J . Nrim,grrieri~.J,1991. Vol. 7. pp. 103-1 14
I-'
Reprint.; available directly from the publisher Photocopying permitted by license only
1991 Harwood Academic Publishers G m b H Printed in the United Kingdom
CHARACTERIZATION OF ANDANTE, A NEW DROSOPHZLA CLOCK MUTANT, AND ITS INTERACTIONS WITH OTHER CLOCK MUTANTS RONALD J. KONOPKA'.?*, RANDALL F. SMITH', and DOMINIC ORR'
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iDepartnient qf Biologj,, Clarkson Universitj., Potsdam, N Y 13699, U S 'Division o f Biologj., Cul$wnia Institute of' Trrhnology, Pasadena, C A 91 125, U S ~ R i w i w iJfu l r ?S, IYYO: rivisivf Aiigiisr 3 / , 19901
A new clock mutant. named Andunre. has been identified on the X chromosome of Drosophilu ttielutiugast i v . dtidunrr lengthens the period of the circadian eclosion and locomotor activity rhythms by 1.52.0 hours. The phase response curves for the eclosion and activity rhythms, indicating light-induced phase shifts. show a similar degree of lengthening. Atidanre also lengthens the periods of other clock mutants, including Clock. and alleles of the periodlocus. Analysis of locomotor activity rhythms reveals that Andanre is semi-dominant. and Anclulirc, rhythms are highly temperature compensated. The sine ocidi.7 mutation, which eliminates the outcr visual system. has no effect on the period of Atidunri,. Deficiency mapping indicates that .4ndunti, is located in the IOEI-2 to lOFl region of the X chromosome, close to the niitiicrrurr-clrr.tk~,locus. Whereas A n h n i r flies have a h k j wing phenotype. ditskj. flies d o not have an .4titfante clock phenotype. K q . i t w d s : ciriudiun rltjtlims. li~cottiotoructivitj, eclosion , period niiitutiis. Clo1.k mutant, visual mutunt
INTRODUCTION Mutations that alter the periodicity of circadian rhythms have been isolated in Drosophila (Konopka & Benzer, I97 l ) , Chlamydomonas (Bruce, 1972), and Neurospora (Feldman & Hoyle, 1973). In the case of Drosophila, two genetic loci on the X chromosome have been described in detail: the period ( p e r ) locus, at which several alleles have been isolated (for review, see Konopka. 1987a, b), and the Clock ( C l k ) locus (Dushay et al., 1990). at which only one allele is known, and which maps very close to the per locus. In this paper we describe a third genetic locus on the X chromosome that is quite separated from, and proximal to, the per and Clk loci. We have named the originally isolated allele Andante ( A n d ) ,as it produces a moderate slowing of the eclosion and adult locomotor activity rhythms. Andante flies also show a dusky wing phenotype, and indeed, Andante maps very close to the miniature-dusky region (see below). Brief descriptions of some of the properties of Andante may be found in Smith (1982) and Konopka (1987b). MATERIALS AND METHODS The Canton-S strain was used in this mutant screen. Flies were raised on cornmeal, sugar, and yeast medium (Lewis, 1960). Males were mutagenized with ethyl methanesulfonate according to the method of Lewis and Bacher (1968). Stocks of mut'Send reprint requests to Ronald J . Konopka, 430 South Santa Anita Ave., Pasadena, CA 91 107, US. I03
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R. J . KONOPKA. R . F. SMITH AND D. ORR
agenized males with identical X chromosomes were maintaiped with attached-)< females. (All attached-)< females used in these experiments also carried the markers j~ellnw(.I>) body and .forked ( . f ) bristles.) These stocks were raised in a 12hours light-I2 hours dark cycle (LD 12:12) at 22°C. At the end of a light period, they were transferred to constant darkness (DD), and given a 12hour, 29” temperature pulse beginning 12 hours after the LD to D D transition. The stocks were then maintained in DD at 22”C., and the relative numbers of males and females emerging over roughly 12 hour periods were determined. The attached-X females, having normal rhythms, served as a reference for detecting males with abnormal emergence patterns. Stocks with apparent abnormalities in the emergence patterns of males were rescreened using automated eclosion monitors known as “bang boxes” (see Konopka & Benzer, 1971). Some stocks were screened using temperature pulses, in attempts to recover putative clock mutants with altered temperature responses. In the case of one of these stocks, the median of the eclosion peak of males, on the third day after a 12 hour, 31°C. temperature pulse, showed a 4 hour phase delay compared to the females. (“Median” = the time at which 50% of the flies in the peak had emerged.) This phase delay was apparently the result of a lengthening of the period of the eclosion clock produced by the Andante mutation that had been induced in the males. Locomotor activity rhythms of individual adult flies were determined in infrared light as described previously (Konopka & Benzer, 1971; Smith & Konopka, 1981). Some of the activity monitor channels were connected to Esterline-Angus event recorders. Other channels were connected to a microprocessor-based unit that kept an hourly count of activity events. These numerical records were then plotted and used for determination of the period length by means of periodogram analysis (Enright, 1965). Unless otherwise stated, activity rhythms were measured at 24°C. Phase response curves (PRCs) for the eclosion rhythm were determined as follows. Fly stocks were raised in LD 12:12 at 18°C. Pupae were harvested during a light period and placed in the “bang boxes” in DD at 18°C. at the end of the 12 hour light period. Experimental cultures were exposed to a light pulse of at least lOOlux in intensity and 40 minutes’ or 80 minutes’ duration at various times after the transition from LD to DD. Control cultures received no light pulse. Phase advances and delays were determined from eclosion peaks that occurred at least three days after the administration of the light pulse, in order to allow the advances and delays to fully develop and stabilize. Phase response curves for adult locomotor activity rhythms were determined as described in Dushay et al. (1990).
RESULTS Phenotypes of the Andante nrutant Figure 1 illustrates the eclosion rhythm of And males (bottom) run concurrently with attached-)< females that have a normal rhythm (top). The And rhythm is longer than that of the control by about I .2 hours, resulting in a substantial phase difference between the males and the females by the sixth and seventh day of the run. The eclosion rhythm periods of Andmales, And females, And heterozygotes, and attachedX females with wild type rhythms are given in Table I. While the periods of the wild type rhythms are, as expected, close to 24 hours (as is also true for other genotypes with wild type eclosion rhythms-see Smith & Konopka, 19811, the periods of And
I05
T H E A N D CLOCK MUTANT
xx
7 = 24.3
7 = 25.5
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And
r
0
I
I
2
I
I
4
I
I
6
I DAYS
FIGURE I Eclosion rhythms of And males (bottom record) and attached-)< v. F And' females (top record) measured concurrently at 18°C. in constant darkness after entrainment in L D 12:12 at 18°C. The beginning of the record (time 0) corresponds to the transition from LD to D D at the end of the light period. T = period estimates in hours.
males and females exceed 25 hours. The periods of Aiidheterozygotes are only slightly longer than the wild type periods. Thus the And mutation does not show quite the degree of semi-dominance for the eclosion rhythm as i t does for the locomotor activity rhythm (see below). Figure 2 shows two examples of event recorder tracings of locomotor activity rhythms of And flies (two bottom records) in comparison with the wild type rhythm of a Canton-S fly (top record). The And rhythms are noticeably longer than normal; this period lengthening is especially obvious in the activity offsets, which are more distinct than the activity onsets. The periods of activity rhythms of And males, And females, And heterozygotes, and control flies are given in Table 11. It can be seen that And flies have significantly longer activity rhythms than d o wild type flies, and that And heterozygotes have periods intermediate between Atid homozygotes and wild TABLE I Periods of the eclosion rhythm of flies bearing the And mutation and controls ~~
Run
Genotype And/Y .4nd+/.4nd+ (XX. y / ) AndlY And'lAnd' (XX. ?' f ) AndlY And/And AndlY Andl And And/ And ( F M 7 ) AtidlAnd ' ( F M 7 ) AndlAtid' ( F M 3 ) +
Period & Std. Dev. 25.1 23.9 25.6 24.3 25.3 25.2 25.3 25.1 24.3 24.4 24.8
_+ 0.6h
k 1.1 h k 0.5 h f 0.6h _+ 0.8 h & 1.0h i 0.8 h f 0.2h f 0.5 h k 0.4h k 0.5 h
The genotype of the females in the second and fourth rows were attached->(. yellor forked. The And+-bearing chromosomes in the last three rows were X-chromosomal balancers [ f n ( I I F M 7 or fn(I!FM3].
