Materials
Protocol
Results
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     Existing genetic transformation systems among fungi are often based upon functional complementation of a selectable auxotrophic marker (Fincham,1989).  Two genes commonly used for this purpose, URA3 and URA5, encode the enzymes orotidine-5'- monophosphate decarboxylase (OMPdecase) and orotidine-5'-monophosphate pyrophosphorylase (OMPpase), respectively.  Both enzymes play integral roles in pyrimidine biosynthesis (Jones and Fink, 1982).  OMPpase utilizes 5'-phosphoribosyl-1- pyrophosphate (PRPP) to convert orotic acid (OA) to orotidine-5'-monophosphate (OMP).  The latter compound is subsequently metabolized by OMPdecase t ouridine-5'-monophosphate (UMP) and carbon dioxide.  Mutants lacking either OMPpase or OMPdecase are characteristically auxotrophic or uracil as well as resistant to the toxic effects of 5-fluoroorotic acid (FOA) (Boeke et al., 1984).

     Genetic transformation systems have been developed in pathogenic fungi using similar methodology .  Of particular note are the systems developed in Cryptococcus neoformans and Histoplasma capsulatum (Edman and Kwon-Chung, 1990; Worsham and Goldman, 1990).  In these systems, mutants lacking OMPpase activity are functionally complemented by a URA5 gene.  The mutants, like the Ura5^- strains of non-pathogenic fungi are also FOA-resistant, uracil auxotrophs (Kwon-Chung et al., 1992; Worsham and Goldman, 1988).  The absence of specific OMPpase activity among these mutants was determined using nutritional and radiometric assays.

     In our attempts to develop a transformation system in Wangiella dermatitidis, OMPpase activity in Ura^-, FOA-resistant mutants was assayed using a spectrophotometric method (Cooper and Szaniszlo, submitted).  The assay is based upon the spectrophotometrically-measured decrease in OA as it is converted to OMP by OMPpase, and subsequently to UMP by OMPdecase.  The major advantages of this methodology include the simplicity of the assay and the need for no specialized equipment other than a spectrophotometer capable of scanning in the range of ultraviolet light.  In contrast to other published methods (Worsham and Goldman, 1988), this assay is non-radiometric and therefore requires no special handling precautions.  With minor changes to the methodology, it is possible to assay OMPdecase activity in the same cell extracts (Silva and Hatfield, 1978)

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Preparation of cell extract

1. Strains to be assayed for OMPpase activity are inoculated into 50ml of YPD broth and grown o/n at 25-30ºC on a rotary shaker operating at 200 rpm.
2. From the culture prepared in Step 1, 10-20 ml are used to inoculate 500 ml YPD broth contained in a 1.0 liter flask.  This culture is incubated overnight under the same conditions.
3. Prior to further processing, the culture is chilled on ice for 30 min.
4. Cells are then collected by centrifugation at 4ºC (4,000xg for 10min.), washed twice in ice-cold 20mM Tris, pH 8.0, and finally resuspended to a volume of 5 to 10 ml using the same buffer.

5. The washed cells are transferred to a chilled screw-cap tube containing 1.0g glass beads (0.5mm dia.) for each ml of cell suspension.  This suspension is then shaken at maximum speed for one-minute intervals using a vortex mixer.  Between intervals, the cell suspension is placed on ice for at least 4 min while an aliquot is examined microscopically.  the mixing is continued for one-minute intervals with 4 min cooling periods until 80-95% of the cells are broken as judged by microscopic examination.

6. Broken and remaining intact cells are removed by centrifuging the homogenate at 40,000xg for 30 min at 4ºC.  The supernatant is then aliquoted in 1 to 2 ml portions and stored at -70ºC.

Orotidine-5'-monophosphate pyrophosphorylase assay

1. For each cell extract to be assayed, prepare 8.9ml of assay reaction mix containing the following components: 1.0 M Tris, pH 8.0, 0.50 ml; 80 mM MgCl₂, 0.375 ml; 7mM OA, 225µl; 0.1M beta-mercaptoethanol, 100 µl; and sterile dH₂O, 7.7 ml. (Note: This volume of assay reaction mix is sufficient for one determination.  To perform replicate assays, prepare the appropriate volume of reaction mix).
2. Distribute 1.78 ml of the above reaction mix to each of five 10 x 75 mm test tubes labeled 1 through 5.  Place the tubes on ice before proceeding to the next step.
3. While still in ice, add the following reagents to these tubes according to the table below:

4. Pre-warm the reaction mixes by placing the tubes at 37ºC for 5 min.
5. Initiate the reaction by adding 100 ml of the cell extract to each tube and incubate the tubes for 30 min at 37ºC.
6. Stop the reactions by placing the tubes on ice.
7. Read the absorbance of the reaction mix from each tube at 295nm.
8. To calculate the amount of OA converted to UMP, first determine the difference between the absorbance of a particular reaction mix (tubes 2-5) and the no reaction control (tube1).  By definition, a decrease in absorbance of 0.395 corresponds to the conversion of 100 nM OA to OMP.  The total number of units of enzyme activity is calculated by determining the nM of OA converted to OMP per min of incubation.  Specific activity is determined by calculating the number of units of enzyme activity per mg protein used.  Protein concentration can be determined using a commercially-available kit (Bio-Rad, Richmond, CA).

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       Ura-, FOA-resistant strains of W. dermatitidis were assayed using the above protocol (Cooper and Szaniszlo, 1993).  The the absence of purified enzyme prepartaions, no OMPpase activity was detected (specific activity < 0.07).  When assays of the same strains included pure preparations of OMPpase, a marked and statistically significant decrease in OA was observed compared to those experimental reaction mixes not containing the enzyme (specific activity < 4.03).  When OMPdecase, but not OMPpase was included in the reaction mixes, no statistically significant increase in the conversion of OA to UMP was observed (specific activity = 0.10).  These results clearly indicate that the Ura-, FOA-resistant strains of W. dermatitidis are defective in OMPpase making them equivalent to ura5 mutants.  In contrast, a Ura-, FOA-sensitive strain of W. dermatitidis exhibited high levels of OMPpase activity without the addition of any enzyme (specific activity = 16.85).  This strain obviously possesses a very active OMPpase but cannot be defective in the URA3-encoded enzyme, OMPdecase, because this strain is readily killed by FOA.

Table 1. Assay for OMPpase Activity Using Purified Preparations of OMPpase and OMPdecase^a

^aThese data represent the average of two independent experiments.

^bThese numbers correspond to the assay tube numbers described in the text (see Methods, Part B, Step 3).  The reaction  mixes do not contain cell extract.

^cThese values are probably artificially high and are based upon the amount of protein present as estimated by the supplier of the enzymes.

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     It is also advisable to include control strains having known defects in OMPpase and OMPdecase.  Appropriate controls might include strains S2021B (ura3) and FL476-1C-1A (ura5) of Saccharomyces cerevisiae.  These strains are available from the Yeast Genetics Stock Center (University of California, Berkeley, California).

     It should be noted that a recent report indicates that a second OMPpase gene, URA10, has been found in S. cerevisiae (de Montigny et al., 1990).  Up to 20% of the OMPpase activity in wild-type cells can be attributed to this gene product.  The above protocol should be able to characterize this gene product, but investigators should be aware that the OMPpase activity in a particular fungus may be due in part to a URA10 homologue.

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Friday, April 30, 2004 01:11:18 PM