0020-71 IX/92 $5.00 + 0.00 Pergamon Press Ltd
hr. J. Biochem. Vol. 24, No. 7, pp. 1141-1150, 1992 Printed in Great Britain
IDENTIFICATION OF TWO CLOCK PROTEINS IN ACETABULARIA CLIFTON11 AND CONSTRUCTION OF cDNA LIBRARIES FROM ACETABULARIA CLIFTONII AND ACETABULARIA MEDITERRANEA* XIAO-PINGYANG? and EC~N J. Max-Planck-Institut
fiir Zellbiologie,
DE GROOT
Abteilung Schweiger, Fed. Rep. Germany
(Received
30 September
Rosenhof,
D-6802
Ladenburg,
1991)
Abstract-l. Two clock proteins were identified in A. clifionii. The first has a molecular weight (mol. wt) of 200 kDa (P200) and its synthesis shows a 24 hr periodicity. The second has mol. wt of 130 kDa (P130) and shows a semicircadian rhythm with a periodicity of about 12 hr. 2. cDNA libraries from A. cliftonii and A. mediferranea were prepared by cloning cDNA in Igt 10 and Lgtl 1, respectively. 3. One clone each of the two libraries hybridized with the human /?-actin pseudogene. One clone of the A. mediterranea and 4 clones of the A. chBonii libraries hybridized to Chlamydomonas heat-shock gene.
INTRODUCTION Biological rhythms play an important role in living organisms. A characteristic feature of these rhythms is their continuation under constant environment conditions. One of the most important rhythms, the circadian rhythm, has a period length of about 24 hr (Edmunds, 1983; Mergenhagen, 1980; Takahashi and Menaker, 1984; Aschoff, 1965; Biinning, 1973; Schweiger, 1980; Schweiger et al., 1986). Circadian rhythms are well documented in a large number of eukaryotes. In Drosophila melanogasters a genetic locus called per- locus has a fundamental role in the expression of circadian rhythms (Konopka and Benzer, 1971). The gene has been cloned and characterized (Bargiello and Young, 1984; Reddy et al., 1984). DNA sequences homologous to the per-locus DNA have been found in the chloroplast genome of Acetabularia mediterranea but not in the nuclear DNA (Li-Weber et al., 1987). Recently, a circadian rhythm in cell division in the prokaryote cyanobacterium Synechococcus sp. was reported (Sweeny and Borgese, 1989). Different models have been proposed to explain the molecular mechanism of circadian rhythms (Hastings and Schweiger, 1976; Sweeney, 1974; Njus et al., 1974; Ehret and Trucco, 1967). Based on the results obtained with Acetabuluriu, the coupled translationmembrane model was postulated (Schweiger and Schweiger, 1977). This model could explain the results from a number of different experiments (for review see Edmunds, 1988) including the data obtained in studies with prokaryotes. According to this model, one or a few essential clock proteins are translated in *This paper is part of the Ph.D. thesis of X.-P. Yang. tAddress correspondence to: Xiao-Ping Yang, Linus Pauling Institute of Science and Medicine, 440 Page Mill Road,
Palo Alto,
CA 96306-2025,
U.S.A.
a circadian manner and integrated into membranes. This alters the properties of the membranes so that the synthesis of the essential clock protein is suspended. Thus, the synthesis of the essential clock protein is under feedback control. A protein that fulfils the requirements of the coupled translation-membrane model for an essential clock protein has been detected and isolated in Acetabuluria (Hartwig et al., 1985, 1986). Recent results obtained in experiments with Chforella show the occurrence of a protein that also fulfils the requirements for an essential clock protein (Walla et al., 1989). Here we report the existence of two proteins in Acetabulariu clifonii whose rate of synthesis oscillates under constant conditions. As a first step in searching for genes involved in the function of the circadian clock, we have constructed cDNA libraries from A. clftonii and A. mediterranea. The human p-actin related pseudogene (HfiAc-) and the Chlamydomonas heat shock gene (HSC) were used as probes to characterize both cDNA libraries.
MATERIALS
AND METHODS
Cells and culture conditions
Cells of Acefubuluriu were cultured under standard conditions in artificial sea medium (Miiller, 1962; Schweiger cl al.. 1977).
