Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster.

Abstract

The pero1 and the pers mutations in Drosophila melanogaster, which seem to eliminate or speed up, respectively, the clocks underlying biological rhythmicity, were mapped to single nucleotides. Chimeric DNA fragments consisting of well-defined wild-type plus mutant DNA subsegments were constructed, introduced into flies by germ-line transformation, and assayed for biological activity. These experiments localized both perol and pers to a 1.7-kilobase DNA fragment that is mostly coding DNA. Sequencing of this subsegment from each mutant showed that per"1 is completely accounted for by a nonsense mutation in the third coding exon of a 4.5-kilobase RNA transcribed from this locus. The pers mutation is also a single nucleotide substitution, in the fourth coding exon, which results in a serine-to-asparagine substitution in the per gene protein product. The functional significance of these changes is discussed with reference to the phenotypes of the two mutations.The period gene of Drosophila melanogaster is defined by some of the most interesting behavioral mutations isolated to date. These mutations affect biological rhythms either by eliminating periodic eclosion or behavior or by causing dramatic changes in rhythmic phenotypes (for review see refs. 1 and 2). The original per mutations were induced by Konopka and Benzer (3). Two of the mutants were per' and perL, isolated with respect to circadian eclosion rhythms whose periods are about 5 hr shorter or longer, respectively, than the normal 24 hr. The per' mutant was also recovered (3), based on aperiodic eclosion. This mutation is now called per"l, because there are currently four independently isolated "arrhythmic" mutations at this locus (refs. 4 and 5; R. J. Konopka and M.H., unpublished results).Detailed genetic and phenotypic analyses of these "clock" mutants suggested that per'1 may be a null mutation, behaving like a deletion of this apparently nonvital locus within segment 3B1-2 of the X chromosome (6, 7). Manipulations of the dosage of the wild-type allele and the pers allele suggested that the increased per activity seemingly associated with pers is caused by a qualitative change in the activity of the gene product (4,8).A 6-kilobase (kb) subset of the per region (9, 10) appears to be necessary for per' activity-i.e., rescues per'-induced arrhythmicity of (i) circadian locomotor activity (5, 11, 12), (ii) eclosion (12), and (iii) variations in the male courtship "singing" behavior (5, 12). This portion of the per region corresponds to a 4.5-kb RNA species (9, 10), allowing one to focus on its informational content (13-15).The results we present here include a determination of the precise sites of the mutations in the original arrhythmic and short-period variants. These experiments were undertaken to address some of the issues raised above and the role of the gene in the development (e.g., refs. 16 and 17) and the function (e.g., refs. 15 and 18) of biological clocks in Drosophila. Thus, we set out to determine ifperol is in fact a null mutant and t...

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

“…1A). This region includes the per short domain and part of the CLK/ CYC inhibition domain (CCID) (Yu et al 1987;Chang and Reppert 2003). It also contains a number of phosphorylation sites that are critical for controlling clock speed, such as T583, S585, S589, and S596 (Chiu et al 2011).…”

Clock mutations alter developmental timing in Drosophila

“…The mutant circadian phenotypes observed in per5, and per° flies are due to changes in the per gene's proteoglycan-like gene product Jackson et a!., 1986;Yu et a!., 1987 andBaylies eta!., 1987). The mutant proteoglycans may alter the pattern of intercellular coupling between cells by affecting gap junctional communication (Spray eta!., 1987 andBargiello eta!., 1987) Gap junctions appear to be important during development, because disturbing them can impaii pattern formation (Warner, 1985).…”

Ontogeny of a biological clock in Drosophila melanogaster.

“…Of particular interest are mutations at the per (period) locus. Molecular analysis ofper has shown that deletion of the gene results in arrhythmicity, while a variety of long-and shortperiod rhythms can be produced by amino acid substitutions in the per protein or by changing the level of per expression (8)(9)(10)(11)(12). per is expressed in many cell types throughout development (13)(14)(15)(16)(17).…”