CHAPTER2

timrit lengthens circadian period in a temperature dependent manner through suppression of PERIOD protein cycling and

nuclear localization.

- p.

30-ABSTRUCT

A fundamental feature of circadian clocks is temperature compensation of period. The freerunning period of

rits

u

(ti�t),

a novel allele of

timeless,

is drastically lengthened in a

temperature dependent manner. PER and TIM protein levels become lower in

timnr

as temperature becomes higher. This mutation reduces

per

mRNA but not

tim

mRNA abundance. PER

constitutively driven by the

rhodopsin]

promoter is lowered in

rit,

indicating that

ti�t

mainly affects the

per

feedback loop at a post-transcriptional level. An excess of

per+

gene dosage can ameliorate all

rit

phenotypes including the weak nuclear localization of PER, suggesting that

timnt

affects circadian rhythms by reducing PER abundance and it's subsequent transportation into nuclei as temperature increases.

INTRODUCTION

The circadian clock keeps its period even when there are no environmental time cues. The clock's freerunning period remains relatively constant with a change in temperature of

1 0°C,

and the temperature quotient,

Q10,

is approximately

1.

The biochemical mechanisms underlying circadian rhythms are clearly distinct from biochemical reactions observed in other physiological and developmental events, because the

Q10

of those reactions is nearly

2�3.

Although the molecular mechanism to generate circadian fluctuation has been extensively studied, there are only a few molecular studies in

Drosophila

on the temperature compensation mechanism. At a behavior level,

per

mutants affect not only period length but also temperature compensation;perr and

per

mutants slightly shorten and lengthen their periods, respectively, as temperature increases (Konopka et al.,

1989;

Ewer et al.,

1990;

Konopka et al.,

1994).

Several molecular studies suggested that the temperature compensation is closely associated with PER.

Huang et al.

(1995)

reported that PER can undergo a temperature independent intramolecular dimerization, while Gekakis et al.

( 1995)

showed that PERL exhibits a temperature dependent defect in binding to TIM although the molecular interaction between TIM and PER is temperature compensated. Moreover, an allele of the

tim

gene,

timsL,

can compensate a temperature dependent

- p.

31

-period lengthening of

per

(Rutila et al., 1996). The length of the Thr-Gly repeat in PER is also reported to affect the temperature compensation (Sawyer et al., 1997).

I isolated

ritsu (rit),

a clock mutant on the second chromosome from a natural population (Murata et al., 1995). I have now investigated features of

rit

and its interaction with

per

and

tim

at both the behavioral and molecular levels.

rit

shows abnormal temperature compensation of period and reduces PER and TIM levels. Molecular genetic analyses show that

rit

has a point mutation in the

tim

gene that leads to a single amino acid change, indicating

rit

is an allele of

tim.

Since an excess of the

per

gene dosage ameliorates the weak and delayed nuclear localization of PER as well as all other phenotypes of

rit

at both the molecular and behavioral levels, PER abundance in nuclei seems to be a key factor in the temperature compensation mechanism.

MATERIALS AND METHODS

Stocks, locomotor rhythms recording and mating procedures. Flies were kept under LD 12: 12 at 24 ^°C^. Can

t

on

-S

was used as wild-type. Double mutants between

rit

and

per

mutants were

synthesized by standard crosses. Flies were grown at 24 ^°C^. Locomotor activity was recorded in the same way described elsewhere (Matsumoto et al., 1994). The period of a locomotor rhythm was calculated by the chi-square periodogram analysis (Sokolove and Bushell, 1978). The mating procedure for the recombination test between

tim

and

rit

were done as follows (see Fig. SA). To produce flies carrying both the

rit

mutation and the

per-

lacZ fusion gene on the second

chromosome, I crossed

rit; ry

females to

per-SG:3; ry

males which carry the

per-

lacZ fusion gene on the second chromosome. These strains carried

ry

mutation on the third chromosome and this eye color should be rescued if a fly has the

per

^-lacZ fusion gene. After two generations I selected

rit

per-lacZhomozygous flies based on the

ry+

eye color and the long period

rit

phenotype. The four lines were selected as a

rit per-

lacZ strain.

rit rh-per

strain was produced according to the standard

mating procedures usingSMJ/Sco;

TM3/Pr

(Lindsley and Zimm, 1992)).