In cico protein labeling A. cliftonii cells were cultured under a 12 hr light/dark regime and labeled with [‘5S]methionine using the method of Hartwig et al. (1985), with slight modifications. Seven to ten days before ‘5S-labeling, cells (at the stage of cap formation) were exposed to a 12 hr light/12 hr dark regime at 2O’C. After this treatment, the cells were kept under constant light and temperature conditions. At defined time intervals after the last dark period, samples of 20 cells were incubated in 2.5 ml of artificial sea water medium containing I .85 MBq
1141
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XIAO-PINGYANGand EG~N J.
of [35S]methionine for 2 hr in the light. The cells were then incubated in 2 ml of artificial sea water that contained 0.1% casamino acid for 30min. Isolation of the labeled cellular contents was performed according to Hartwig ef al. (1985). After fluorography the developed films were scanned with a LKB Ultro-Scan XL-laser densitometer (Pharmacia-LKB, Freiburg, Fed. Rep. Germany). isolation of total cellular RNA and poiy(A) + RNA
Total cellular RNA was prepared by the method of Li-Weber and Schweieer (1985) with slight modifications. Poly(A) + RNA was i&la&d b; oligo(dTj-cellulose affinity chromatography (Maniatis el al., 1982). In vitro translation of Poly(A)+ RNA
The quality of the poly(A)+ RNA preparations was determined by in vitro translation in a commercially available rabbit reticulocyte lysate system (Pelham and Jackson, 1976). Construction of the cDNA libraries
The cDNA was synthesized using reverse transcriptase and E. coli ribonuclease H (Gubler and Hoffman, l983), and cloned in the phage vectors lgtl0 and lgtll (Huynh et al., 1985).
Ah Timethrs]
DE GROOT
DNA labeling and hybridization
A commercially available random primer system (Amersham) was used for labeling the probes. Molecular weight standards for electrophoresis were end-labeled using the Klenow fragment of E. coli DNA polymerase and cl[32P]dNTP (Maniatis et al., 1982). Phage plaques were transferred to nitrocellulose filters and hybridized in situ as described in Maniatis et al. (1982). DNA was transferred from agarose gel to nitrocellulose filter or Hybond-N membranes (Amersham Buchler, Braunschweig, Fed. Rep. Germany) as described by Southern (1975). The filters were prehybridized in 5 x Denhardt’s solution (0. I % polyvinylpirrolidon. 0.1% bovine serum albumin and 0.1% Ficoll 400), 4 x SET solution (0.6 M NaCl, 0.12 M Tris-HCl, pH 8.0, 4 mM EDTA), at 42°C (H/IAc and HSC genes were used as probes) or at 68°C (cDNA of Acetabularia was used as a probe). Hybridization was carried out in 20ml of hybridization buffer (1 x Denhardt, 5 x SET, 0.5% SDS) at 42 or 68”C, for 16-24 hr. In each hybridization, 2-4 x 105cpm/ml of the labeled DNA probe were used. After hvbridization the filters were washed twice (30 min each) in 5do ml of 2 x SET, 0.2% SDS at 42 or 68°C and finally in 500 ml of 0.2 x SET, 0.2% SDS for 30min at room temperature.
I AAJ
Time 0
3
0
6
3
192 15 6 9 ::
18 21
24 27 / I
1.5
Distance
1
20
1
I
2.5
of migration
(cm1
Fig. 2. Densitometric tracing of the individual lanes shown in Fig. 1. The molecular weight range from 160 to 224 kDa (distance 2.5-1.5 cm) is displayed. The vertical arrow indicates the position of P200.
r,
L.5
1
5
1
5.5
Distance of migration (cm 1 Fig. 4. Densitometric tracing of the lanes shown in Fig. 3. The molecular weight range from 110 to 150 kDa (distance 5.7-3.9 cm) is displayed. The vertical arrow indicates the position of P130.