R. J. KONOPKA, R. F. SMITH A N D D. ORR
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WT
And
FIGURE 2 Event recorder representations of the locomotor activity rhythms of individual Drosophilu adults, monitored in infrared light at 24°C. The top record shows the activity rhythm of a Canton-S wild type (WT) female, and the bottom two records illustrate the activity rhythms of two different And females. All records are plotted modulo 24 hours. In addition, all records are double plotted in order to facilitate visual inspection of the data. The And records are noticeably delayed each day in comparison with the wild type record. reflecting the longer free-running period of And.
type, illustrating the semi-dominance of the Andmutation. Partial dominance appears to be a general characteristic of Drosophifu clock mutations (Konopka, 1987b). The And mutation also has a visible wing phenotype, similar to that of dusky (dy). Complementation tests with dy confirmed that the wing phenotype is indeed due to a dy mutation associated with And. However, analysis of the activity rhythms of dv TABLE I1 Activity rhythm periods of flies bearing the And mutation alone and in combination with controls Genotype And/Y Andl And And+/Y And ' 1 And ' dvldy niD/mD
AndlAnd And/dy Atid/niD
+
d ~and . m", and
Period & Std. Dev.
N
25.5 & 0.9 h' 25.7 & 0.3 h' 24.0 & 0.4 h 24.2 f 0.3 h 24.6 & 0.4h 23.7 k 0.4 h 24.8 & 0.2 h' 25.0 & 0.3 h' 24.8 k 0.S h'
18 10 12 7 9 9
9
7 5
'These genotypes are significantly longer than the wild type controls (below) ( p < 0.01, t-tests). 'There is no significant difference among these genotypes ( p 0.10. t-tests). However, these And heterozygotes have periods significantly longer than wild type (above) and significantly shorter than And homozygotes ( p i0.01. t-tests).
T H E .4ND CLOCK MUTANT
107
TABLE 111 Temperature compensation of activity rhythms of And and wild type Hies Temp. ("C.)
Period i Std. Dev.
N
Andante
17 22 25
25.90 F 0.84 h 25.99 2 0.80 h 25.86 2 0.44h
15 I? 21
Canton-S
17 22 25
24.15 k 0.40h 23.89 f 0.34 h 23.82 k 0.45h
75 27 17
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Genotype
and another mutation in the same region, m D ,indicated that these two alleles did not show an And rhythm phenotype (Table 11). In particular, the periods of dy and mD were in the normal range, and in addition, the periods of Ar1dld.v and And/mD heterozygotes were indistinguishable from the period of And/ heterozygotes, and significantly shorter than the period of And/And homozygotes. A well known property of circadian clocks is the temperature compensation of period length; that is, the steady-state period length of a rhythm shows little variation with temperature in a physiological range. Thus, the Q,,,of period length is usually close to 1 .O. The periods of locomotor activity rhythms of And mutant flies over the range 17°C. to 25°C. are given in Table 111. The total amount of variation of the mean periods in this range is less than 0.2 hours. In this respect, And is very similar to wild type; for wild type flies, the mean periods vary about 0.3 hours in the same temperature range (Table 111; Konopka, Pittendrigh & Orr, 1989). In contrast, for the same temperature range, the variation in mean period lengths for perS flies is about 0.8 hours, and for perL flies it is about 2.7 hours (Konopka, Pittendrigh & Orr. 1989).
+
Recombination and mapping e.uperiments
In order to localize the And mutation on the X chromosome, a mapping stock containing the markers yellm. ( J'), chocolate (cho), crossveinless (cv), vermillion (v). and forked ( f ) was crossed to an And stock, and F, recombinant males were isolated and mated to attached-)< females to establish stocks such that the males in each stock had identical X chromosomes. The activity rhythm periods of the males in these stocks were then determined. In Table IV, it can be seen that the And rhythm TABLE IV Recombination mapping of Anti: Activity rhythm phenotypes Recombinant Types
Period
Std. Dev.
25.6 & 0.6h 23.9 2 0.311 23.8 f 0.2h 25.4 f 0.3 h 25.4 & 0.3h 24.0 & 0.2 h
24.4 & 0.4 h 25.2 2 0.3 h
N
I08
R. J. KONOPKA, R. F. SMITH A N D D. ORR
TABLE V Deficiency mapping of '4nd:Activity rhythm phenotypes. Numbers of flies are indicated in parentheses Genotype of X Chromosome B: And Anti+ Genotype of X Chromosome A
Of( 1)KA7 Df ( I ) K A 6 qf(l)H.48S Of( 1)m"V-J
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D f ( 1)NIOS
And And
+
Period f Std. Dev. 26.6 25.8 25.9 25.1 24.6 24.3 25.7 24.8
Period & Std. Dev.
& 0.3 h (6) & O.2h (7) & O.2h (6) F 0.2h (6) f 0.2 h (7) f 0.3 h (5) f 0.3 h (10) & 0.2h (9)
24.4 24.0 25.2 24.5 24.3 23.8 24.8 24.2
f 0.4h & 0.2 h f 0.4 h & 0.2 h & 0.3 h f 0.8 h & 0.2 h f 0.3 h
Breakpoints 10A9; IOF6-7 IOEI: llA7-8 IOCI-2: IIAI-2 IOC2-3: IOEI-2 IOFI: IOF9-I0 IOF7: I l D l
(10) (6) (8) (7) (8)
(9) (9) (7)
Refst a. b a. c a a. b a, c a, c
tReferences: a, Craymer & Roy (1980): b. Mortin & Lefevre (I981 ); c. Wieschaus. Nusslein-Volhard & Jurgens (19x4).
phenotype maps within the vermillion-jorked interval, and that the And rhythm phenotype segregates with the 419wing phenotype. Deficiencies that involve the nziniarure-dzttskj3 region were then used to further localize the And mutation. The results are shown in Table V. The And rhythm phenotype is uncovered by the deficiencies KA6 and KA7, but not by the deficiencies NlU5, RA47, or nz?"-'. In particular, the period of KA6/And is not significantly different from that of And/Anci ( p > 0.1, t-test), while the period of K A 7 / A n d is significantly longer than that of And/And ( p < 0.01, t-test). On the other hand, the periods of NlUS/And, RA47/And, and n~*~'-'/And are all significantly shorter than that of Atzd/And ( p < 0.01, t-tests). The H A 8 5 result is somwhat ambiguous. Although HA85 does appear to uncover And, as it should, (the period of HA85/And is not significantly different from that of And/And, as a t-test yields p > O . l ) , the period of the H A 8 5 / + heterozygote is significantly longer than normal ( p < 0.01, t-test). These results map the rhythm phenotype of And to the region 10E1-2 to lOFl on the X chromsome, which is very close to the miniarure-dusky region. TABLE VI Lengthening of activity rhythm periods produced by And in combination with other clock mutations. measured at 22°C. Genotype
Period & Std. Dev.
N
prrS perS And
19.1 f 0.Sh 20.2 & 0.Sh
21 19
+ I.Oh*
Clock Clock And
22.6 f 0.4h 23.7 f 0.3 h
I04 12
+ 1 . 1 h*
Wild type Antl
23.5 & 0.4h 25.3 i 0.5 h
23 35
+ 1.8 h*
perL1 perL1 And
29.6 _+ 1.1 h 31.2 & 0.7h
21 28
+ 1.6h*
perL' pwL' And
2 9 2 f 1.6h 32.2 f 0.6 h
12 II
+ 3.0 h*
*All lengthenings are significant (t-tests. p < 0.01)
Lengthening
T H E ,4ND CLOCK MUTANT
109
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Interactions qf' Andante with per alleles and with Clock Table VI shows the periods of activity rhythms of double mutant, male flies that bear . or Clock mutations, in comparison the Andante mutation and the per", p e r L 1perL2, with males carrying only a per allele or Clock. Also shown are period values for control Andante and wild type males calculated from activity records of sibling males obtained from the cross that produced the double mutants. These results show that the And mutation significantly lengthens the period of each of the four mutations tested. The lengthening is least for the two mutations with periods shorter than 24hours; And lengthens perS by 1.0 hours and Clock by 1.1 hours. The period lengthening produced by And is greatest for perL2,at 3.0 hours. perL2appears to be a weakerper allele thanperL'in terms ofper product activity (see Konopka, 1988). Both wild type and perL1males show an intermediate degree of lengthening, at 1.8 hours and 1.6 hours respectively. Activitj. rhj~thnlperiods of' the Andante, sine oculis double mitatit Although the outer portions of the fly's visual system are not necessarily for entainment or maintenance of circadian rhythmicity of locomotor activity (Helfrich, I986), it is possible that input from the visual system might modify the period of the activity rhythm (see discussion in Hall, 1990). Histochemical staining of photoreceptor cells and optic lobe cells was observed using antibodies to the per gene product (Siwicki et al.. 1988). In addition, Siwicki et al. found that the intensity of per-specific staining in the visual system showed an oscillation that was apparently controlled by an endogenous, circadian oscillator (also see Zerr et al., 1990). In order to determine whether the absence of the outer portions of the visual system affected the period of Andante flies, the activity rhythms of flies carrying Andante and sine oculis were measured. The expression of sine oculis is somewhat variable, but flies can be selected that have no compound eye facets at all. Histological examination of these flies shows that such flies have no ommatidial cells; the lamina and part of the medulla are missing as well (Helfrich. 1986 & Konopka, unpublished observations). Table VII shows that there is no significant difference in the mean activity rhythm period of eyeless And;so flies as compared with And flies. In addition, the mean activity rhythm period of so flies is not significantly different from that of wild type flies ( p > 0.1. t-tests). Phase response curves for the eclosiori and uctirity rliythms of Andante und wild type pies Figure 3 shows the amount of phase shift of the eclosion rhythm produced by light pulses administered to pupae of And males and attached-)< females. As is the usual TABLE VII Activity rhythm periods of flies carrying the Atidutirr and sinc oculis mutations alone and in combination Genotype And And:so SO
Wild type
Period f Std. Dev. 25.5 25.6 23.5 23.8
f 0.9h' f 1.6h' & 0.8h' & 0.9h'
"There is no significant dimerence between each set of genotypes ( p > 0. I . t-tests).
N 18 34 18 15
R. J . KONOPKA. R. F. SMITH A N D D. ORR
I10
A
And/Y
A
+/+
[YfX^x/Y,
A
4
f
A f n
A A
A
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-6 ~
0
I
8
4
12
I
I
16
20
Time of L i g h t P u l s e
,
32
28
24
( h a f t e r DL-DD
I
,
~
36
I
FIGURE 3 The eclosion rhythm phase response curves of Atid males (dark symbols) and attached-)< .4nd+ females (open symbols). determined concurrently. Flies were raised in LD 12: 12 at 18°C.: light pulses (white light at least IOOlux in intensity) were administered at various times after the transition from LD to D D at 18°C. The LD to D D transition occurred at the end of a light period. The duration of the light pulses was 40 minutes (triangles) or 80 minutes (squares); the points are plotted at the pulse endpoints.
case for circadian rhythms, phase delays occur during the early subjective night, and phase advances during the late subjective night (Winfree, 1980). The maximum phase delay in the And response curve (about 4 hours) is slightly greater than that for the wild type rhythm of attached-)< females (about 2 hours). The maximum phase advance in the And and wild type response curves is similar (about 2 hours). Both 6.00
3
And
4.00 A
v,
'YI t
2.00
W
0.00
-
I
v,
- 2 00
W
v,
2
-4.00
a -6.00
1
3,
-8 00 0.00
5.00
10.00
15.00
20.00
25.00
30.00
TIME OF LIGHT PULSE (HRS) FIGURE 4 The locomotor activity rhythm phase response curve of Atid male adults. Flies were raised in constant light (LL) at 22°C.. then transferred to constant darkness at the same temperature a few days after eclosion. Flies were maintained for several days in D D in order to determine each fly's free-running period. Light pulses of about 2000 lux wcre administered for 10 minutes at various phases, determined after normalizing for the free-running period. The resulting phase shifts were then calculated. relative to the initial phase of the rhythm before the light pulse. after several more days in DD. The error bars indicate the standard deviations from the mean phase shift, computed from measurements on several flies.
T H E .4ND CLOCK MUTANT
1
4’00
2.00
A
v,
w
I
w
0.00
-t
LL
WT
I
€
1-2.00
v, W
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fl -4.00
5 I
-8.00 7
-
0.00
5.00
10.00
15.00
20.00
25.00
30.00
TIME OF LIGHT PULSE (HRS) FIGURE 5 The locomotor activity rhythm phase response curve of Canton-S wild type (WT) male adults. Protocols are the same as described in the legend to Figure 4.
response curves are classified as Winfree Type I (Winfree, 1980), indicating “weak” resetting by light. In the second cycle of the response curves, there is a noticeable lag (on the order of 2 hours) in the And curve relative to that of wild type, reflecting the longer free-running period of the And rhythm in constant darkness. The second phase delay maximum occurs at hour 28 for the wild type rhythm, and at hour 30 for the And rhythm. Likewise, a t hour 32, the light pulse produces a phase delay of about an hour in the Atid eclosion rhythm, while the wild type rhythm is phase advanced by about 2 hours. A similar indication of the lengthened Atidoscillation can be seen in comparing the phase response curve for the locomotor activity rhythm of And (Figure 4) with that of wild type (Figure 5). At hour 26, the And curve shows no phase shift, indicating that the oscillator is still in the subjective day, while the wild type curve shows a phase delay of more than 3 hours, indicating that the wild type oscillator is in the subjective night. Again, the Atid response curve is about 2 hours longer than that of wild type. The response curves for the activity rhythms of And and wild type have similar maximum phase delays (more than 3 hours). The maximum phase advance in the And curve is about 3 hours, while in the wild type curve it is about 2 hours. Both the And and wild type activity rhythms show Type 1 (weak) resetting behavior, as was observed for the eclosion rhythms. DISCUSSION The Andunre locus is the third genetic locus on the rnelunogaster X chromosome that significantly increases or decreases the period of the circadian eclosion and locomotor activity rhythms, the other two being per (Konopka & Benzer, 1971) and Clock (Dushay et al., 1990). The disco mutation, which is also on the X chromosome, disrupts rhythmicity but does not produce a stable change in period (Dushay, Ros-
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I I2
R. J . KONOPKA. R. F. SMITH AND D. ORR
bash & Hall, 1989). Interestingly, the Clock mutation decreases the period by about I .5 hours, while the Anduntr mutation increases the period by a similar amount. It is not known at this time whether these two genes are related in some way, or whether 1.5 hours represents some type of quantization or temporal subunit. In the case of activity rhythms, both And and Clock are semi-dominant; the periods of heterozygotes are intermediate between mutant and wild-type (Table 11). The semi-dominance is less obvious in the case of eclosion rhythms (Table I). The perS mutation is also semi-dominant (Konopka & Benzer. 1971). as the period of the p e r S / + heterozygote (21.9h) is intermediate between pers (19.5 h) and wild type (24.4 h). However, the perL/+ and pero/+ heterozygotes are only about 0.5 hours longer then wild type (Smith & Konopka, 198 I ) . The disco mutation is recessive (Dushay, Rosbash & Hall. 1989). Although the Andmutant also has a dzrskj, phenotype, the d.v and mD alleles do not have an And phenotype, either as homozygotes or as heterozygotes with And (Table 11). As And has been shown to map close to the nziniature-dusk?’ region (Table V), the mutation event may have affected both the dusky gene and a clock gene, And. Alternatively, And may represent a double mutant. The relationship between the period-lengthening phenotype and the dusky phenotype of And remains to be clarified. The periods of both Clock and And activity rhythms are very well temperature compensated; the QI,,for each is very close to I .O. This is in contrast to perS and p e r L , whose periods have a greater variation over the 17OC. to 25°C. range; perS shows a variation of 0.8 hours, while perL has a 2.7 hour variation in this temperature range (Konopka, Pittendrigh & Orr, 1989). If the amount of temperature-induced variation in period length was proportional to the amount of lengthening induced by the mutation, then, using the perL characteristics as a basis for calculation, a mutation that produces a lengthening of about 2 hours, similar to And, should show one-third the amount of temperature-induced variation ofperL,or about 0.9 hours. In fact, the variation of And is much less; it is less than 0.2 hours (Table 111). Thus the variation of period with temperature is not proportional to the amount of period lengthening, but is different for the two period-lengthening mutants, A n d and perL, reflecting a difference in the homeostatic control of period in these two mutations. The Andmutation also has a lengthening effect on period when in combination with other clock mutations (Table VI). The amount of lengthening is in the range of 1.0 to I .8 hours, except in the case of p e r L 2 ,which is lengthened by 3.0 hours. The perL2 allele apparently represents the “weakest” per allele that still shows a period, without being completely arrhythmic. For example, a substantial fraction (about 50%) of perL2/perL’heterozygotes were weakly rhythmic or arrhythmic, while all perL’/perL’ heterozygotes were strongly rhythmic (Konopka, 1988). In addition, perL’/perL’ heterozygotes were 0.9 hours longer in period than perL’ flies, while perL2/perL’ heterozygotes were 3.3 hours longer than perL2flies. Thus the 3.0 hour lengthening produced by And in combination with perL’ may be attributable to the weak nature of the perL’ allele. The results in Table VTI indicate that flies lacking a substantial part of the visual system, including the photoreceptors, lamina, and at least part of the medulla, still show a locomotor activity rhythm with a normal free-running period (so flies), thus confirming the results of Helfrich (1986). The so mutation likewise has no effect on the expression of And, as And;so flies have the same free-running period as And flies (Table VII). Eyeless so flies can also entrain to light cycles (Helfrich. 1986 & Konopka, unpublished experiments), indicating the existence of an extraretinal photoreceptor for entrainment of the activity rhythm. In view of these results, the significance of
