Molecular analysis of a Drosophila melanogaster sn-glycerol-3-phosphate dehydrogenase allozyme variant that has cold labile activity.

A rare naturally occurring allele, GpdhACb62, at the sn-glycerol-3-phosphate dehydrogenase locus in Drosophila melanogaster, encodes an enzyme with an electrophoretic mobility that is more cathodal than that produced by the common slow electrophoretic allele. After electrophoresis and staining of extracts of single adult flies there is a single band of activity corresponding in position to GPDH-1, but, using highly concentrated extracts, a faint band corresponding to GPDH-3 is observed. In GpdhACb62 homozygotes there is about 26% of the normal level of activity in adults, and less than 6% in third instar larvae. The reduction in activity is significantly greater than the decrease in GPDH immunologically cross-reacting material (CRM). Northern analyses, and rapid amplification of the cDNA ends (RACE) of the 3' regions of the transcripts, show that the levels and structures of the poly(A)+RNAs are similar in homozygotes for GpdhACb62 and for a normal activity allele GpdhAC8. Hybridization to oligonucleotide probes specific for the GPDH-1 and GPDH-3 transcripts was of a similar intensity in GpdhACb62 and GpdhAC8 adult flies. In third instar larvae the main transcript is for GPDH-3 and again the hybridization signals were similar in each line. The activity of the enzyme produced by GpdhACb62 was unstable both at 50 degrees C and at 0 degrees C. The activity lost at 0 degrees C was recovered by incubation at 20 degrees C. The complete GpdhACb62 gene, and the partial Gpdh tandem duplication 3' to this gene, were cloned and sequenced. Comparisons with two normal activity GpdhF genes revealed 31 unique changes in the first copy of GpdhACb62. In exon 4, a T to G substitution changes cysteine to glycine and may disrupt a disulphide bond and be responsible for the distinctive properties of GPDH-ACb62.