Two clock proteins hr
3
0
6
1143
in Acetabularia
9
12
15
18
21
24
MW
94
68
45
Fig. 1. Fluorogram of proteins prepared from the chloroplast fraction (supernatant). Twenty cells were labeled with [ZsS]methionine for 2 hr at different times after the last dark period. After lysis of the chloroplasts and centrifugation at lO,OOOg, the proteins from the supernatant fraction were separated by SDS-polyacrylamide gel electrophoresis. The proteins with a molecular weight of 200,000 (P200), which is synthesized with a circadian rhythm, is marked with an arrow. The following ‘%-labeled molecular weight markers were run in the first line to the left: myosin heavy chain (200,000); phosphorylase B (94,000); bovine serum albumin (68,000); and ovalbumin (45,000).
hr
0
3
6
9
12
15
21
24
21
MW x lo-
200
-
130
94
-
68
-
45
-
Fig. 3. Fluorogram of proteins prepared from the pellet of the chloroplast fraction. The protein with a molecular weight of 130,000 (P130), which is synthesized with a rhythm of about 12 hr, is indicated. The lane to the left represents “C-labeled molecular weight markers as in Fig. 1.
XIAO-PING YANG and EC~N J. DE GREAT
1144
Base
pair
315 227
-
181-
Fig.
5.
Alkaline
1
gel
2
electrophoresis of ds-cDNA from A. cltyfonii. Lane pBR329%HaeII/TaqI digest; lane 2: 32P-labeled ds-cDNA.
3
4
5
6
7
8
9
1: DNA
10
11
markers,
12
KbD
3.1 9.4
6.6
-
4.4
-
2.3
-
1.4
-
0.9 0.6
-
0.3
-
2.0
1.1
-
-
Fig. 6. Estimate EcoRI digestion
of the size of cDNA inserts of positive clones from the A. cliftonii cDNA library after by hybridization with 32P-labeled A. clifronii ss-cDNA. The position of the lgtl0 arms are marked (arrows).
Two clock proteins
1
2
Kbp
23.1 -
9.4
-
6.7
-
4.4
-
in Acetabularia
Kb P
23.1
9.4
-
6.7
-
-
2.2 -
2.2
1.8 -
1.8
1.2 -
1.2
1.0 -
1.0
-
0.6
-
0.5
-
Fig. 7. DNA of a positive clone from the A. clifronii cDNA library was restricted with JZcoRI and hybridized with ‘ZP-labeled Hj3Ac (lane 2).
1145
Fig. 8. DNA of a positive clone from the A. mediterranea cDNA library was restricted with EcoRI and hybridized with 3ZP-labeled HSC (lane 2).
XIAO-PING YANG and EGON J. DE GROUT
1146
Kbp
9.4
-
67-
Fig, 9. DNA
4.4
-
2.2
-
1.8
-
1.2
-
1.0
-
0.6
-
0.5
-
of 4 positive
clones from the A. clzftonii cDNA library were restricted hybridized with “P-labeled HSC (lanes 2-5).
with EcoRI
and
Two clock proteins in Acetabularia
DNA sequencing The 400 bp EcoRI fragment containing the sequence homologous to HBAc - was isolated, subcloned into plasmid pUC 1%and sequenced using the dideoxynucleotidd chaintermination method (Sanger et al., 1977; Chen and Seeburg, 1985).