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T H E AND CLOCK MUTANT
I13
per-specific staining in the outer visual system by antibodies to the per gene product (Siwicki et al., 1988) is difficult to interpret. Siwicki et at. (1988) suggest that the per protein in the visual system may be involved in non-circadian visual behaviors, or in a circadian system separate from that controlling eclosion or activity. The possibility that per may be involved in other aspects of light-mediated timekeeping is suggested by the results of Saunders et al. (1989). who have shown that while arrhythmic per mutants have a photoperiodic ovarian diapause, the critical daylength of pero mutants is shifted by about two hours. Thus, while per is essential for rhythmicity of eclosion and locomotor activity, it may also have a non-essential role in modulating other timekeepers and/or behaviors. The And gene product may also have essential and non-essential roles in timekeeping and other behavioral and developmental processes. The null phenotype of the A d locus has yet to be determined. A comparison of the expression patterns of the A n d product and the per product may aid in the localization of the cells responsible for the control of the locomotor activity rhythm. The phase response curves for both the locomotor activity and eclosion rhythms show the characteristic period lengthening of And in comparison with the wild type controls. Otherwise, the shapes of the Andand wild type curves are fairly similar. The maximum phase delay for the Atid eclosion rhythm and the maximum phase advance for the And activity rhythm are slightly greater than those of wild type. The eclosion and activity rhythm response curves for And and wild type are Winfree Type 1 ("weak" resetting) (Winfree, 1980), as is the activity rhythm response curve of Clock (Dushay et al., 1990). In contrast, the perS mutation changes the Type I response curve for the wild type eclosion rhythm to a Type 0 ("strong" resetting) curve, in addition to shortening the period of the response curve by about 5 hours (Konopka, 1979; Winfree & Gordon, 1977). As the And response curves are only aboat 2 hours longer than those of wild type, it is not possible to unequivocally determine whether And selectively affects the duration of the subjective day or night. In the case of perS, the 5 hour decrease in free-running period was shown to result from a decrease in the duration of the subjective day, while the duration of the subjective night remained at 12 hours as in wild type (Konopka & Orr, 1980). Although And represents the third clock gene identified on the X chromosome, it is likely that not all clock genes in the Drosophilu genome have been identified (see also Jackson, 1983). Genes involved in determining the fundamental parameters of biological clocks, such as the free-running period. may be expected to have counterparts expressed in the timekeeping cells of other organisms, as is the case with the per gene (Siwicki et al., 1989). Further analysis of And will determine whether it too has a counterpart in other organisms, or whether And is a clock gene unique to Drosop/zilu. ACKNOWLEDGMENTS We thank Steve Wells and Carolyn Hax for excellent technical assistance. This work was supported by grants to R.K. from the USPHS-NIH.
REFERENCES Bruce. V. G. (1972). Mutants of the biological clock in Ch/uni~,donioncrsri+/iurdi. Ge.rir/ics. 70, 537-548. Craymer. L. & Roy. E. ( 1980). New mutants. D,o.rop/ii/r r,ir/NiioRcr.\.cc,r.. Droaophilu //+7nir. Seri,., 55, 200-204. Dushay. M. S.. Konopka. R. J . . Orr. D.. Grccnacrc, M. L., Kyrincou. C . P.. Rosbash, M. & Hall, J . C. ( 1990). Phenotypic and genetic analysis of Clock. ii new circadian rhythm mutant in Drosop/iih iiic,/~iiio~u.srt~r. Gcncrics. 125, 551 -5 78.
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R. J. KONOPKA, R. F. SMITH A N D D. ORR
Dushay, M., Rosbash. M. & Hall, J. C. (1989). The Disconnecied visual system mutantions in Drosophila me/unogu.yrer drastically disrupt circadian rhythms. J . Biol. Rhyrhmi, 4 . 1-27. Enright. J. (1965). The search for rhythmicity in biological time-series. J . Theorei. Biol., 8. 426-468. Feldman. J. F. & Hoyle, M. N. (1973). Isolation ofcircadian clock mutants of Neurospora crassu. Genetics. 75. 605-613. Hall. J. C. (1990). Genetics of circadian rhythms. .4nn. Rev. Genei.. in press. Helfrich, C . (1986). Role of the optic lobes in the regulation of the locomotor activity rhythm of Drosophilu melutiogasrer: behavioural analysis of neural mutants. J . Neurogener.. 3. 321-343. Jackson. F. R. (1983). The isolation of biological rhythm mutations on the autosomes of Drosophilu nielatiogasrer. J . Neurogerief.. I , 3- 15. Konopka, R. J . (1979). Genetic dissection of the Drosophilu circadian system. Fed. Proc.. 38, 2602-2605. Konopka. R. J. (1987a). Genetics of biological rhythms in Drosophilu. Ann. Rev. Genei.. 21, 227-236. Konopka, R. J . (1987b). Neurogenetics of Drosuphilu circadian rhythms. In Evoluiionury Generics of Itiverrehrure Beliuvior. ed. M. D. Huettel (New York: Plenum Press), pp. 215-221. Konopka, R. (1988). A variegating long-period clock mutant of Drosophilu mrlanogusrer. Gener. ( L f e Sci. Adv. I , 7. 3 9 4 I . Konopka. R . J. & Benzer, S . (1971). Clock mutants of Drosophilu nielanogusrer. Proc. Nail. Arud. Sci. U S A , 68, 21 12-21 16. Konopka. R. & Orr, D. (1980). Effects of a clock mutation on the subjective day-implications for a membrane model of the Drosophilu circadian clock. In Developnienr and Neurobiology of Drosophila, eds. 0. Siddiqi, P. Babu, L.M. Hall & J.C. Hall (New York: Plenum Press), pp.409-416. Konopka, R. J., Pittendrigh, C. & Orr, D. (1989). Reciprocal behaviour associated with altered homeostasis and photosensitivity of Drosophilu clock mutants. J . Neurugener., 6, 1-10, Lewis. E. B. (1960). A new standard food medium. Drosophilu I n f o . Serv., 34, 117-1 18. Lewis. E. B. & Bacher, F. (1968). Method of feeding ethyl methane sulfonate (EMS) lo Drosuphiku males. Drosophila Info. Serv., 43, 193. . Mortin. M. & Lefevre, G . (1981). An RNA polymerase 11 mutation in Drosophih niekanogusier that mimics Ultrabithorax. Clironio,somu,82, 237-248. Saunders, D. S., Henrich. V . C. & Gilbert, L. 1. ( 1989). Induction ofdiapause in Drosophilu nielunopsrer: photoperiodic regulation and the impact of arrhpthmic clock mutations on time measurement. Proc. Null. Acad. Sri. LISA. 86, 3748-3152. Siwicki. K.. Eastman. C.. Petersen, G.. Rosbash. M. & Hall. J. (1988). Antibodies to the period gene product of Drosopliilu reveal diverse tissue distribution and rhythmic changes in the visual system. Neuron. I . 141-150. Siwicki, K., Strack. S.. Rosbash, M., Hall, J. & Jacklet. J . (1989). An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla. Neuron. 3, 5 1-58, Smith, R. ( 1982). Genetic analysis of the circadian clock system of Drosipliila nielunogusiei. Ph.D. Thesis, California Institute of Technology. Smith. R. & Konopka, R. (1981). Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus. Molec. Gen. Getiei.. 183. 243-251. Wieschaus, E., Nusslein-Volhard. C. & Jurgens. G . (1984). Mutations affecting the pattern of the larval cuticle of Dro.wpliilu nielanoga.c/er: 3. Zygotic loci on the X-chromosome and 4th chromosome. Wilhelm Ro1r.r Arch. Dev. Biol., 193. 296-307. Winfree, A. T. (1980). The Geomerrj, of Biolugicd Tinie. (New York: Springer-Verlag). Winfree. A. T. & Gordon, H. (1977). The photosensitivity of a mutant circadian clock. J . Conip. Physiol.. 122, 87-109. Zerr. D. M.. Hall, J. C.. Roshash. M. & Siwicki. K. K . (1990). Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drusophilu. J . Neurosci. 10. 2749-2762.
