At Least Four Distinct Circadian Regulatory Mechanisms Required for All Phases of Rhythms in mRNA Amount

Since the advent of techniques to investigate gene expression on a large scale, numerous circadian rhythms in mRNA abundance have been reported. These rhythms generally differ in amplitude and phase. First studies on circadian rhythms of transcription on a large scale are also emerging. We investigated to what extent the same circadian regulatory mechanism of transcription can give rise to rhythms in RNA amount that differ in phase solely based on a parameter that is not regulated by the circadian clock. Using a discrete-time approach, we modeled a sinusoidal rhythm in transcription with various constant exponential RNA decay rates. We found that the slower the RNA is degraded the later the phase of the RNA amount rhythm compared to the phase of the transcriptional rhythm. However, we also found that the phase of the rhythm in RNA amount is limited to a time frame spanning the first quarter of the period following the phase of the transcriptional rhythm. This finding is independent of the amplitude and vertical shift of the transcriptional rhythm or even of the way RNA degradation is modeled. We confirmed our results with a continuous-time model, which also allowed us to derive a simple formula that relates the phase of a rhythm in mRNA amount solely to the phase and period of its sinusoidal transcriptional rhythm and its constant mRNA half-life. This simple formula even holds true for the best sinusoidal approximations of a non-sinusoidal rhythm of transcription and mRNA amount. When using our discrete-time approach to model constant rates of transcription with a sinusoidal RNA half-life, on the other hand, we found that varying the constant component of the system, i.e. the rate of transcription, does not change the phase of the rhythm in RNA amount. In summary, our data show that at least four distinct circadian regulatory mechanisms are required to allow for all phases in rhythms of RNA amount, one for each quarter of the period

Improved automated monitoring and new analysis algorithm for circadean phototaxis rhythms in Chlamydomonas

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computercontrolled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotopes

Genomic analysis of a large set of phages infecting the common hostMycobacterium smegmatis mc2155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode