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Although we hypothesize that the short isoform is responsible for the period altering effect, our results do not rule out the possibility that these higher molecular weight complexes contribute to the observed phenotypes. As demonstrated by Western analysis, the novel isoform has a slightly lower molecular weight compared to the known isoform of DBT, indicating a shorter amino acid sequence. At present, we do not know which dbt transcript expresses the short DBT isoform although all four transcripts are capable of encoding it. We also note that our results do not rule out an alternative but unlikely possibility that LARK OE results in DBT proteolytic cleavage resulting in the smaller isoform.

Thus, it is possible that the short DBT isoform has no kinase activity but rather plays a structural role. A non-catalytic role of DBT has been suggested by others in a recent study. Yu et. These results suggest that the short-isoform and full-length kinase may interact to set the speed of the clock. A plausible hypothesis is that the short DBT isoform serves as a non-catalytic subunit which modulates full-length DBT kinase.

Among these LARK-associated mRNAs, only three others encode proteins that are known to be involved in circadian function: flapwing flw , no receptor potential A norpA , and dunce dnc. Thus, the known clock regulation of LARK [47] may, in part, depend on pmediated phosphorylation of the protein. Alterations in DBT expression are predicted to modulate circadian period, by affecting either the accumulation or degradation of PER. Chiu et al. Lack of per-short domain phosphorylation leads to faster degradation of PER and short-period behavioral rhythms [40].

These results are consistent with a previous study suggesting that the per-short domain regulates the activity of DBT against PER [75]. Thus, enhanced or prolonged phosphorylation of this domain may lengthen period. We postulate that increased LARK expression and production of a short, non-catalytic DBT isoform leads to delayed PER degradation and lengthened circadian period by altering the timing of DBT-mediated phosphorylation of the per short domain. The observation that dbt P , which is a hypomorphic allele of dbt , enhances the period lengthening effect of LARK OE compare Figure 6 with Figure 5 , also see Table S1 suggests that alteration of the short to full-length DBT ratio may be responsible for period lengthening.

Interestingly, a mutant form of DBT DBT AR that was suggested to play a non-catalytic, auxiliary role — similar to our proposal for the DBT short isoform — also causes period lengthening in heterozygotes [75]. This novel transcript, dbt - RE , shows a translational oscillation that is in phase with LARK abundance in LD conditions and it can be induced by light in dark conditions. Together with the observation that LARK abundance is highest at the beginning of the day [47] , these results suggest that this RNA-binding protein may be light inducible in addition to showing circadian variation.

Although a role for LARK and DBT in pacemaker light sensitivity has not been reported, our study suggests it may be of interest to explore this possibility. The following stocks were obtained from the Bloomington Stock Center stock number in parenthesis : w , elav-gal4 , uas-dbt and dbt P and uas-dicer2 Justin Blau, pdf-gal4 was obtained from Dr. Paul Hardin, uas-dbt DN was provided by Dr.

double time

Marek Mlodzik. The antibodies were coupled to Dynabeads Invigrogen according to manufacturer's instruction. Joel D. RNA-protein binding reactions were carried out according to [53]. Heads of the frozen flies were harvested and ground into fine powder in liquid Nitrogen. Cleared tissue lysate was obtained after the centrifugation and the concentration of total protein was determined. Electrophoresis and western blotting were carried out according to standard protocols. Details of the TRAP method are described in [55].

Data were collected with Strategene Mx or Mx A pair of primers specific for the Ribosomal Protein 49 Rp49 gene, which is known to be transcribed and translated at a constant rate throughout the circadian cycle Huang and Jackson, unpublished observation , was used as an internal reference to account for variation in the input cDNA amount.

Visualization of actograms and the analysis of rhythmicity and period length were performed using a signal processing toolbox [76] within the MATLAB software package MathWorks. The toolbox analyzes circadian rhythmicity of fly locomotor activity by applying an autocorrelation analysis.

