catalyses the utilisation of ethanol which, under the in vitro conditions tested, would result in production of acetaldehyde.
00001573_114405_64303.docx