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Reprint.; available directly from the publisher Photocopying permitted by license only
1991 Harwood Academic Publishers G m b H Printed in the United Kingdom
CHARACTERIZATION OF ANDANTE, A NEW DROSOPHZLA CLOCK MUTANT, AND ITS INTERACTIONS WITH OTHER CLOCK MUTANTS RONALD J. KONOPKA'.?*, RANDALL F. SMITH', and DOMINIC ORR'
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iDepartnient qf Biologj,, Clarkson Universitj., Potsdam, N Y 13699, U S 'Division o f Biologj., Cul$wnia Institute of' Trrhnology, Pasadena, C A 91 125, U S ~ R i w i w iJfu l r ?S, IYYO: rivisivf Aiigiisr 3 / , 19901
A new clock mutant. named Andunre. has been identified on the X chromosome of Drosophilu ttielutiugast i v . dtidunrr lengthens the period of the circadian eclosion and locomotor activity rhythms by 1.52.0 hours. The phase response curves for the eclosion and activity rhythms, indicating light-induced phase shifts. show a similar degree of lengthening. Atidanre also lengthens the periods of other clock mutants, including Clock. and alleles of the periodlocus. Analysis of locomotor activity rhythms reveals that Andanre is semi-dominant. and Anclulirc, rhythms are highly temperature compensated. The sine ocidi.7 mutation, which eliminates the outcr visual system. has no effect on the period of Atidunri,. Deficiency mapping indicates that .4ndunti, is located in the IOEI-2 to lOFl region of the X chromosome, close to the niitiicrrurr-clrr.tk~,locus. Whereas A n h n i r flies have a h k j wing phenotype. ditskj. flies d o not have an .4titfante clock phenotype. K q . i t w d s : ciriudiun rltjtlims. li~cottiotoructivitj, eclosion , period niiitutiis. Clo1.k mutant, visual mutunt
INTRODUCTION Mutations that alter the periodicity of circadian rhythms have been isolated in Drosophila (Konopka & Benzer, I97 l ) , Chlamydomonas (Bruce, 1972), and Neurospora (Feldman & Hoyle, 1973). In the case of Drosophila, two genetic loci on the X chromosome have been described in detail: the period ( p e r ) locus, at which several alleles have been isolated (for review, see Konopka. 1987a, b), and the Clock ( C l k ) locus (Dushay et al., 1990). at which only one allele is known, and which maps very close to the per locus. In this paper we describe a third genetic locus on the X chromosome that is quite separated from, and proximal to, the per and Clk loci. We have named the originally isolated allele Andante ( A n d ) ,as it produces a moderate slowing of the eclosion and adult locomotor activity rhythms. Andante flies also show a dusky wing phenotype, and indeed, Andante maps very close to the miniature-dusky region (see below). Brief descriptions of some of the properties of Andante may be found in Smith (1982) and Konopka (1987b). MATERIALS AND METHODS The Canton-S strain was used in this mutant screen. Flies were raised on cornmeal, sugar, and yeast medium (Lewis, 1960). Males were mutagenized with ethyl methanesulfonate according to the method of Lewis and Bacher (1968). Stocks of mut'Send reprint requests to Ronald J . Konopka, 430 South Santa Anita Ave., Pasadena, CA 91 107, US. I03
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I04
R. J . KONOPKA. R . F. SMITH AND D. ORR
agenized males with identical X chromosomes were maintaiped with attached-)< females. (All attached-)< females used in these experiments also carried the markers j~ellnw(.I>) body and .forked ( . f ) bristles.) These stocks were raised in a 12hours light-I2 hours dark cycle (LD 12:12) at 22°C. At the end of a light period, they were transferred to constant darkness (DD), and given a 12hour, 29” temperature pulse beginning 12 hours after the LD to D D transition. The stocks were then maintained in DD at 22”C., and the relative numbers of males and females emerging over roughly 12 hour periods were determined. The attached-X females, having normal rhythms, served as a reference for detecting males with abnormal emergence patterns. Stocks with apparent abnormalities in the emergence patterns of males were rescreened using automated eclosion monitors known as “bang boxes” (see Konopka & Benzer, 1971). Some stocks were screened using temperature pulses, in attempts to recover putative clock mutants with altered temperature responses. In the case of one of these stocks, the median of the eclosion peak of males, on the third day after a 12 hour, 31°C. temperature pulse, showed a 4 hour phase delay compared to the females. (“Median” = the time at which 50% of the flies in the peak had emerged.) This phase delay was apparently the result of a lengthening of the period of the eclosion clock produced by the Andante mutation that had been induced in the males. Locomotor activity rhythms of individual adult flies were determined in infrared light as described previously (Konopka & Benzer, 1971; Smith & Konopka, 1981). Some of the activity monitor channels were connected to Esterline-Angus event recorders. Other channels were connected to a microprocessor-based unit that kept an hourly count of activity events. These numerical records were then plotted and used for determination of the period length by means of periodogram analysis (Enright, 1965). Unless otherwise stated, activity rhythms were measured at 24°C. Phase response curves (PRCs) for the eclosion rhythm were determined as follows. Fly stocks were raised in LD 12:12 at 18°C. Pupae were harvested during a light period and placed in the “bang boxes” in DD at 18°C. at the end of the 12 hour light period. Experimental cultures were exposed to a light pulse of at least lOOlux in intensity and 40 minutes’ or 80 minutes’ duration at various times after the transition from LD to DD. Control cultures received no light pulse. Phase advances and delays were determined from eclosion peaks that occurred at least three days after the administration of the light pulse, in order to allow the advances and delays to fully develop and stabilize. Phase response curves for adult locomotor activity rhythms were determined as described in Dushay et al. (1990).
RESULTS Phenotypes of the Andante nrutant Figure 1 illustrates the eclosion rhythm of And males (bottom) run concurrently with attached-)< females that have a normal rhythm (top). The And rhythm is longer than that of the control by about I .2 hours, resulting in a substantial phase difference between the males and the females by the sixth and seventh day of the run. The eclosion rhythm periods of Andmales, And females, And heterozygotes, and attachedX females with wild type rhythms are given in Table I. While the periods of the wild type rhythms are, as expected, close to 24 hours (as is also true for other genotypes with wild type eclosion rhythms-see Smith & Konopka, 19811, the periods of And
I05
T H E A N D CLOCK MUTANT
xx
7 = 24.3
7 = 25.5
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And
r
0
I
I
2
I
I
4
I
I
6
I DAYS
FIGURE I Eclosion rhythms of And males (bottom record) and attached-)< v. F And' females (top record) measured concurrently at 18°C. in constant darkness after entrainment in L D 12:12 at 18°C. The beginning of the record (time 0) corresponds to the transition from LD to D D at the end of the light period. T = period estimates in hours.
males and females exceed 25 hours. The periods of Aiidheterozygotes are only slightly longer than the wild type periods. Thus the And mutation does not show quite the degree of semi-dominance for the eclosion rhythm as i t does for the locomotor activity rhythm (see below). Figure 2 shows two examples of event recorder tracings of locomotor activity rhythms of And flies (two bottom records) in comparison with the wild type rhythm of a Canton-S fly (top record). The And rhythms are noticeably longer than normal; this period lengthening is especially obvious in the activity offsets, which are more distinct than the activity onsets. The periods of activity rhythms of And males, And females, And heterozygotes, and control flies are given in Table 11. It can be seen that And flies have significantly longer activity rhythms than d o wild type flies, and that And heterozygotes have periods intermediate between Atid homozygotes and wild TABLE I Periods of the eclosion rhythm of flies bearing the And mutation and controls ~~
Run
Genotype And/Y .4nd+/.4nd+ (XX. y / ) AndlY And'lAnd' (XX. ?' f ) AndlY And/And AndlY Andl And And/ And ( F M 7 ) AtidlAnd ' ( F M 7 ) AndlAtid' ( F M 3 ) +
Period & Std. Dev. 25.1 23.9 25.6 24.3 25.3 25.2 25.3 25.1 24.3 24.4 24.8
_+ 0.6h
k 1.1 h k 0.5 h f 0.6h _+ 0.8 h & 1.0h i 0.8 h f 0.2h f 0.5 h k 0.4h k 0.5 h
The genotype of the females in the second and fourth rows were attached->(. yellor forked. The And+-bearing chromosomes in the last three rows were X-chromosomal balancers [ f n ( I I F M 7 or fn(I!FM3].