+/w+Y; rit,

C(J)DX,

y w f lw+Y; rit, Dp(2;Y)odd4·31; rit, Dp(2;Y)odd2.31; rit

and

w; rit

strains were produced the according to the mating procedures described elsewhere (Matsumoto et al., 1994) with the minor changes. The

p. 32

-recombination test was done by two different mating procedures. The rit/tim females were mated to tim01 males in one cross and mated to SMJ!Pm males in the other crosses. SMJ/Pm flies show a normal rhythmicity, and were designated by+/+ in Fig. 3A (right). In both cases, progenies from these crosses were then monitored for locomotor rhythms at 30°C. If the recombination between tim and rit occurs, there should be rif tim+ /tim01 progenies whose rhythm is normal in the former cross.

In the latter cross, the double mutant ofrit tim!SMJ or rit tim/Pm whose rhythm is abnormal could be obtained if recombination occurs.

RNase protection assay. Flies were entrained in LD12:12 for five days before they were collected. Total RNA were extracted from 50 fly heads in 500 Jll extraction buffer (15 mM NaOAc,

5 mM EDT A, 1% SDS, 0.01% diethyl pyrocarbonate, 50 mM Tris, pH 9.0). RNase free DNase (Boehringer) were used to remove contaminated DNA. RNase protection assays were done as described elsewhere (Hardin et al., 1990) with minor modifications. I used the per5 and TIMAX1 probe and rp49 as a control. The per5 probe is a genomic fragment of the per gene containing about 210bp (per5849-6060) of the per exon5. TIMAXI is a eDNA fragment of the tim gene from 4963 to 5192nt (Sehgal et al., 1995). I found that the abundance of rp49 in samples obtained at 30°C was half as much as that at 24 OC, although the reason was unknown. So each measurement was

nonnalized by the value of the peak level in wild-type at each temperature.

Immunoblot analyses. Protein extracts were made from 50 fly heads for each time point as

in Edery et al. (1994) with minor modifications. E ach sample was homogenized in 15 Jll ice-cold extraction buffer, and the tip of homogenizing pestle was rinsed twice with another 15 Jll extraction buffer. Amount of proteins in total of 45 Jll extraction buffer were measured by Bradford protein assay system (Bio-Rad). After the measurement, extraction buffer was added to make protein concentration to 5 Jlg/Jll in each sample. Five microliters of 3 X SDS sample buffers were add to 10

�-tl of samples and boiled for 5 min. Supernatants were loaded on 5% SDS-PAGE gel. Western blotting was done as described (Edrey et al., 1994) with minor modifications. I used anti-TIM antibody gifted by J. Blau diluted in 1:5000. HRP-conjugated anti-Rat lgG antibody (Cappel) was used as a secondary antibody with diluting 1:3000. For quantitating of chemiluminesence

(SuperSignal CL-HRP Substrate system, Pierce), an exposure on X-lay film (FUn FILM) was

p. 33

-digital-imaged by Densito Graph (ATTO) and quantified by Nlli-image (Nlli) software. After the exposure, the membrane used was incubated for 3 hrs in the substrate buffer in order to eliminate theHRP activity. Then the membrane was used to detect PER abundance as follows. I used anti­

PER antibody gifted by R. Stanewsky diluted in 1:10000. HRP-conjugated anti-Rabbit IgG antibody (Cappel) was used as a secondary antibody with diluting 1:3000. The exposure and quantification were done as described above.

Histology. Expression of the per gene in rit flies were assayed histologically using the per-lacZ fusion gene as a reporter. Flies at ZT18 and ZT6 were frozen in a liquid nitrogen and mounted into O.C.T. compound (Tissue-Tek). Sections of 10 �m were cut and stained by X-gal. The staining procedure was done according to Liu et al. (1988). Head sections of wild-type and rit flies were embedded side-by-side on the same slide to compare the staining profiles of the two strains.

Photographs were taken with a Zeiss AxioPhot microscope. For fluorescent immunostaining with anti-PER antibody, the white eyed strain was used as wild-type to eliminate a background

fluorescence of eye pigment. For the same purpose the double mutant ofw; rit was used. The sections (14 Jlm) of two strains were embedded side-by-side on the same slide to ease to compare the strength of signals. Anti-PER antibody was applied at dilution of 1:15,000. Anti-rabbit IgG conjugated to peroxidase labelled-dextran polymer (En Vision+ , Dako) was applied as a secondary antibody. Signals were enhanced with FITC-Tyramide (NEN). TSA reaction was usually done for 7.5 min. In order to obtain a maximum amplification, it was extended to 15 min. Counter staining of nuclei was done by propidium iodide (1 �g/�1, Sigma) after RNase treatment (10 �g/�1,

Boehringer) for 30 min. The double staining images were visualized with a Zeiss LSM41 0 conforcal laser scan microscope equipped with a Krypton/ Argon laser.

