Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans.

Substantial progress has been made in understanding the genetic basis of temperature-compensated circadian clocks. Ultradian rhythms, with a period shorter than 24 h, are at least as widespread as circadian rhythms. We have initiated genetic analysis of defecation behavior, which is controlled by an ultradian clock in Caenorhabditis elegans. The defecation motor program is activated every 45 sec, and this rhythm is temperature compensated. We describe mutations in 12 genes that either shorten or lengthen the cycle period. We find that most of these mutations also disrupt temperature compensation, suggesting that this process is an integral part of the clock. These genes open the way for molecular genetic dissection of this ultradian clock.

Molecular coevolution within a Drosophila clock gene

The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract. Similar repeats are found in the frq and wc2 clock genes of Neurospora crassa and in the mammalian per homologues, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster, whose repetitive regions differ in length by about 150 amino acids. The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock’s temperature compensation.

Circadian rhythms in Neurospora crassa: Lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq9 and frq10) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq9, chol-1 frq10, chol-1 wc-1, chol-1 wc-2, cel frq9, cel frq10, and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.

The oxygen and carbon dioxide compensation points of C3 plants: possible role in regulating atmospheric oxygen.

The O2 and CO2 compensation points (O2 and CO2) of plants in a closed system depend on the ratio of CO2 and O2 concentrations in air and in the chloroplast and the specificities of ribulose bisphosphate carboxylase/oxygenase (Rubisco). The photosynthetic O2 is defined as the atmospheric O2 level, with a given CO2 level and temperature, at which net O2 exchange is zero. In experiments with C3 plants, the O2 with 220 ppm CO2 is 23% O2; O2 increases to 27% with 350 ppm CO2 and to 35% O2 with 700 ppm CO2. At O2 levels below the O2, CO2 uptake and reduction are accompanied by net O2 evolution. At O2 levels above the O2, net O2 uptake occurs with a reduced rate of CO2 fixation, more carbohydrates are oxidized by photorespiration to products of the C2 oxidative photosynthetic carbon cycle, and plants senesce prematurely. The CO2 increases from 50 ppm CO2 with 21% O2 to 220 ppm with 100% O2. At a low CO2/high O2 ratio that inhibits the carboxylase activity of Rubisco, much malate accumulates, which suggests that the oxygen-insensitive phosphoenolpyruvate carboxylase becomes a significant component of the lower CO2 fixation rate. Because of low global levels of CO2 and a Rubisco specificity that favors the carboxylase activity, relatively rapid changes in the atmospheric CO2 level should control the permissive O2 that could lead to slow changes in the immense O2 pool.