R. J. KONOPKA, R. F. SMITH A N D D. ORR
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WT
And
FIGURE 2 Event recorder representations of the locomotor activity rhythms of individual Drosophilu adults, monitored in infrared light at 24°C. The top record shows the activity rhythm of a Canton-S wild type (WT) female, and the bottom two records illustrate the activity rhythms of two different And females. All records are plotted modulo 24 hours. In addition, all records are double plotted in order to facilitate visual inspection of the data. The And records are noticeably delayed each day in comparison with the wild type record. reflecting the longer free-running period of And.
type, illustrating the semi-dominance of the Andmutation. Partial dominance appears to be a general characteristic of Drosophifu clock mutations (Konopka, 1987b). The And mutation also has a visible wing phenotype, similar to that of dusky (dy). Complementation tests with dy confirmed that the wing phenotype is indeed due to a dy mutation associated with And. However, analysis of the activity rhythms of dv TABLE I1 Activity rhythm periods of flies bearing the And mutation alone and in combination with controls Genotype And/Y Andl And And+/Y And ' 1 And ' dvldy niD/mD
AndlAnd And/dy Atid/niD
+
d ~and . m", and
Period & Std. Dev.
N
25.5 & 0.9 h' 25.7 & 0.3 h' 24.0 & 0.4 h 24.2 f 0.3 h 24.6 & 0.4h 23.7 k 0.4 h 24.8 & 0.2 h' 25.0 & 0.3 h' 24.8 k 0.S h'
18 10 12 7 9 9
9
7 5
'These genotypes are significantly longer than the wild type controls (below) ( p < 0.01, t-tests). 'There is no significant difference among these genotypes ( p 0.10. t-tests). However, these And heterozygotes have periods significantly longer than wild type (above) and significantly shorter than And homozygotes ( p i0.01. t-tests).
T H E .4ND CLOCK MUTANT
107
TABLE 111 Temperature compensation of activity rhythms of And and wild type Hies Temp. ("C.)
Period i Std. Dev.
N
Andante
17 22 25
25.90 F 0.84 h 25.99 2 0.80 h 25.86 2 0.44h
15 I? 21
Canton-S
17 22 25
24.15 k 0.40h 23.89 f 0.34 h 23.82 k 0.45h
75 27 17
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Genotype
and another mutation in the same region, m D ,indicated that these two alleles did not show an And rhythm phenotype (Table 11). In particular, the periods of dy and mD were in the normal range, and in addition, the periods of Ar1dld.v and And/mD heterozygotes were indistinguishable from the period of And/ heterozygotes, and significantly shorter than the period of And/And homozygotes. A well known property of circadian clocks is the temperature compensation of period length; that is, the steady-state period length of a rhythm shows little variation with temperature in a physiological range. Thus, the Q,,,of period length is usually close to 1 .O. The periods of locomotor activity rhythms of And mutant flies over the range 17°C. to 25°C. are given in Table 111. The total amount of variation of the mean periods in this range is less than 0.2 hours. In this respect, And is very similar to wild type; for wild type flies, the mean periods vary about 0.3 hours in the same temperature range (Table 111; Konopka, Pittendrigh & Orr, 1989). In contrast, for the same temperature range, the variation in mean period lengths for perS flies is about 0.8 hours, and for perL flies it is about 2.7 hours (Konopka, Pittendrigh & Orr. 1989).
+
Recombination and mapping e.uperiments
In order to localize the And mutation on the X chromosome, a mapping stock containing the markers yellm. ( J'), chocolate (cho), crossveinless (cv), vermillion (v). and forked ( f ) was crossed to an And stock, and F, recombinant males were isolated and mated to attached-)< females to establish stocks such that the males in each stock had identical X chromosomes. The activity rhythm periods of the males in these stocks were then determined. In Table IV, it can be seen that the And rhythm TABLE IV Recombination mapping of Anti: Activity rhythm phenotypes Recombinant Types
Period
Std. Dev.
25.6 & 0.6h 23.9 2 0.311 23.8 f 0.2h 25.4 f 0.3 h 25.4 & 0.3h 24.0 & 0.2 h
24.4 & 0.4 h 25.2 2 0.3 h
N
I08
R. J. KONOPKA, R. F. SMITH A N D D. ORR
TABLE V Deficiency mapping of '4nd:Activity rhythm phenotypes. Numbers of flies are indicated in parentheses Genotype of X Chromosome B: And Anti+ Genotype of X Chromosome A
Of( 1)KA7 Df ( I ) K A 6 qf(l)H.48S Of( 1)m"V-J
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D f ( 1)NIOS
And And
+
Period f Std. Dev. 26.6 25.8 25.9 25.1 24.6 24.3 25.7 24.8
Period & Std. Dev.
& 0.3 h (6) & O.2h (7) & O.2h (6) F 0.2h (6) f 0.2 h (7) f 0.3 h (5) f 0.3 h (10) & 0.2h (9)
24.4 24.0 25.2 24.5 24.3 23.8 24.8 24.2
f 0.4h & 0.2 h f 0.4 h & 0.2 h & 0.3 h f 0.8 h & 0.2 h f 0.3 h
Breakpoints 10A9; IOF6-7 IOEI: llA7-8 IOCI-2: IIAI-2 IOC2-3: IOEI-2 IOFI: IOF9-I0 IOF7: I l D l
(10) (6) (8) (7) (8)
(9) (9) (7)
Refst a. b a. c a a. b a, c a, c
tReferences: a, Craymer & Roy (1980): b. Mortin & Lefevre (I981 ); c. Wieschaus. Nusslein-Volhard & Jurgens (19x4).
phenotype maps within the vermillion-jorked interval, and that the And rhythm phenotype segregates with the 419wing phenotype. Deficiencies that involve the nziniarure-dzttskj3 region were then used to further localize the And mutation. The results are shown in Table V. The And rhythm phenotype is uncovered by the deficiencies KA6 and KA7, but not by the deficiencies NlU5, RA47, or nz?"-'. In particular, the period of KA6/And is not significantly different from that of And/Anci ( p > 0.1, t-test), while the period of K A 7 / A n d is significantly longer than that of And/And ( p < 0.01, t-test). On the other hand, the periods of NlUS/And, RA47/And, and n~*~'-'/And are all significantly shorter than that of Atzd/And ( p < 0.01, t-tests). The H A 8 5 result is somwhat ambiguous. Although HA85 does appear to uncover And, as it should, (the period of HA85/And is not significantly different from that of And/And, as a t-test yields p > O . l ) , the period of the H A 8 5 / + heterozygote is significantly longer than normal ( p < 0.01, t-test). These results map the rhythm phenotype of And to the region 10E1-2 to lOFl on the X chromsome, which is very close to the miniarure-dusky region. TABLE VI Lengthening of activity rhythm periods produced by And in combination with other clock mutations. measured at 22°C. Genotype
Period & Std. Dev.
