Glucose-6-phosphate dehydrogenase : construction of yeast DNA libraries and screening for its gene
The aim of the project was to determine the sequence of the gene encoding glucose-6-phosphate dehydrogenase (G6PDH) in the industrially used, but genetically little explored yeast Candida utilis. In the first strategy, a C. utilis DNA library was constructed in the expression vector phage lambda gt11, and was screened immunologically with antiserum raised against C. utilis G6PDH. The second strategy involved construction of libraries in the plasmid vector pTZ18R and screening with a mixed oligonucleotide probe. Two methods were developed that allow DNA, of appropriate quality for the construction of DNA libraries, to be prepared from C. utilis cells. Both involve centrifugation of C. utilis lysates through caesium trifluoroacetate gradients. A rabbit antiserum was raised against a commercial preparation of C. utilis G6PDH. It reacted with Western blotted C. utilis G6PDH, and non-specific binding of the antiserum to Western blotted E. coli protein was reduced by treatment with an E. coli extract. Antiserum pretreated in this way was used for immunological screening. Sequence information was determined for the inserts of 7 phage identified during immunological screening. None of the insert sequences was considered to contain part of the C. utilis G6PDH coding sequence. One putative positive colony was identified during the screening of the pTZ18R libraries. Sequence determination showed the insert to contain a region complementary to 16 bases in one of the 17-base long probe oligonucleotides. This insert was considered not to contain part of the C. utilis G6PDH coding sequence. Time did not permit further work which, using the methods developed, should now permit determination of the C. utilis G6PDH gene sequence.