RESULTS IdentiJication of proteins which are synthesized with circadian or semicircadian rhythm in A. cliftonii A. cftftonii cells, in the stage of cap formation, were labeled in uivo with [35S]methionine as described in Materials and Methods. The chloroplasts were lysed by several cycles of freezing and thawing and the sample was separated by centrifugation into two parts: supernatant (mainly chloroplast stroma fraction) and pellet (mainly chloroplast membrane fraction). In all experiments, the incorporation of [35S]methionine at different times exhibited relatively constant rates both in the supernatant and in the pellet. The electrophoretic analysis revealed that the supernatant was selectively enriched in many high molecular protein bands, while the pellet was depleted of them (Figs 1 and 3). Kinetics of methionine incorporation in the different protein bands revealed the presence of a band (P200) in the supernatant whose synthesis oscillated with a circadian rhythm. Its molecular weight was about 200 kDa and its synthesis reached a maximum 3 hr after the last dark period (Figs 1 and 2). A second protein (Pl30) was identified in the pellet whose synthesis also oscillated rhythmically. The protein has a molecular weight of 130 kDa and it exhibited maximum rate of [35S]methionine incorporation 6-9 hr after the last dark period (Figs 3 and 4). Poly(A)+ RNA and cDNA ,from A. chftonii and A. mediterranea
Approximately 1IO-150 pg of total cellular RNA were obtained from 1 g wet weight of A. clifonii and A. mediterranea. This corresponds to about 225 cells with a length of 2.5-3.5 cm. The yield of poly(A)+ RNA was in the range of 1.9-2.2% of the total RNA. The double-stranded cDNAs (ds-cDNA) were synthesized from the poly(A)+ RNAs. In different experiments, the percentage of poly(A) + RNA transcribed into first strand cDNA was 13.4-26.2%. The percentage of first strand cDNA transcribed into second strand cDNA was 61-92%. Alkaline agarose gel electrophoresis of small aliquots under denaturing condition revealed that ds-cDNA size range was from 0.13 to 6.5 kb (Fig. 5). Characterization of the cDNA libraries of A. cliftonii and A. mediterranea
Titration of recombinants and plaque hybridization with ss-cDNA as a probe indicated that the percentage of recombinants was 65% in the A. cltftonii cDNA library and 56% in the A. mediterranea cDNA library. The A. cliftonii library contained a total of 3.75 x lo5 clones. The A. mediterranea library contained a total of 5 x lo5 clones. The insert size of cDNA fragments ranges from 0.1 to 2.18 kb in the A. clifronii cDNA library (Fig. 6). In the case of
1147
A. mediterranea, the size of the inserted cDNAs was up to 5.5 kb (Fig. 8). The detection of several full length SSU sequences in both cDNA libraries confirmed the quality of the two cDNA libraries. Twenty-eight of 2 x IO4plaques in the A. cliftonii cDNA library and 29 of 1.8 x lo4 plaques in the A. mediterranea cDNA library contained sequences homologous to the SSU cDNA (Schneider, 1989). Several of these DNAs were sequenced. Three clones from the A. cliftonii and 4 clones from the A. mediterranea libraries contained the complete sequence of the SSU gene together with the transit peptide (Schneider et al., 1989). Two further gene probes, the human /I-actinrelated pseudogene (Moos and Gallwitz, 1982, 1983) and the heat-shock gene from Chlamydomonas (Grimm et al., 1989) were used to screen for homologous sequences in the constructed cDNA libraries. When H/IAc- was used as a probe 1 positive clone was found in each of the two libraries. The positive clone from the A. mediterranea library was not further analyzed. The size of the cDNA fragment of the positive clone from A. cltftonii was about 400 bp (Fig. 7). The sequencing data showed that this positive clone consists of 383 bp (Fig. 10). The homology of this sequence to the HP AC - gene was 49%. One HSC positive clone with a size of 5500 bp was found in the A. mediterranea cDNA library (Fig. 8). Three HSC positive clones were found in the A. clifronii library. The sizes of these inserts were 900, 700 and 500 bp (Fig. 9).