RT -PCR and eDNA sequence. Total RNA from 50 heads obtained from rit flies at ZT18 at 24 OC were reverse-transcripted by Ready-To-Go T -primed First-Strand Kit (Pharmacia). Using four

tim-specific primer sets (tim237-258 and tim1245-1226, tim914-933 and tim1835-1816, timl813-1834 and tim3404-3383, and tim 3122-3142 and tim4474-4453; these numbers are based on the nucleotide position of the tim eDNA according to Myers et al., 1994), fragments were amplified and cloned into the pCRll vector (Invitorogen). When amplified fragments were too large to be

p. 34

-sequenced, they were digested by restriction enzymes and subcloned. Both strands of each clone were sequenced at least twice by ALFred DNA sequencer (Pharmacia). Experiments were repeated at least twice in order to avoid PCR errors. Fragments from tim3122 to 4553 amplified from the wild-type or rit, which includes the nucleotide substitution from CCG to GCG, were digested by EcoRI at 37°C for 3 hrs. The digestion occurred in the wild-type fragment but not in the rit fragment.

RESULTS

rit alters free-running periods in a temperature-dependent manner. Locomotor activity rhythms of individual flies were recorded under 12 hrs light and 12 hrs dark conditions (LD12: 12) for three days followed by constant darkness (DD). rit was entrained to LD 12:12 at 24 OC and showed a lengthened circadian rhythm under DD and its period was about two hours longer than that of wild-type (Fig. 1A and C).

I

then measured the period ofrit at different temperatures (Fig.

IA).

When the temperature was lower than 24°C, the period ofrit was only slightly lengthened, with a Q10 of 0.93, which was comparable to wild-type flies (Q10=1). When temperature was above 24 OC, the period of rit lengthened remarkably to about 10 hrs longer than wild-type flies at 30°C (Fig. 1 A). Thus, above 24 OC, the Q10 of rit was 0.62, which is significantly different from the wild-type value of 1. This phenotype is recessive since the period of heterozygous rit/+ flies were well temperature compensated (Fig. 1 C).

Genetic interaction between rit and per. rit strongly interacts with per with respect to period lengthening. The double mutant ofrit (25.5 hr at 24°C) with per (28.5 hr at 24°C) showed a period of 32.9 hr at 24 OC (Fig. 1B). This is �2 hrs longer than the value expected (31 hr) on the basis of an additive effect of the two mutants. At 27°C,per;rit showed an extremely long period of 44.4 hr (Fig. 1B). Although these periods are extraordinary long, the rhythmicity itself was still clear and stable with a punctual onset and offset of the active phase. While only two out of 25 flies were arrhythmic at 27°C, most of rit became arrhythmic at 30°C (Table

I).

One fly that was rhythmic at

p. 35

-30°C revealed an extremely long period of 50.4 ^{hr (Fig.} 1B). Even in this case,

I

observed a stable

rhythmicity. The Q10 of the double mutant was drastically lower (Q10=0.46) than that of

pel

(Q10=0.88) ^above 24 OC ^(Table

II).

The period lengthening also occurred in the double mutant with

per

(Fig. 1 C). The period

of

per; rit

was 6 hrs longer than

pers

at 27°C, which corresponds to the value expected on the basis of an additive effect. Only two out of 15 flies were found to be rhythmic at 30°C. The periods were ca. 24 hr, which is 5 hrs longer than the period in

per::,

at this temperature. The Q10 ^of

per;

ritwas 0.92, not significantly different from that

ofpers.

Thus, there is an allele specificity in the

interaction between

rit

and

per;

a drastic effect in

pe�

and a minimal effect in

pers.

rit induces arrhythmicity at higher temperature. I observed that a majority of

rit

flies become arrhythmic at temperatures higher than 24 OC ^(Table

1).

When the temperature was 24 °C, flies were quite rhythmic in all the strains used in this study. At the lowest and highest temperatures tested (16 ^or 30°C), a part of wild-type flies and of

per

mutants showed arrhythmicity (Table I).

This is because overall locomotor activity tends to be reduced at 16°C (data not shown). At high temperatures (30°C), some flies were statistically arrhythmic, although a weak but stable

rhythmicity could be observed in all plots by eye.