N
prrS perS And
19.1 f 0.Sh 20.2 & 0.Sh
21 19
+ I.Oh*
Clock Clock And
22.6 f 0.4h 23.7 f 0.3 h
I04 12
+ 1 . 1 h*
Wild type Antl
23.5 & 0.4h 25.3 i 0.5 h
23 35
+ 1.8 h*
perL1 perL1 And
29.6 _+ 1.1 h 31.2 & 0.7h
21 28
+ 1.6h*
perL' pwL' And
2 9 2 f 1.6h 32.2 f 0.6 h
12 II
+ 3.0 h*
*All lengthenings are significant (t-tests. p < 0.01)
Lengthening
T H E ,4ND CLOCK MUTANT
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Interactions qf' Andante with per alleles and with Clock Table VI shows the periods of activity rhythms of double mutant, male flies that bear . or Clock mutations, in comparison the Andante mutation and the per", p e r L 1perL2, with males carrying only a per allele or Clock. Also shown are period values for control Andante and wild type males calculated from activity records of sibling males obtained from the cross that produced the double mutants. These results show that the And mutation significantly lengthens the period of each of the four mutations tested. The lengthening is least for the two mutations with periods shorter than 24hours; And lengthens perS by 1.0 hours and Clock by 1.1 hours. The period lengthening produced by And is greatest for perL2,at 3.0 hours. perL2appears to be a weakerper allele thanperL'in terms ofper product activity (see Konopka, 1988). Both wild type and perL1males show an intermediate degree of lengthening, at 1.8 hours and 1.6 hours respectively. Activitj. rhj~thnlperiods of' the Andante, sine oculis double mitatit Although the outer portions of the fly's visual system are not necessarily for entainment or maintenance of circadian rhythmicity of locomotor activity (Helfrich, I986), it is possible that input from the visual system might modify the period of the activity rhythm (see discussion in Hall, 1990). Histochemical staining of photoreceptor cells and optic lobe cells was observed using antibodies to the per gene product (Siwicki et al.. 1988). In addition, Siwicki et al. found that the intensity of per-specific staining in the visual system showed an oscillation that was apparently controlled by an endogenous, circadian oscillator (also see Zerr et al., 1990). In order to determine whether the absence of the outer portions of the visual system affected the period of Andante flies, the activity rhythms of flies carrying Andante and sine oculis were measured. The expression of sine oculis is somewhat variable, but flies can be selected that have no compound eye facets at all. Histological examination of these flies shows that such flies have no ommatidial cells; the lamina and part of the medulla are missing as well (Helfrich. 1986 & Konopka, unpublished observations). Table VII shows that there is no significant difference in the mean activity rhythm period of eyeless And;so flies as compared with And flies. In addition, the mean activity rhythm period of so flies is not significantly different from that of wild type flies ( p > 0.1. t-tests). Phase response curves for the eclosiori and uctirity rliythms of Andante und wild type pies Figure 3 shows the amount of phase shift of the eclosion rhythm produced by light pulses administered to pupae of And males and attached-)< females. As is the usual TABLE VII Activity rhythm periods of flies carrying the Atidutirr and sinc oculis mutations alone and in combination Genotype And And:so SO
Wild type
Period f Std. Dev. 25.5 25.6 23.5 23.8
f 0.9h' f 1.6h' & 0.8h' & 0.9h'
"There is no significant dimerence between each set of genotypes ( p > 0. I . t-tests).
N 18 34 18 15
R. J . KONOPKA. R. F. SMITH A N D D. ORR
I10
A
And/Y
A
+/+
[YfX^x/Y,
A
4
f
A f n
A A
A
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-6 ~
0
I
8
4
12
I
I
16
20
Time of L i g h t P u l s e
,
32
28
24
( h a f t e r DL-DD
I
,
~
36
I
FIGURE 3 The eclosion rhythm phase response curves of Atid males (dark symbols) and attached-)< .4nd+ females (open symbols). determined concurrently. Flies were raised in LD 12: 12 at 18°C.: light pulses (white light at least IOOlux in intensity) were administered at various times after the transition from LD to D D at 18°C. The LD to D D transition occurred at the end of a light period. The duration of the light pulses was 40 minutes (triangles) or 80 minutes (squares); the points are plotted at the pulse endpoints.
case for circadian rhythms, phase delays occur during the early subjective night, and phase advances during the late subjective night (Winfree, 1980). The maximum phase delay in the And response curve (about 4 hours) is slightly greater than that for the wild type rhythm of attached-)< females (about 2 hours). The maximum phase advance in the And and wild type response curves is similar (about 2 hours). Both 6.00
3
And
4.00 A
v,
'YI t
2.00
W
0.00
-
I
v,
- 2 00
W
v,
2
-4.00
a -6.00
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TIME OF LIGHT PULSE (HRS) FIGURE 4 The locomotor activity rhythm phase response curve of Atid male adults. Flies were raised in constant light (LL) at 22°C.. then transferred to constant darkness at the same temperature a few days after eclosion. Flies were maintained for several days in D D in order to determine each fly's free-running period. Light pulses of about 2000 lux wcre administered for 10 minutes at various phases, determined after normalizing for the free-running period. The resulting phase shifts were then calculated. relative to the initial phase of the rhythm before the light pulse. after several more days in DD. The error bars indicate the standard deviations from the mean phase shift, computed from measurements on several flies.
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TIME OF LIGHT PULSE (HRS) FIGURE 5 The locomotor activity rhythm phase response curve of Canton-S wild type (WT) male adults. Protocols are the same as described in the legend to Figure 4.
response curves are classified as Winfree Type I (Winfree, 1980), indicating “weak” resetting by light. In the second cycle of the response curves, there is a noticeable lag (on the order of 2 hours) in the And curve relative to that of wild type, reflecting the longer free-running period of the And rhythm in constant darkness. The second phase delay maximum occurs at hour 28 for the wild type rhythm, and at hour 30 for the And rhythm. Likewise, a t hour 32, the light pulse produces a phase delay of about an hour in the Atid eclosion rhythm, while the wild type rhythm is phase advanced by about 2 hours. A similar indication of the lengthened Atidoscillation can be seen in comparing the phase response curve for the locomotor activity rhythm of And (Figure 4) with that of wild type (Figure 5). At hour 26, the And curve shows no phase shift, indicating that the oscillator is still in the subjective day, while the wild type curve shows a phase delay of more than 3 hours, indicating that the wild type oscillator is in the subjective night. Again, the Atid response curve is about 2 hours longer than that of wild type. The response curves for the activity rhythms of And and wild type have similar maximum phase delays (more than 3 hours). The maximum phase advance in the And curve is about 3 hours, while in the wild type curve it is about 2 hours. Both the And and wild type activity rhythms show Type 1 (weak) resetting behavior, as was observed for the eclosion rhythms. DISCUSSION The Andunre locus is the third genetic locus on the rnelunogaster X chromosome that significantly increases or decreases the period of the circadian eclosion and locomotor activity rhythms, the other two being per (Konopka & Benzer, 1971) and Clock (Dushay et al., 1990). The disco mutation, which is also on the X chromosome, disrupts rhythmicity but does not produce a stable change in period (Dushay, Ros-
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R. J . KONOPKA. R. F. SMITH AND D. ORR
bash & Hall, 1989). Interestingly, the Clock mutation decreases the period by about I .5 hours, while the Anduntr mutation increases the period by a similar amount. It is not known at this time whether these two genes are related in some way, or whether 1.5 hours represents some type of quantization or temporal subunit. In the case of activity rhythms, both And and Clock are semi-dominant; the periods of heterozygotes are intermediate between mutant and wild-type (Table 11). The semi-dominance is less obvious in the case of eclosion rhythms (Table I). The perS mutation is also semi-dominant (Konopka & Benzer. 