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Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila.

Levine JD, Funes P, Dowse HB, Hall JC - BMC Neurosci (2002)

Bottom Line: Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing.Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and NSF Center for Biological Timing, Brandeis University, Waltham, MA 02454, USA. jlev@brandeis.edu

ABSTRACT

Background: Previously, we reported effects of the cry(b) mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.

Results: Isolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cry(b) alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions.

Conclusions: These analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

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Effect of detrending and normalization on the analysis of luciferase-reported rhythms. This example is taken from an antennal pair isolated from a BG-luc; cry+ adult. luc activity was recorded for 8 days in constant darkness and at a constant temperature of 27°C. a) The upper panel in this column shows a plot of the raw data given in counts per second of bioluminescence plotted on the ordinate. Samples were recorded hourly as indicated on the abcissa. The dashed line illustrates a trend line defined by the application of a 72 hour low pass filter (see [27] for more detail about this use of filters). The lower panel shows the autocorrelation function for the raw data plotted immediately above. We use the autocorrelation function to detect rhythmicity[23,27]. Correlation coefficients with range from -1 to 1 appear on the ordinate and the lag is plotted on the abscissa. The shaded area centered around 0 defines a 95% confidence interval. In this case, the shape of the function indicates one rhythmic cycle followed by a decrease, a pattern that suggests the signal is not rhythmic, b) The upper panel in this column shows the detrended and normalized signal obtained from the raw data shown in a). The signal was obtained by dividing each data value by the corresponding value on the trend line for a given time point. The values on the ordinate are given in arbitrary units and vary around a mean of 1 (as described in [27]). The lower panel shows the autocorrelation function for the detrended and normalized signal shown immediately above it. The application of these procedures reveals the presence of significant rhythmicity within the data set shown in a) above. The RI value is the value of the autocorrelation function at the third peak (marked by the asterisk); it indicates the strength of rhythmicity (see Table 1, Materials and Methods and [27] for more detail). The RS value is obtained from the ratio of the RI value to the 95% confidence line. Thus when RS is ≥ 1, the rhythm is statistically significant (see Materials and Methods for more detail). The absence of an asterisk in the lower panel of a) indicates the absence of a third peak in the autocorrelation function for the raw data set.
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Figure 1: Effect of detrending and normalization on the analysis of luciferase-reported rhythms. This example is taken from an antennal pair isolated from a BG-luc; cry+ adult. luc activity was recorded for 8 days in constant darkness and at a constant temperature of 27°C. a) The upper panel in this column shows a plot of the raw data given in counts per second of bioluminescence plotted on the ordinate. Samples were recorded hourly as indicated on the abcissa. The dashed line illustrates a trend line defined by the application of a 72 hour low pass filter (see [27] for more detail about this use of filters). The lower panel shows the autocorrelation function for the raw data plotted immediately above. We use the autocorrelation function to detect rhythmicity[23,27]. Correlation coefficients with range from -1 to 1 appear on the ordinate and the lag is plotted on the abscissa. The shaded area centered around 0 defines a 95% confidence interval. In this case, the shape of the function indicates one rhythmic cycle followed by a decrease, a pattern that suggests the signal is not rhythmic, b) The upper panel in this column shows the detrended and normalized signal obtained from the raw data shown in a). The signal was obtained by dividing each data value by the corresponding value on the trend line for a given time point. The values on the ordinate are given in arbitrary units and vary around a mean of 1 (as described in [27]). The lower panel shows the autocorrelation function for the detrended and normalized signal shown immediately above it. The application of these procedures reveals the presence of significant rhythmicity within the data set shown in a) above. The RI value is the value of the autocorrelation function at the third peak (marked by the asterisk); it indicates the strength of rhythmicity (see Table 1, Materials and Methods and [27] for more detail). The RS value is obtained from the ratio of the RI value to the 95% confidence line. Thus when RS is ≥ 1, the rhythm is statistically significant (see Materials and Methods for more detail). The absence of an asterisk in the lower panel of a) indicates the absence of a third peak in the autocorrelation function for the raw data set.

Mentions: While carrying out those studies, we became aware that there are interesting features of these peripheral rhythms, like the phase of peak expression, for example, that we were unable to compare rigorously amongst body parts. Such concerns motivated us to develop a set of tools for the analysis of circadian rhythms [27]. Here we apply two key features of this analytic package that were unavailable for our earlier report on the effects of cryb in the periphery [23]. First is a method to eliminate nonlinear trends in the data, a potential source of artifact that could affect whether luciferase activity is scored as rhythmic or arrhythmic. Figure 1 illustrates how rhythmicity generated by one antennal pair is extracted from rather weak output by the application of appropriate analytic tools. In addition, the normalization procedure facilitates comparisons between rhythms from different tissues by reducing the scale of the output to a mean of one for each case (see Results). Second, we were not previously able to examine effects of a given variable (genotype, body part) on circadian phase. The application of circular statistics to these data allows us to evaluate such effects.


Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila.