The proportion of arrhythmic flies homozygous for the

rit

mutation was not different from that in wild-type and

per

mutants in the range from 16 to 24 °C. On the contrary, the proportion of arrhythmic flies at 27 ^and 30°C was remarkably high in all strains homozygous for the

rit

mutation (Table

1).

While

per;rit

flies exhibited extraordinarily long periods at 27°C, 29 ^{out of} 30 flies were arrhythmic at 30°C. Such arrhythmicity would occur when the period lengthened beyond the circadian range. Alternatively, the arrhythmicity would be directly induced by

rit

at higher

temperatures. The latter possibility is supported by the result that

per; rit

showed a period of ca. 24 hr at 30°C ^but 76.5 ^o/o of these flies are arrhythmic.

An excess of the per gene dosage ameliorates the rit phenotype.

rit

males and females, carrying a

per+

gene translocation on theY chromosome (w+Y), were produced to test if an

- p.

36-additional

per+

dosage affects periodicity in

rit.

In

+/w+Y; rit

male flies, the

per

gene dosage is

double that of a wild-type male, while attached-X

(C(J)DX)Iw+Y; rit

females have 1.5 times more per gene dosage than wild-type males because

per

is on the X chromosome (Cooper et al., 1994).

A

per gene dosage of

2

completely rescued the

rit

phenotype behaviorally; its period was normal and temperature compensated (Fig.

2A).

When the

per

gene dosage was 1.5 in

C(J)DX!w+Y; rit

flies, the

rit

phenotype was rescued at lower temperature, but incompletely rescued at higher temperature (Fig.

2A).

Complementation and recombination tests with tim The

rit

locus was roughly mapped near the cl gene (Murata et al., 1995). Since this position is near the

tim

locus,

rit

could be an allele of

tim.

To determine if this is the case, I tested whether

rit

could complement

tim01

(a null allele of

tim;

Myers et al., 1995). Locomotor activity rhythms of

tim01/rit

flies were recorded at various

temperatures from 19 to 30°C (Fig.

2B). rit/tim01

heterozygotes phenocopied the

rit

phenotype. Its period at each temperature was, however, about one hour shorter than the period in

rit

homozygotes (t-test, p<0.05). I obtained a similar result using

Df(2L)tim02

(data not shown) , which deletes the entire

tim

gene (Myers et al., 1995). Since the temperature dependency of period in

rit/tim

phenocopied homozygous

rit

and the phenotype of

rit

is rescued by duplication of the

per

locus as described above, I expected that the phenotype in

rit/tim

could be also rescued by the

per

duplication. Male flies having the

per

gene dosage of

2

showed normal periods at various temperatures (Fig.

2B).

A

duplication of the

tim+

gene was tested to determine if it could complement the temperature dependent period lengthening caused by the

rit

mutation (Fig.

2C). Dp(2; Y)odd 4"31;

ritlrit

and

Dp(2; Y)odd 2"31; ritlrit

flies, both of which carry a

tim+

duplication on theY chromosome, showed the periods of about

26

hr at every temperature (Fig.

2

^C)

.

Given that null alleles of

tim

did not complement

rit

and duplications bearing

tim

corrected the

rit

phenotype with respect to the temperature compensation on period, I suspected

rit

is an allele of

tim.

However, it remains a fonnal possibility that trans heterozygotes between a null mutant of

tim

and a clock mutant closely mapped near the

tim

gene will show a similar phenotype.

- p.

37-I then examined whether recombination occurs between rit and tim. Females having the rit

mutation on one second chromosome and the tim01 mutation on the other were mated to tim01 males (Fig. 3A, Cross 1 ). Progenies from this cross were then monitored for their locomotor rhythms at 30°C. If the rit mutation is not allelic to tim, there should be recombinant progenies that show a nearly normal period among majority of arrhythmic flies. Total of 546 progenies were tested and I failed to obtain any significant circadian rhythmicity by the chi-square periodogram (Sokolove and Bushell, 1978) ranged from 19 to 29 hrs. Furthermore, I crossed tim01/rit females to males which are phenotypically wild-type with respect to circadian rhythm (Fig. 3A, Cross2). If a recombination betweenrit and tim occurs, rit tim01/+ progeny would be expected to show a very long period or an arrhythmic phenotype like ritltim01 flies at 30°C. Circadian rhythms of about seven hundred flies obtained were recorded, and none was categorized as a recombinant. These data support the result of the complementation test which indicates that rit is an allele of tim.

