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cryptochrome genes form an oscillatory loop independent of the per / tim loop in the circadian clockwork of the cricket Gryllus bimaculatus

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ABSTRACT

Background: Animals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood.

Results: In the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb’cry1) and mammalian-type cry2 (Gb’cry2). Gb’cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb’cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb’cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb’cry1RNAi. In Gb’cry1/Gb’cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb’cry1 and Gb’cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells.

Conclusion: Based on these results, we propose a new model of the cricket’s circadian clock, including a molecular oscillatory loop for Gb’cry2, which can operate independent of the Gb’per/Gb’tim loop.

No MeSH data available.


A model for the cricket circadian clock. The clock includes two negative feedback loops, one for Gb’per/Gb’tim like in Drosophila and the other for Gb’cry2. The latter is comprised of two Gb’cry2 variants, Gb’cry2c and Gb’cry2f, or Gb’cry2c and Gb’cry1, and their product proteins that form a complex to suppress the transcription mediated by Gb’CLK/Gb’CYC complex. These two loops may be able to operate independently but interact with each other through influence on the Gb’CLK/Gb’CYC. In addition, there are two other loops for rhythmic expression of Gb’Clk and Gb’cyc [12]
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Fig8: A model for the cricket circadian clock. The clock includes two negative feedback loops, one for Gb’per/Gb’tim like in Drosophila and the other for Gb’cry2. The latter is comprised of two Gb’cry2 variants, Gb’cry2c and Gb’cry2f, or Gb’cry2c and Gb’cry1, and their product proteins that form a complex to suppress the transcription mediated by Gb’CLK/Gb’CYC complex. These two loops may be able to operate independently but interact with each other through influence on the Gb’CLK/Gb’CYC. In addition, there are two other loops for rhythmic expression of Gb’Clk and Gb’cyc [12]

Mentions: We have shown that the cricket circadian clock includes a loop for rhythmic expression of Gb’per and Gb’tim [9, 10], of which transcription is regulated by Gb’CLK/Gb’CYC [11, 12]. It has been suggested that CRY2 forms a complex with PER or the PER/TIM complex to enter the nucleus and repress the CLK/CYC transcriptional activity in other insects [5, 6]. However, the results of the present study suggest that Gb’CRY2 composes a loop to repress Gb’CLK/Gb’CYC activity by forming complex between its variants or with Gb’CRY1, and that the loop can operate independent of the Gb’per/Gb’tim loop to some degree (Fig. 8). This hypothesis is based on the following observations. When Gb’tim was knocked down by RNAi or Gb’tim and Gb’per stopped their oscillation by Gb’cycRNAi [12], Gb’cry2 maintains its oscillation within a normal range but with a slightly attenuated amplitude (Fig. 7). Alternatively when Gb’cry2 is knocked down, Gb’tim maintained its oscillation. Our data suggest that the circadian locomotor rhythm is expressed when either Gb’cry2 or Gb’tim is rhythmically expressed.Fig. 8


cryptochrome genes form an oscillatory loop independent of the per / tim loop in the circadian clockwork of the cricket Gryllus bimaculatus
A model for the cricket circadian clock. The clock includes two negative feedback loops, one for Gb’per/Gb’tim like in Drosophila and the other for Gb’cry2. The latter is comprised of two Gb’cry2 variants, Gb’cry2c and Gb’cry2f, or Gb’cry2c and Gb’cry1, and their product proteins that form a complex to suppress the transcription mediated by Gb’CLK/Gb’CYC complex. These two loops may be able to operate independently but interact with each other through influence on the Gb’CLK/Gb’CYC. In addition, there are two other loops for rhythmic expression of Gb’Clk and Gb’cyc [12]
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Fig8: A model for the cricket circadian clock. The clock includes two negative feedback loops, one for Gb’per/Gb’tim like in Drosophila and the other for Gb’cry2. The latter is comprised of two Gb’cry2 variants, Gb’cry2c and Gb’cry2f, or Gb’cry2c and Gb’cry1, and their product proteins that form a complex to suppress the transcription mediated by Gb’CLK/Gb’CYC complex. These two loops may be able to operate independently but interact with each other through influence on the Gb’CLK/Gb’CYC. In addition, there are two other loops for rhythmic expression of Gb’Clk and Gb’cyc [12]
Mentions: We have shown that the cricket circadian clock includes a loop for rhythmic expression of Gb’per and Gb’tim [9, 10], of which transcription is regulated by Gb’CLK/Gb’CYC [11, 12]. It has been suggested that CRY2 forms a complex with PER or the PER/TIM complex to enter the nucleus and repress the CLK/CYC transcriptional activity in other insects [5, 6]. However, the results of the present study suggest that Gb’CRY2 composes a loop to repress Gb’CLK/Gb’CYC activity by forming complex between its variants or with Gb’CRY1, and that the loop can operate independent of the Gb’per/Gb’tim loop to some degree (Fig. 8). This hypothesis is based on the following observations. When Gb’tim was knocked down by RNAi or Gb’tim and Gb’per stopped their oscillation by Gb’cycRNAi [12], Gb’cry2 maintains its oscillation within a normal range but with a slightly attenuated amplitude (Fig. 7). Alternatively when Gb’cry2 is knocked down, Gb’tim maintained its oscillation. Our data suggest that the circadian locomotor rhythm is expressed when either Gb’cry2 or Gb’tim is rhythmically expressed.Fig. 8

View Article: PubMed Central - PubMed

ABSTRACT

Background: Animals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood.

Results: In the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb’cry1) and mammalian-type cry2 (Gb’cry2). Gb’cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb’cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb’cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb’cry1RNAi. In Gb’cry1/Gb’cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb’cry1 and Gb’cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells.

Conclusion: Based on these results, we propose a new model of the cricket’s circadian clock, including a molecular oscillatory loop for Gb’cry2, which can operate independent of the Gb’per/Gb’tim loop.

No MeSH data available.