Levine JD, Funes P, Dowse HB, Hall JC - BMC Neurosci (2002)

Effect of detrending and normalization on the analysis of luciferase-reported rhythms. This example is taken from an antennal pair isolated from a BG-luc; cry+ adult. luc activity was recorded for 8 days in constant darkness and at a constant temperature of 27°C. a) The upper panel in this column shows a plot of the raw data given in counts per second of bioluminescence plotted on the ordinate. Samples were recorded hourly as indicated on the abcissa. The dashed line illustrates a trend line defined by the application of a 72 hour low pass filter (see [27] for more detail about this use of filters). The lower panel shows the autocorrelation function for the raw data plotted immediately above. We use the autocorrelation function to detect rhythmicity[23,27]. Correlation coefficients with range from -1 to 1 appear on the ordinate and the lag is plotted on the abscissa. The shaded area centered around 0 defines a 95% confidence interval. In this case, the shape of the function indicates one rhythmic cycle followed by a decrease, a pattern that suggests the signal is not rhythmic, b) The upper panel in this column shows the detrended and normalized signal obtained from the raw data shown in a). The signal was obtained by dividing each data value by the corresponding value on the trend line for a given time point. The values on the ordinate are given in arbitrary units and vary around a mean of 1 (as described in [27]). The lower panel shows the autocorrelation function for the detrended and normalized signal shown immediately above it. The application of these procedures reveals the presence of significant rhythmicity within the data set shown in a) above. The RI value is the value of the autocorrelation function at the third peak (marked by the asterisk); it indicates the strength of rhythmicity (see Table 1, Materials and Methods and [27] for more detail). The RS value is obtained from the ratio of the RI value to the 95% confidence line. Thus when RS is ≥ 1, the rhythm is statistically significant (see Materials and Methods for more detail). The absence of an asterisk in the lower panel of a) indicates the absence of a third peak in the autocorrelation function for the raw data set.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC103668&req=5

Figure 1: Effect of detrending and normalization on the analysis of luciferase-reported rhythms. This example is taken from an antennal pair isolated from a BG-luc; cry+ adult. luc activity was recorded for 8 days in constant darkness and at a constant temperature of 27°C. a) The upper panel in this column shows a plot of the raw data given in counts per second of bioluminescence plotted on the ordinate. Samples were recorded hourly as indicated on the abcissa. The dashed line illustrates a trend line defined by the application of a 72 hour low pass filter (see [27] for more detail about this use of filters). The lower panel shows the autocorrelation function for the raw data plotted immediately above. We use the autocorrelation function to detect rhythmicity[23,27]. Correlation coefficients with range from -1 to 1 appear on the ordinate and the lag is plotted on the abscissa. The shaded area centered around 0 defines a 95% confidence interval. In this case, the shape of the function indicates one rhythmic cycle followed by a decrease, a pattern that suggests the signal is not rhythmic, b) The upper panel in this column shows the detrended and normalized signal obtained from the raw data shown in a). The signal was obtained by dividing each data value by the corresponding value on the trend line for a given time point. The values on the ordinate are given in arbitrary units and vary around a mean of 1 (as described in [27]). The lower panel shows the autocorrelation function for the detrended and normalized signal shown immediately above it. The application of these procedures reveals the presence of significant rhythmicity within the data set shown in a) above. The RI value is the value of the autocorrelation function at the third peak (marked by the asterisk); it indicates the strength of rhythmicity (see Table 1, Materials and Methods and [27] for more detail). The RS value is obtained from the ratio of the RI value to the 95% confidence line. Thus when RS is ≥ 1, the rhythm is statistically significant (see Materials and Methods for more detail). The absence of an asterisk in the lower panel of a) indicates the absence of a third peak in the autocorrelation function for the raw data set.
Mentions: While carrying out those studies, we became aware that there are interesting features of these peripheral rhythms, like the phase of peak expression, for example, that we were unable to compare rigorously amongst body parts. Such concerns motivated us to develop a set of tools for the analysis of circadian rhythms [27]. Here we apply two key features of this analytic package that were unavailable for our earlier report on the effects of cryb in the periphery [23]. First is a method to eliminate nonlinear trends in the data, a potential source of artifact that could affect whether luciferase activity is scored as rhythmic or arrhythmic. Figure 1 illustrates how rhythmicity generated by one antennal pair is extracted from rather weak output by the application of appropriate analytic tools. In addition, the normalization procedure facilitates comparisons between rhythms from different tissues by reducing the scale of the output to a mean of one for each case (see Results). Second, we were not previously able to examine effects of a given variable (genotype, body part) on circadian phase. The application of circular statistics to these data allows us to evaluate such effects.

Bottom Line: Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing.Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and NSF Center for Biological Timing, Brandeis University, Waltham, MA 02454, USA. jlev@brandeis.edu

ABSTRACT

Background: Previously, we reported effects of the cry(b) mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.

Results: Isolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cry(b) alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions.

Conclusions: These analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

Show MeSH
Related in: MedlinePlus