MOLECULAR ANALYSIS OF A DROSOPHILA-MELANOGASTER SN-GLYCEROL-3-PHOSPHATE DEHYDROGENASE ALLOZYME VARIANT THAT HAS COLD LABILE ACTIVITY

A rare naturally occurring allele, Gpdh(ACb62), at the sn-glycerol-3-p hosphate dehydrogenase locus in Drosophila melanogaster, encodes an en zyme with an electrophoretic mobility that is more cathodal than that produced by the common slow electrophoretic allele, After electrophore sis and staining of extracts of single adult flies there is a single b and of activity corresponding in position to GPDH-1, but, using highly concentrated extracts, a faint band corresponding to GPDH-3 is observ ed. In Gpdh(ACb62) homozygotes there is about 26% of the normal level of activity in adults, and less than 6% in third instar larvae, The re duction 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)(+) RNA s are similar in homozygotes for Gpdh(ACb62) and for a normal activity allele Gpdh(AC8). Hybridization to oligonucleotide probes specific fo r the GPDH-1 and GPDH-3 transcripts was of a similar intensity in Gpdh (ACb62) and Gpdh(AC8) adult flies, In third instar larvae the main tra nscript is for GPDH-3 and again the hybridization signals were similar in each line. The activity of the enzyme produced by Gpdh(ACb62) 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 Gpdh(ACb62) gene, and the partial Gpdh tandem duplication 3' to this gene, were cloned and sequenced, Comparisons with two normal activity Gpdh(F) genes revealed 31 unique changes in the first copy of Gpdh(ACb 62). In exon 4, a T to G substitution changes cysteine to glycine and may disrupt a disulphide bond and be responsible for the distinctive p roperties of GPDH-ACb62.