DISCUSSION Detection of clock proteins in A. cliftonii
Previous studies on the mechanism of the circadian clock show that unicellular organisms play an important role. Among these organisms the unicellular and uninuclear marine green alga Acetabularia has been the most valuable. A protein (P230) has been detected in Acetabularia whose synthesis oscillated with a period length of about 24 hr under endogenous conditions (Hartwig et al., 1985). This result is in accordance with the coupled translation-membrane model that predicts the existence of one or a few essential clock proteins (Schweiger and Schweiger, 1977). Moreover, it has been shown that the circadian rhythm of the synthesis of this protein in Acetabularia was retained in the absence of the nucleus (Hartwig et al., 1986). The results presented here demonstrated that in Acetabularia cliftonii, a high molecular weight protein (P200), is synthesized rhythmically, in contrast to the behaviour of almost all other proteins. It is remarkable that a high molecular weight protein plays an important role in the function of the circadian clock in at least two different species of Dasycladaceae. Recently published results demonstrated the occurrence of an essential clock protein in the unicellular green alga Chlorella (Walla et al., 1989). Further evidence for the existence of a protein whose synthesis oscillates in a circadian manner comes from investigations on Chlamydomonas reinhardii. In this alga, a protein from the soluble cell fraction with a molecular weight of about 64,OOODa is synthesized
1148
XIAO-PING YANG and EG~N J. 10 ITT’lTCCTGA
AATTCCTTTT
20 60
ACAATTGGTT 90
ACAGATTTCT
30 AACGGGCTAT
50 TGAATTGAAT
DE GROOT
70 GATTTTAGTT
100 CATTAATTTT
110 ATATTTTTTC
130 140 150 TAAATCAGGA TAACACTTTA ATTTGGATAA
40 TTCCTKTTT 80 TATTTGTTGG 120 CCCATGAATA 160 CATTTTAATT
170 180 190 200 TGGACTATTT TAATTTAGAC TTTTGAAGTT GGTACAAATT 210 AAACACCGGTTAAGTGTTTA
220
230 240 AGTGTTTTAC AGTATGTTAT
250 260 270 280 AAACTTAGGA CAGAATTATT TTAATGGTTT GTTTGTTTGT 290 300 TTTGGCTGGC ATGTTGTTGG
310 320 TTAGAAAACA GATGATGATA
330 GTTAATTTTGTTTCGAAATG
350 GGGTGTAATAATTCAGATCT
340
360
370 380 TAATTCAGAG AGAAAAAAAA AAG
Fig. 10. DNA sequence of the EcoRI-fragment of A. clifroniiobtained after hybridization with (Hfi AC-) as a probe.
in a circadian rhythm under constant conditions (Weidmann and Schweiger, 1990). These results show that in different organisms a particular protein is synthesized with a circadian rhythm. This observation is in agreement with the coupled translation-membrane model, which predicts that such essential clock proteins are elements of the circadian clock. Further biochemical characterization is required to answer the question of whether the clock proteins identified in different organisms show similar biochemical features. After microsequencing of the isolated clockproteins and synthesis of the corresponding oligonucleotides, the next step would be to carry out hybridization studies in the corresponding cDNA libraries. Therefore, we constructed and characterized cDNA libraries of A. mediterranea and A. clifonii. Construction and characterization of’ cDNA libraries
The isolation of RNA from Acetabularia is complicated by the presence of large amounts of high molecular weight polyphosphates (Thilo et al., 1956; Niemeyer, 1977). Therefore, Baltimore (1966) suggested the use of LiCl for the precipitation of RNA. Components of the cell wall and of the vacuole also interfere with the isolation of total cellular RNA (Shoeman and Schweiger, 1982). This problem was solved by gently homogenizing the cells to prevent large amounts of contaminations being released from cell wall or vacuole. The number of clones that must be screened to ensure isolation of a particular sequence is given by the formula N = ln(1 -P)/ln(l
-n)
where N is the number of clones required; p is the desired probability of isolating the clone (usually set
at 99%); and n is the fractional proportion of the total mRNA population represented by a single mRNA species (the clone that is sought) (Jendrisak et al., 1987). Generally, not all mRNAs are present in the same proportion in cells. In the case of mouse hepatocytes, evidence has been presented for existence of two or three mRNA abundance classes (Ryffel and McCarthy, 1975; Young et al., 1976; Hostie and Bishop, 1976; Tse et al., 1978). A total clone bank of cDNA synthesized from mouse liver poly(A) + RNA of 4 x 10’ clones was obtained (Norgard et al., 1980). In case of a typical vertebrate cell with about l-3 x IO4different mRNA sequences, an initial library of lo6 recombinants ensures statistically that a very rare mRNA will be contained within that library (Norgard et al., 1980; Williams, 1981; Jendrisak et al., 1987). A comparison of the ratio of genome size to the size of the complete cDNA library in mouse hepatocyte, in a typical vertebrate cell and in the two species of Acetabularia, indicated that the Acetabularia libraries we have constructed of 3.75 x lo5 and 5 x lo5 clones, respectively, were large enough to contain sequences representing even the rarest mRNAs. Detection of‘fuil length cDNA genes demonstrated by the SSU sequence
To check the completeness of our libraries we screened them with several different genes. Genetic analysis indicated that the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase is located in the large single copy region of the chloroplast genome of most higher plants (Bedbrook et al., 1979). The gene for the small subunit (SSU) is present as a multigene family in the nuclear genome (Berry-Lowe et al., 1982).