rit carries an amino acid substitution in TIM. The coding region of tim eDNA in rit flies was

amplified by RT -PCR. The fragments amplified were sequenced and compared to the tim+ eDNA previously described (Myers et al, 1995 and 1997). To avoid PCR errors, experiments were independently repeated twice and sequence data were confirmed for each repeat. There were 16 mutations between rit and tim+ at the nucleotide level. Among these, 14 were silent mutations that do not cause a change in the amino acid sequence. I found a single base nucleotide deletion at position 294 (according to Myers et al, 1995 and 1997) in the noncoding region (Fig. 3B). This deletion has been described as a mutation which has no effect on circadian rhythm in three

Drosophila species including D. melanogaster (Rosato et al., 1997). There is a missense mutation which produced an amino acid substitution at 3492nt from the start point of the tim eDNA (Fig.

3B). At this point, the nucleotide change from CCG to GCG yields the amino acid substitution from proline to alanine (Fig. 3B) at 1 093aa in TIM protein (according to Myers et al, 1997). Since there is an EcoRI site at this position (Fig. 3B), I confirmed that this mutation abolishes the restriction of the timnr eDNA by EcoRI at this region (data not shown). Taken together, I judged that rit is an allele of tim. Hereafter, I use "rit" as an allele name and "tinf1" as a mutation.

p. 38

-rit lowered expression and protein abundances in tim and per. I measured

per

and

tim

mRNA cycling in fly heads by RNase protection assay. Flies entrained in LD12:12 were collected every 4 hrs. RNA abundance was normalized by the peak value (at ZT13) of the wild-type

per

and

tim

mRNA at 24 °C, respectively (Fig. 4A). Wild-type flies at 24 OC exhibited robust mRNA cyclings with the peak at ZT13-17 and a trough at ZT1, meaning that these

mRNA

cyclings are in phase. The

tim mRNA

also cycled in

rit

at 24 OC but with a delayed peak. The levels of

tim

mRNA in

rit

were not significantly different from those in wild-type at each time, while the peak value of

permRNA

in

rit

was reduced to about 70% (t-test, p<0.05, Fig. 4B). At 30°C, the peak of

per

and

tim

mRNA cyclings in wild-type was delayed if compared to that at 24 OC (Fig. 4C). The shape of

tim

mRNA cycling in

rit

at 30°C was quite similar to that at 24 OC (Fig. 4D). The abundance of

per

mRNA at the peak (ZT17) decreased to 60o/o (t-test, p<0.05). Thus the amplitude of

per

mRNA cycling becomes smaller as temperature increases, while

tim

mRNA is not affected in

rit.

I next analyzed the levels of TIM and PER protein abundance using Western blot analyses.

PER and TIM abundance cycle in

rit

under LD at 24 °C, where the peak was at ZT18 and the trough was at ZT6-1 0 (Fig. 5A). The shape and phase of these protein fluctuations were similar to those in wild-type except that the peak level was reduced �30o/o (Fig. 5B). At 27°C, when

rit

shows a longer freerunning period of locomotor activity rhythms, the amplitude of PER and TIM cycling was reduced. These peaks reduced to nearly half the level of wild-type (Fig. 5A and B). At 30°C, where 80o/o

ofrit

flies become behaviorally arrhythmic,

rit

showed two types of protein cycling with respect to peak levels. One is similar to the result observed at 27°C; the peak abundance of PER and TIM became about a half level of wild-type and their amplitudes were reduced (Fig. 5A, 30°C-1

and

B, rit-1).

Four out of ten experiments were classified into this type. The remaining six were categorized into other type (Fig. 5A, 30°C-2). In this type, TIM fluctuated with a nearly normal shape with the peak level of 80%, while amplitude of PER fluctuation was reduced. The peak and trough levels of PER were to 80o/o and 50% of the wild-type peak level, respectively (Fig. SB,

rit-2).

In either case, the amplitude of PER cycling in

rit

became smaller as temperature became higher. The rhythmic mobility shift by phosphorylation is reported to occur in PER band (7, 31,

- p.

39-32).

_A nearly normal phosphorylation of PER occurs both at 24 and 27°C while PER nearly always (but not always) seems to be hypophosphorylated at 30°C without regard to that PER abundance

cycled or not.