Double Time

The Rythmicity Index RI is defined as the height of the third peak in the correlogram resulting from this analysis counting the peak at lag 0 as the first peak. Period length is determined by Fourier analysis [76]. Flies were considered rhythmic if they had a high RI value generally greater than 0. Immunohistochemistry was carried out according to standard procedure for staining whole mount fly brains. The section with the largest cell diameter, i. Average pixel intensity within the ROI was calculated for each individual l-LNv cell in a brain hemisphere.

The value obtained for individual cells were then further averaged among all cells in a same brain hemisphere to get a value for each individual animal. Different versions of genome annotation of the dco i. A, Previous annotation from Release 5. Current annotation from Release 5. Existing EST sequences aligning to the region of the genome. Location of primers used to specifically amplify individual dbt transcripts in Q-RTPCR experiments are indicated by red arrows.


The forward primers for RA and RB each span a splice junction, thus were drawn across the respective introns, although their sequences does not include any intronic sequence. Abundance profile of dbt-RC in the first day of DD. Abundance profile of dbt-RE in LD. Abundances in the time series are normalized to that of the first time point. Each individual image was scored by two different observers and the two scores were then averaged. Scores of all images for the same genotype at the same time point were averaged and plotted.

OE: overexpression. OC: control for overexpression. Times of sample collections ZT2 or ZT14 are indicated.

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Black arrow: known DBT isoform. Red arrow: novel short DBT isoform. Arrow head: high molecular weight DBT-immunoreactive bands. Molecular weight standards in KD are shown on the left side of the image. Western blot showing that LARK overexpression does not induce the smaller isoform at pupal stage. Time of sample collections are indicated. Overexpression of LARK was achieved by driving uas-lark with elav-gal4. Samples extracted from whole pupae are on the left lanes 1—4 , sample extracted from adult heads as a positive control are on the right lanes 6—7.

Lane 5: molecular weight ladder.

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Upper and lower panels show the same blot with different exposure times. Exposure time in the lower panel was reduced to allow a clear view of the novel short DBT isoform in the adult OE sample. Period and Rythmicity Index RI for all characterized genotypes. Abbreviated labels are the same as those used in Figures. The table shows genotypes, number of flies tested, the rhythmic fraction of flies tested, rhythmicity index, and circadian period. We thank the Bloomington Stock Center and Drs.

We thank Mary Roberts for assistance with locomotor activity experiments, Dr. Introduction The Drosophila doubletime dbt , a. Download: PPT. Circadian or diurnal changes in the translation of two low-abundance dbt transcripts In wild-type flies, LARK shows a circadian oscillation in abundance; the level of LARK is high during the day and low at night [47]. Figure 3. Light induced translation of dbt-RE The observations that translation of dbt-RE displays a robust cycle under LD but not DD, and that peak translation occurs shortly after lights-on suggest that its translation might be induced by light.

Figure 4. Figure 5.

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Figure 6. Interactions between lark and dbt modulate circadian period. DBT kinase activity is required to mediate the effect of LARK overexpression Our previous research found that high level LARK overexpression, using two copies each of pdf-gal4 and uas-lark , resulted in complete arrythmicity [50]. Figure 7. Figure 8.

Discussion Despite many studies of DBT function in cellular signaling pathways and circadian period determination, little is known about the regulation of DBT itself. Materials and Methods Drosophila strains, rearing conditions and genetic crosses The following stocks were obtained from the Bloomington Stock Center stock number in parenthesis : w , elav-gal4 , uas-dbt and dbt P and uas-dicer2 Supporting Information. That joy lasted until her first jumping pass. She two-footed the landing of her triple Lutz, then squeaked out only a single toe loop in combination.