Two clock proteins in Acerabularia
Several clones containing the DNA sequence corresponding to the SSU were found in the cDNA libraries of A. cliftonii and A. mediterranea. The complete sequence was located in 3 clones in A. clifionii and in 4 clones of A. mediterranea (Schneider et al., 1989). The occurrence of the full length cDNA genes demonstrated the high quality of the libraries in the sense that they contained full length cDNA copies. The fact that several different SSU clones were found in each of the two libraries indicates that a high diversity of mRNAs is represented in the libraries. The detection of clones containing sequences homologous to the H/I Ac gene and the HSC gene in both Acetabularia cDNA libraries is additional evidence of the high quality of the libraries. The construction of the two cDNA libraries of Acetabzdaria gives us the possibility to screen these libraries with oligonucleotides based on the protein sequences of essential clock proteins from Acetabuiaria. Furthermore, it is feasible to screen the libraries with oligonucleotides derived from clock proteins from different organisms. One example is the identified clock protein isolated from Chlorella (Walla, unpublished results). The sequence of another clock protein isolated from Chlamydomonas (Wiedmann, unpublished results) may also be used to construct oligonucleotides. Furthermore, these libraries are a prerequisite to search for sequences homologous to genes such as per or frq that play an essential role in the function of the circadian clock. We hope information derived from such hybridization studies will help us to further elucidate the biochemical mechanism of the circadian clock.
SUMMARY
Two proteins have been identified in the unicellular and uninucleate green alga Acetabularia clifronii, whose synthesis exhibit a pronounced endogenous rhythm. The first of these proteins has a molecular weight of 200,000 Da(P200) and is synthesized with a period of 24 hr. The second protein has a molecular weight of 130,000 Da(Pl30) and shows a semicircadian rhythm with a period length of 12 hr. To search for genes involved in the mechanism of the circadian clock, cDNA libraries from A. clzytonii and A. mediterranea were prepared. The cDNA of A. cl$tonii was cloned in the vector Igt 10, the cDNA of A. mediterranea was cloned in 1gt 11. The size of the cDNA inserts ranged up to 2.18 kb in the case of A. clifronii and up to 5.5 kb in the case of A. mediterranea. A total of 3.75 x lo5 clones was obtained from the original cDNA library of A. cliftonii. The A. mediterranea library yielded 5 x lo5 clones. Different genes were used as probes to characterize the cDNA libraries. The quality of both cDNA libraries was confirmed by the detection of full length SSU sequences. One clone of each of the two Acetabularia libraries was found to hybridize with the human /?-actin related pseudogene. The size of the insert of the positive clone identified in the A. mediterranea library was about 400 bp. The sequence homology of this clone to the human /I-actin-related pseudogene was 49%.
1149
One clone containing
sequences homologous
to
Chlamydomonas heat-shock gene was found in the library of A. mediterranea. Four clones were found in the library from A. cliftonii. The size of the insert from A. mediterranea was about 5.5 kb, and the size of the inserts from A. cliftonii were between 500 and
900 bp. Acknowledgemenrs-We wish to thank Dr Michael Leible and Dr Diedrick Menzel for invaluable advice and many fruitful discussions, and Professor Dieter Gallwitz and Professor Klaus Kloppstech for kindly supplying the HPAC- and HSC clones. The skilful technical assistance of Mrs Brigitte Seib and Mrs Ute Rath is appreciated. We also thank Mrs Erika Schindler for preparing the photographs and Dr Robert Shoeman for critical reading of the manuscript. This investigation was partly supported by the Deutsche Forschungsgemeinschaft.
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Li-Weber M. and Schweiger H.-G. (1985) Molecular cloning of cDNA from Acetabularia mediterranea: comparative studies with other Dasycladaceae. Eur. J. Cell Biol. 38, 67-72.
