In order to characterize three per mutations that eliminate (pero1), shorten (pers), or lengthen (perl) the circadian period of Drosophila melanogaster, the authors sequenced DNA from the per locus in all three strains.
Sequencing revealed the presence of unique single nucleotide substitutions in each of the mutant strains when compared to the wildtype sequence. In perl flies, the mutation was found to be caused by a T-to-A transversion resulting in a valine to aspartic acid amino acid change in the protein. The authors also independently identified the point mutations in pers and pero1 flies. The circadian arrhythmicity in pero1 flies was reported to be attributable to a truncated protein with loss of function, while the rhythm alterations in pers and perl flies were proposed to be attributable to hyper- or hypoactive per protein, respectively, or an increase or decrease in per stability.
Two mutant strains of Drosophila melanogaster, pero1 (arrhythmic) and pers (short period), were mapped to determine the location and nature of these mutations. The authors isolated and digested the mutated DNA fragments from pero1 and pers flies via restriction mapping and vector cloning. Mutant or wildtype vectors were then transduced into pero1 flies to assess changes in circadian behaviour.
Cloning of different sized fragments of per DNA mapped both mutations to a 1.7kb region of the per gene. Sequencing of these fragments revealed the pero1 mutation site as a single nucleotide C-to-T substitution, resulting in a stop codon in exon 4 and a truncated protein. The pers mutation was identified as a G-to-A substitution, resulting in a single amino acid missense mutation.
Using mutagenesis by ethyl methane sulfonate, the authors investigated potential genes controlling the circadian clock in Drosophila melanogaster. Mutagen-exposed males were mated to attached-X females resulting in offspring carrying the father’s mutated X chromosome. These offspring were then screened for alterations in the circadian rhythm of eclosion and locomotor activity.
From the resulting offspring, 3 mutants with altered rhythms were found. The first mutant identified was arrhythmic for both eclosion and locomotor activity. The second mutant displayed a short circadian period in eclosion and activity of approximately 19 hours. The third mutant produced long period rhythms in circadian eclosion and locomotor activity of about 28 hours. The locations of these mutations were mapped by assessing genetic recombination with known markers on the Drosophila X chromosome. The authors found that all 3 rhythm mutations mapped to a similar position on the left end of the X chromosome.