Since most ofrit flies were behaviorally arrhythmic in DD at 30°C, I investigated whether the cyclings of PER and TIM were also abolished inrit in DD (Fig. 5C). The level of these protein expressions increased to half of the wild-type peaks. TIM and PER levels do not show rhythmic fluctuations, though some random variability in their levels is apparent.

rit affects per abundance at a post-transcriptional level. rit appears to lower per mRNA abundance and the amplitude of PER protein cycling, while tim mRNA abundance is not affected but TIM protein abundance decreases. One possibility for this reason is that rit primarily affects the

per mRNA transcription level. The other possibility is that rit reduces PER abundance and in turn this may lead per mRNA abundance lower through the per feedback loop. To solve this problem, I

produced arit;rh-per strain and measured its PER abundance. In rh-per flies, per is strongly driven by rhodopsin] promoter in eyes independent of the innate per gene expression (Zeng et al., 1994).

If PER is reduced in rit, rh-per flies, rit should affect PER at a post-transcriptional level. PER level in the strain carrying rh-per was lowered to about 80% in rit flies than in rit flies. This is obvious at 30°C (Fig. 6A). This suggests that rit affects the per feedback loop at a post-transcriptional level.

PER seems to be hypophosphorylated in rit background at 30°C when the mobility shifts were compared at ZT 2 to be run side-by-side (Fig. 6A).

At a behavioral level, the excess of per gene dosage rescued the rit phenotype. In order to determine if PER and TIM protein cycling is also rescued by the per duplication, I measured their abundances at 30°C. TIM and PER protein showed a clear cycling with the peak level of twice and three times as much as wild-type, respectively (Fig. 6B). The lower amplitude of PER cycling shown in rit at 30°C was rescued to be normal in +/w+Y; rit flies. Interestingly, TIM abundance was

also increased in +/w+Y; rit even though only per dosage was increased.

Levels of PER translocated into the nucleus are lowered in a rit background. The - p.

40-spatial pattern of the per expression can be monitored by lacZ expression in transform ant flies carrying a per-lacZ fusion gene (Liu et al., 1988). This fusion gene contains one-half of the per coding region, ca. 4 kb 5' flanking region and the entire coding region of the lacZ gene derived from E,� coli. per-lacZ and rit per-lacZ flies were entrained under LD12: 12 at 24 OC or 30°C for 3 to 5 days. Sections of wild-type flies and rit were incubated with X-gal for 2 hrs at 37°C. In both

strains at 24 OC (Fig. 7 A and

B),

there were /acZ-positive cells in lamina, medulla and central brain.

The expression pattern was principally coincident with the previous study (Liu et al., 1988). Nuclei in photoreceptor cells were strongly stained in wild-type but only weakly in rit at 24 °C.

While the pattern of theper-lacZ expression at 30°C was similar to that at 24 OC in wild-type (Fig. 7C), there were few /acZ-positive cells in the optic lobe and the brain ofrit (Fig.

7D).

^The

lacZ expression in nuclei of the eyes was very weak in rit at 30°C (Fig. 7D) suggesting that PER localization in nuclei is much lower in rit than in wild-type.

I next checked whether the weak staining of PER-l3GAL could be rescued in +/w+Y; rit strain, because not only the aberrant locomotor rhythm but also the weak cycling of PER was rescued by the excess of the per gene dosage in rit. The level of staining ofPER-BGAL in brain and photoreceptors and their nuclear localization especially in photoreceptor cells were rescued (Fig.

7E).

Nuclear localizations of PER in lateral neurons (LNs) were examined by anti-PER antibody with fluorescent probes (FITC; green color in Fig. 8) at ZT19, 21 and 23.5 at 30°C. Counter

staining of nuclei was done with propidium iodide (red color in Fig. 8). Sections from about fifty heads of each strain were observed at each time point of day. Nuclei in photoreceptor cells were clearly stained in wild-type through ZT19 to ZT23 .5 (Fig. 8A,

B

^{and C).} PER was cytoplasmic in LNs at ZT19, and in nuclei at ZT21 and 23.5 (Fig. 8D, E and F). This temporal regulation of PER nuclear entry is consistent with the previous report (Curtin et al., 1995). The strength of PER signal increases through a time course of day. Compared with wild-type, signals of PER in photoreceptors and LNs in rit were weak although the staining pattern is comparable to wild-type (Fig. 8G, Hand

I).

To reveal the cellular localization of PER in rit, I lengthened the incubation time of the FITC­

Tyramide reaction to 15 min. PER was cytoplasmic in LNs at ZT19 (Fig. 8J and

M).

At ZT21, the p. 41

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