Her jumps went downhill from there. In the second half, she singled her second triple Lutz in more ways than one. Her future self was late or maybe her present self was rushing ahead of the music. Only one of her was on the ice to attempt the jump when she popped it, turning only one revolution in the air before landing. Older Shelly landed a triple. Younger Shelly landed a double instead. Even with the bonus for elements completed in the second half of the program, she was hemorrhaging points.

Her lungs burned. Her body stung from the falls. The ice felt like mud beneath her skates. Their applause thundered across the rink even when someone skated like a human Zamboni. Flowers and product placements dotted the kiss and cry area. Shelly and Mr. Song sat on a bench waiting for her score. The backdrop showed mountains and a camera was trained on them, capturing their reactions. Her humiliation had been and would continue to be broadcast worldwide. However, she refused to cry. Song rubbed his hands together. Song crossed his arms over his chest. Her score boomed over the PA system.

Skating in double time was stupid. Only two other skaters had attempted it here. Mom sat by the aisle reading some engineering journal on her ereader. Shelly sat by the window staring at her tablet. Words gathered in dense blocks covered half the screen while a keyboard covered the other half. Shelly kind of wished it would. She exchanged her ereader for an eye mask and neck pillow. When you were four and decided you wanted to skate, I was so happy, but skaters like her come once in a lifetime.

Song when we get home. With that, Mom shut off her overhead light then went to sleep. The drone of engines covered up any other whispered conversations, isolating Shelly within her pool of light. Tears welled in her eyes. For once, Mom had relented. Shelly should have been relieved, so why did she feel so awful? It had been at least a decade since Mr. Song skated competitively but, in skates, he always looked as though he could land quad Lutzes as a warm up. On the ice, Mr. Song might have been a student waiting for his coach rather than a coach waiting for his student.

Only Senior-level skaters trained this early in the morning and, as they warmed up on the ice, Shelly took her time lacing her skates. She was stalling and she knew it. Nationals was only six weeks away. Do you have time elsewhere in the day to lose? Song grinned as he rubbed his hands and Shelly started cross-stroking around the rink. The less Shelly thought about her short program at Nationals, the better. The judges had rather generously placed her sixth, about ten points behind a still recovering Emily Takahashi in first.

For the free skate, Shelly had drawn last in the skate order.

Her free skate seemed more like a formality they had to plow through than anything to do with deciding who would advance to either Four Continents or Worlds. The crowd cheered as she skated to center ice. Emily Takahashi had backed off skating her entire routine in double time to spare her healing toe, skating only the back half that way as Shelly had also planned. Future her had gone too far back in time. Years seemed to pass before the world stopped teetering for either of them. They struck their opening poses then the music started.

Michelle Kwan once said that to skate Lyra Angelica, she just went onto the ice, then thought of angels. The two Shellys skated as one, hitting every jump and spin in unison. They glided across the rink etching intricate patterns in swift arcs on the ice. Her lungs burned and her legs grew rubbery. For three minutes and fifty-nine seconds, she was the avenging angel.

Her every edge and gesture was determined to prove that she could too skate. She refused to implode on the ice. Not again. Finally, the two Shellys hit their final pose to the last beat of the music and the younger disappeared with a flash of gold into the past. She might have under-rotated her triple Salchow and skipped some steps in her footwork.

The Technical Caller would sort that out via slow-motion video replay after her skate. The audience exploded into generous, even for Nationals, applause. Flowers wrapped in cellophane, teddy bears, and other stuffed animals fell like thick hail on the ice. Tiny girls, ten years old at most, swarmed the rink collecting it all. Everyone in the audience appeared to be. Shelly bowed.

Every breath flayed her lungs and her legs felt like water. Improve This Listing. Show options.

Double-Time Bra – Outdoor Voices

Reviews 6. Write a review. Filter reviews. Traveler rating. Excellent 6. Very good 0. Average 0. Poor 0. Terrible 0. Traveler type. Time of year. Language English. All languages. English 6. See what travelers are saying:. Selected filters. Updating list Reviewed June 20, Excellent fishing trip.