Li-Weber M., de Groot E. J. and Schweiger H.-G. (1987) Sequence homology to the Drosophila per locus in higher plant nuclear DNA and in Acefabularia chloroplast DNA. Molec. gen. Genet. 209, l-7. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning. A Laboratory, Manual. Cold Spring Harbor Laboratory, New York. Mergenhagen D. (1980) Circadian rhythms in unicellular organisms. Curr. Topics Microbial. Immun. 90, 1233147. Moos M. and Gallwitz D. (1982) Structure of a human /I-actin-related pseudogene which lacks intervening sequences. Nucleic Acids Res. 10, 784337849. Moos M. and Gallwitz D. (1983) Structure of two human fi-actin-related processed genes one of which is located next to a simple repetitive sequence. EMBO J. 2,757-76 I. Miiller D. (1962) Uber jahres-und lunarperiodische Erscheinungen bei einigen Braunalgen. Bor. Mar. 4, 140-155.
Niemeyer R. (1977) On the biosynthesis of condensed phosphates. In Progress in Acetabularia Research (Edited by Woodcock C. L. F.), pp. 955104. Academic Press, New York. Njus D., Sulzman F. M. and Hastings J. W. (1974) Membrane model for the circadian clock. Nature, Land. 248, 116-120.
Norgard M. V., Tocci M. J. and Monahan J. J. (1980) On the cloning of eukaryotic total poly(A)-RNA populations in Escherichia coli. J. biol. Chem. 255 766557672. Pelham H. R. B. and Jackson R. J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 61, 247-256. Reddy P., Zehring W. A., Wheeler D. A., Pirrotta V., Hadfield C., Hall J. C. and Rasbach M. (1984) Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms. Cell 38 701-710. Ryffel G. U. and McCarthy B. J. (1975) Complexity of cytoplasmic RNA in different mouse tissues measured by hybridization of polyadenylated RNA to complementary DNA. Biochemistry 14, 137991385. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 14, 5436-5467.
Schneider S. U. (1989) Das Gensystem der kleinen Untereinheit der Ribulose-1,5-bisphosphat Carboxylase/ Oxygenase von Acetabularia mediterranea, Acetabularia
Applications to Shifrs in Schedules. NATO Advanced Study Institutes Series, Ser.D: Behavioral and Social Sciences
J. molec. Biol. 98, 503-517.
Sweeney B. M. (1974) A physiological model for circadian rhythms derived from the Acetabularia rhythm paradoxes. Int. J. Chronobiol. 2, 25-33.
Sweeney B. M. and Borgese M. B. (1989) A circadian rhythm in cell division in a prokaryote, the cyanobacterium Synechococcus. WH 780. J. Phycol. 25, 183-186. Takahashi J. S. and Menaker M. (1984) Circadian rhythmicity: regulation in the time domain. In Biological Regulation- and-Development (Edited by Goldberg R. F. and Yamamoto U. K. R.). Vol. 3B. DD. . _ 285-303. Plenum Press, New York. Thilo E., Grunze H., Hiimmerling J. and Werz G. (1956) tiber Isolierung und Identifizierung der Polyphosphate aus Acetabularia mediterranea. Z. Naturf llb, 266-270. Tse T. P. H., Morris H. P. and Taylor J. M. (1978) Molecular basis of reduced albumin synthesis in morris hepatoma 7777. Biochemistry 17, 3121-3128. Walla 0. J., de Groot E. J. and Schweiger M. (1989) Identification of a polypeptide in Chlorella that apparently is involved in circadian rhythm. Eur. J. Cell Biol. SO, 181-186.
Wiedemann 1. and Schweiger M. (1990) A 64 kD protein may be part of the biological clock in Chlamydomonas reinhardk Eur. J. Biochem. 30, Suppl., 54. Williams J. G. (1981) The preparation and screening of a cDNA clone bank. In Genetic Engineering (Edited by Williamson R.), Vol. I, pp. l-55. Academic Press, New York. Young B. D., Birnie G. D. and Paul J. (1976) Complexity and specificity of polysomal poly(A)+ RNA in mouse tissues. Biochemistry 15, 2823-2829.