1971). as the period of the p e r S / + heterozygote (21.9h) is intermediate between pers (19.5 h) and wild type (24.4 h). However, the perL/+ and pero/+ heterozygotes are only about 0.5 hours longer then wild type (Smith & Konopka, 198 I ) . The disco mutation is recessive (Dushay, Rosbash & Hall. 1989). Although the Andmutant also has a dzrskj, phenotype, the d.v and mD alleles do not have an And phenotype, either as homozygotes or as heterozygotes with And (Table 11). As And has been shown to map close to the nziniature-dusk?’ region (Table V), the mutation event may have affected both the dusky gene and a clock gene, And. Alternatively, And may represent a double mutant. The relationship between the period-lengthening phenotype and the dusky phenotype of And remains to be clarified. The periods of both Clock and And activity rhythms are very well temperature compensated; the QI,,for each is very close to I .O. This is in contrast to perS and p e r L , whose periods have a greater variation over the 17OC. to 25°C. range; perS shows a variation of 0.8 hours, while perL has a 2.7 hour variation in this temperature range (Konopka, Pittendrigh & Orr, 1989). If the amount of temperature-induced variation in period length was proportional to the amount of lengthening induced by the mutation, then, using the perL characteristics as a basis for calculation, a mutation that produces a lengthening of about 2 hours, similar to And, should show one-third the amount of temperature-induced variation ofperL,or about 0.9 hours. In fact, the variation of And is much less; it is less than 0.2 hours (Table 111). Thus the variation of period with temperature is not proportional to the amount of period lengthening, but is different for the two period-lengthening mutants, A n d and perL, reflecting a difference in the homeostatic control of period in these two mutations. The Andmutation also has a lengthening effect on period when in combination with other clock mutations (Table VI). The amount of lengthening is in the range of 1.0 to I .8 hours, except in the case of p e r L 2 ,which is lengthened by 3.0 hours. The perL2 allele apparently represents the “weakest” per allele that still shows a period, without being completely arrhythmic. For example, a substantial fraction (about 50%) of perL2/perL’heterozygotes were weakly rhythmic or arrhythmic, while all perL’/perL’ heterozygotes were strongly rhythmic (Konopka, 1988). In addition, perL’/perL’ heterozygotes were 0.9 hours longer in period than perL’ flies, while perL2/perL’ heterozygotes were 3.3 hours longer than perL2flies. Thus the 3.0 hour lengthening produced by And in combination with perL’ may be attributable to the weak nature of the perL’ allele. The results in Table VTI indicate that flies lacking a substantial part of the visual system, including the photoreceptors, lamina, and at least part of the medulla, still show a locomotor activity rhythm with a normal free-running period (so flies), thus confirming the results of Helfrich (1986). The so mutation likewise has no effect on the expression of And, as And;so flies have the same free-running period as And flies (Table VII). Eyeless so flies can also entrain to light cycles (Helfrich. 1986 & Konopka, unpublished experiments), indicating the existence of an extraretinal photoreceptor for entrainment of the activity rhythm. In view of these results, the significance of
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T H E AND CLOCK MUTANT
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per-specific staining in the outer visual system by antibodies to the per gene product (Siwicki et al., 1988) is difficult to interpret. Siwicki et at. (1988) suggest that the per protein in the visual system may be involved in non-circadian visual behaviors, or in a circadian system separate from that controlling eclosion or activity. The possibility that per may be involved in other aspects of light-mediated timekeeping is suggested by the results of Saunders et al. (1989). who have shown that while arrhythmic per mutants have a photoperiodic ovarian diapause, the critical daylength of pero mutants is shifted by about two hours. Thus, while per is essential for rhythmicity of eclosion and locomotor activity, it may also have a non-essential role in modulating other timekeepers and/or behaviors. The And gene product may also have essential and non-essential roles in timekeeping and other behavioral and developmental processes. The null phenotype of the A d locus has yet to be determined. A comparison of the expression patterns of the A n d product and the per product may aid in the localization of the cells responsible for the control of the locomotor activity rhythm. The phase response curves for both the locomotor activity and eclosion rhythms show the characteristic period lengthening of And in comparison with the wild type controls. Otherwise, the shapes of the Andand wild type curves are fairly similar. The maximum phase delay for the Atid eclosion rhythm and the maximum phase advance for the And activity rhythm are slightly greater than those of wild type. The eclosion and activity rhythm response curves for And and wild type are Winfree Type 1 ("weak" resetting) (Winfree, 1980), as is the activity rhythm response curve of Clock (Dushay et al., 1990). In contrast, the perS mutation changes the Type I response curve for the wild type eclosion rhythm to a Type 0 ("strong" resetting) curve, in addition to shortening the period of the response curve by about 5 hours (Konopka, 1979; Winfree & Gordon, 1977). As the And response curves are only aboat 2 hours longer than those of wild type, it is not possible to unequivocally determine whether And selectively affects the duration of the subjective day or night. In the case of perS, the 5 hour decrease in free-running period was shown to result from a decrease in the duration of the subjective day, while the duration of the subjective night remained at 12 hours as in wild type (Konopka & Orr, 1980). Although And represents the third clock gene identified on the X chromosome, it is likely that not all clock genes in the Drosophilu genome have been identified (see also Jackson, 1983). Genes involved in determining the fundamental parameters of biological clocks, such as the free-running period. may be expected to have counterparts expressed in the timekeeping cells of other organisms, as is the case with the per gene (Siwicki et al., 1989). Further analysis of And will determine whether it too has a counterpart in other organisms, or whether And is a clock gene unique to Drosop/zilu. ACKNOWLEDGMENTS We thank Steve Wells and Carolyn Hax for excellent technical assistance. This work was supported by grants to R.K. from the USPHS-NIH.
REFERENCES Bruce. V. G. (1972). Mutants of the biological clock in Ch/uni~,donioncrsri+/iurdi. Ge.rir/ics. 70, 537-548. Craymer. L. & Roy. E. ( 1980). New mutants. D,o.rop/ii/r r,ir/NiioRcr.\.cc,r.. Droaophilu //+7nir. Seri,., 55, 200-204. Dushay. M. S.. Konopka. R. J . . Orr. D.. Grccnacrc, M. L., Kyrincou. C . P.. Rosbash, M. & Hall, J . C. ( 1990). Phenotypic and genetic analysis of Clock. ii new circadian rhythm mutant in Drosop/iih iiic,/~iiio~u.srt~r. Gcncrics. 125, 551 -5 78.
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Dushay, M., Rosbash. M. & Hall, J. C. (1989). The Disconnecied visual system mutantions in Drosophila me/unogu.yrer drastically disrupt circadian rhythms. J . Biol. Rhyrhmi, 4 . 1-27. Enright. J. (1965). The search for rhythmicity in biological time-series. J . Theorei. Biol., 8. 426-468. Feldman. J. F. & Hoyle, M. N. (1973). Isolation ofcircadian clock mutants of Neurospora crassu. Genetics. 75. 605-613. Hall. J. C. (1990). Genetics of circadian rhythms. .4nn. Rev. Genei.. in press. Helfrich, C . (1986). Role of the optic lobes in the regulation of the locomotor activity rhythm of Drosophilu melutiogasrer: behavioural analysis of neural mutants. J . Neurogener.. 3. 321-343. Jackson. F. R. (1983). The isolation of biological rhythm mutations on the autosomes of Drosophilu nielatiogasrer. J . Neurogerief.. I , 3- 15. Konopka, R. J . (1979). Genetic dissection of the Drosophilu circadian system. Fed. Proc.. 38, 2602-2605. Konopka. R. J. (1987a). Genetics of biological rhythms in Drosophilu. Ann. Rev. Genei.. 21, 227-236. Konopka, R. J . (1987b). Neurogenetics of Drosuphilu circadian rhythms. In Evoluiionury Generics of Itiverrehrure Beliuvior. ed. M. D. Huettel (New York: Plenum Press), pp. 215-221. Konopka, R. (1988). A variegating long-period clock mutant of Drosophilu mrlanogusrer. Gener. ( L f e Sci. Adv. I , 7. 3 9 4 I . Konopka. R . J. & Benzer, S . (1971). Clock mutants of Drosophilu nielanogusrer. Proc. Nail. Arud. Sci. U S A , 68, 21 12-21 16. Konopka. R. & Orr, D. (1980). Effects of a clock mutation on the subjective day-implications for a membrane model of the Drosophilu circadian clock. In Developnienr and Neurobiology of Drosophila, eds. 0. Siddiqi, P. Babu, L.M. Hall & J.C. Hall (New York: Plenum Press), pp.409-416. Konopka, R. J., Pittendrigh, C. & Orr, D. (1989). Reciprocal behaviour associated with altered homeostasis and photosensitivity of Drosophilu clock mutants. J . Neurugener., 6, 1-10, Lewis. E. B. (1960). A new standard food medium. Drosophilu I n f o . Serv., 34, 117-1 18. Lewis. E. B. & Bacher, F. (1968). Method of feeding ethyl methane sulfonate (EMS) lo Drosuphiku males. Drosophila Info. Serv., 43, 193. . Mortin. M. & Lefevre, G . (1981). An RNA polymerase 11 mutation in Drosophih niekanogusier that mimics Ultrabithorax. Clironio,somu,82, 237-248. Saunders, D. S., Henrich. V . C. & Gilbert, L. 1. ( 1989). Induction ofdiapause in Drosophilu nielunopsrer: photoperiodic regulation and the impact of arrhpthmic clock mutations on time measurement. Proc. Null. Acad. Sri. LISA. 86, 3748-3152. Siwicki. K.. Eastman. C.. Petersen, G.. Rosbash. M. & Hall. J. (1988). Antibodies to the period gene product of Drosopliilu reveal diverse tissue distribution and rhythmic changes in the visual system. Neuron. I . 141-150. Siwicki, K., Strack. S.. Rosbash, M., Hall, J. & Jacklet. J . (1989). An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla. Neuron. 3, 5 1-58, Smith, R. ( 1982). Genetic analysis of the circadian clock system of Drosipliila nielunogusiei. Ph.D. Thesis, California Institute of Technology. Smith. R. & Konopka, R. (1981). Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus. Molec. Gen. Getiei.. 183. 243-251. Wieschaus, E., Nusslein-Volhard. C. & Jurgens. G . (1984). Mutations affecting the pattern of the larval cuticle of Dro.wpliilu nielanoga.c/er: 3. Zygotic loci on the X-chromosome and 4th chromosome. Wilhelm Ro1r.r Arch. Dev. Biol., 193. 296-307. Winfree, A. T. (1980). The Geomerrj, of Biolugicd Tinie. (New York: Springer-Verlag). Winfree. A. T. & Gordon, H. (1977). The photosensitivity of a mutant circadian clock. J . Conip. Physiol.. 122, 87-109. Zerr. D. M.. Hall, J. C.. Roshash. M. & Siwicki. K. K . (1990). Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drusophilu. J . Neurosci. 10. 2749-2762.
