Academic journal article Genetics

Rhythm Defects Caused by Newly Engineered Null Mutations in Drosophila's Cryptochrome Gene

Academic journal article Genetics

Rhythm Defects Caused by Newly Engineered Null Mutations in Drosophila's Cryptochrome Gene

Article excerpt

ABSTRACT

Much of the knowledge about cryptochrome function in Drosophila stems from analyzing the cry^sup b^ mutant. Several features of this variant's light responsiveness imply either that CRY^sup b^ retains circadian-photoreceptive capacities or that additional CRY-independent light-input routes subserve these processes. Potentially to resolve these issues, we generated cry knock-out mutants (cry^sup 0^'s) by gene replacement. They behaved in an anomalously rhythmic manner in constant light (LL). However, cry^sup 0^ flies frequently exhibited two separate circadian components in LL, not observed in most previous cry^sup b^ analyses. Temperature-dependent circadian phenotypes exhibited by cry^sup 0^ flies suggest that CRY is involved in core pacemaking. Further locomotor experiments combined cry^sup 0^ with an externally blinding mutation (norpA^sup P24^), which caused the most severe decrements of circadian photoreception observed so far. cry^sup b^ cultures were shown previously to exhibit either aperiodic or rhythmic eclosion in separate studies. We found cry^sup 0^ to eclose in a solidly periodic manner in light:dark cycles or constant darkness. Furthermore, both cry^sup 0^ and cry^sup b^ eclosed rhythmically in LL. These findings indicate that the novel cry^sup 0^ type causes more profound defects than does the cry^sup b^ mutation, implying that CRY^sup b^ retains residual activity. Because some norpA^sup P24^ cry^sup 0^ individuals can resynchronize to novel photic regimes, an as-yet undetermined light-input route exists in Drosophila.

CIRCADIAN clocks evolved in most organisms in part to mediate the internal "temporal order" that is associated with various biological phenomena, and, furthermore, such that an animal, plant, or microbe can anticipate everyday periodic changes of light and temperature. Light comprises the principal clock resetting cue, which keeps the process running at a 24.0-hr pace in natural conditions (DUNLAP et al. 2004). Thermal cycles can also "entrain" circadian clocks; and another aspect of how these processes respond to temperature changes is to compensate such that the pacemaker operates at the same velocity in different thermal conditions (DUNLAP et al. 2004).

One way by which Drosophila melanogaster's version of a cryptochrome (CRY) protein was implicated in circadian rhythms was by screening for mutations that would perturb luciferase (luc)-reported expression of a canonical clock gene called period (per). The seminal cry^sup b^ mutation was thus recognized because it eliminated the normal daily cycles of luminescence mediated by a per-luc transgene in real-time monitorings of whole flies (STANEWSKY et al. 1998). This mutation entails an amino-acid substitution within the fly's CRY at a site believed to be involved in binding a flavin moiety that participates in the protein's capacity to absorb blue light (STANEWSKY et al. 1998; cf. SANCAR 2000).

Most of the "glow cycling" emanating from cry^sup +^ (and otherwise rhythm-normal) Drosophila comes from peripheral tissues (STANEWSKY et al. 1997), including several appendages projecting from the adult animal (PLAUTZ et al. 1997; LEVINE et al. 2002b). The effects of the cry^sup b^ mutation on suchmolecular rhythmicity implied that the normal gene is expressed in the periphery, but cry1 was found also to make its products in the central nervous system (EMERY et al. 2000b; KLARSFELD et al. 2004). In one test of cry^sup b^ 's effects on whole-animal rhythm-related phenotypes, mutant individuals re-synchronized their behavior in a largely normal manner to shifted light:dark (LD) cycles; "L" in the postshift regime was dim blue light (STANEWSKY et al. 1998). However this mutant type required longer-than-normal times to resynchronize (EMERY et al. 2000b). Much poorer photic sensitivity was observed when a no-receptor-potential A mutation (norpA^sup P41^), which probably causes blindness at the level of all external photoreceptors, was added to a cry^sup b^ genetic background. …

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