hr. J. Biochem. Vol. 24, No. 7, pp. 1141-1150, 1992 Printed in Great Britain
IDENTIFICATION OF TWO CLOCK PROTEINS IN ACETABULARIA CLIFTON11 AND CONSTRUCTION OF cDNA LIBRARIES FROM ACETABULARIA CLIFTONII AND ACETABULARIA MEDITERRANEA* XIAO-PINGYANG? and EC~N J. Max-Planck-Institut
fiir Zellbiologie,
DE GROOT
Abteilung Schweiger, Fed. Rep. Germany
(Received
30 September
Rosenhof,
D-6802
Ladenburg,
1991)
Abstract-l. Two clock proteins were identified in A. clifionii. The first has a molecular weight (mol. wt) of 200 kDa (P200) and its synthesis shows a 24 hr periodicity. The second has mol. wt of 130 kDa (P130) and shows a semicircadian rhythm with a periodicity of about 12 hr. 2. cDNA libraries from A. cliftonii and A. mediferranea were prepared by cloning cDNA in Igt 10 and Lgtl 1, respectively. 3. One clone each of the two libraries hybridized with the human /?-actin pseudogene. One clone of the A. mediterranea and 4 clones of the A. chBonii libraries hybridized to Chlamydomonas heat-shock gene.
INTRODUCTION Biological rhythms play an important role in living organisms. A characteristic feature of these rhythms is their continuation under constant environment conditions. One of the most important rhythms, the circadian rhythm, has a period length of about 24 hr (Edmunds, 1983; Mergenhagen, 1980; Takahashi and Menaker, 1984; Aschoff, 1965; Biinning, 1973; Schweiger, 1980; Schweiger et al., 1986). Circadian rhythms are well documented in a large number of eukaryotes. In Drosophila melanogasters a genetic locus called per- locus has a fundamental role in the expression of circadian rhythms (Konopka and Benzer, 1971). The gene has been cloned and characterized (Bargiello and Young, 1984; Reddy et al., 1984). DNA sequences homologous to the per-locus DNA have been found in the chloroplast genome of Acetabularia mediterranea but not in the nuclear DNA (Li-Weber et al., 1987). Recently, a circadian rhythm in cell division in the prokaryote cyanobacterium Synechococcus sp. was reported (Sweeny and Borgese, 1989). Different models have been proposed to explain the molecular mechanism of circadian rhythms (Hastings and Schweiger, 1976; Sweeney, 1974; Njus et al., 1974; Ehret and Trucco, 1967). Based on the results obtained with Acetabuluriu, the coupled translationmembrane model was postulated (Schweiger and Schweiger, 1977). This model could explain the results from a number of different experiments (for review see Edmunds, 1988) including the data obtained in studies with prokaryotes. According to this model, one or a few essential clock proteins are translated in *This paper is part of the Ph.D. thesis of X.-P. Yang. tAddress correspondence to: Xiao-Ping Yang, Linus Pauling Institute of Science and Medicine, 440 Page Mill Road,
Palo Alto,
CA 96306-2025,
U.S.A.
a circadian manner and integrated into membranes. This alters the properties of the membranes so that the synthesis of the essential clock protein is suspended. Thus, the synthesis of the essential clock protein is under feedback control. A protein that fulfils the requirements of the coupled translation-membrane model for an essential clock protein has been detected and isolated in Acetabuluria (Hartwig et al., 1985, 1986). Recent results obtained in experiments with Chforella show the occurrence of a protein that also fulfils the requirements for an essential clock protein (Walla et al., 1989). Here we report the existence of two proteins in Acetabulariu clifonii whose rate of synthesis oscillates under constant conditions. As a first step in searching for genes involved in the function of the circadian clock, we have constructed cDNA libraries from A. clftonii and A. mediterranea. The human p-actin related pseudogene (HfiAc-) and the Chlamydomonas heat shock gene (HSC) were used as probes to characterize both cDNA libraries.
MATERIALS
AND METHODS
Cells and culture conditions
Cells of Acefubuluriu were cultured under standard conditions in artificial sea medium (Miiller, 1962; Schweiger cl al.. 1977).
In cico protein labeling A. cliftonii cells were cultured under a 12 hr light/dark regime and labeled with [‘5S]methionine using the method of Hartwig et al. (1985), with slight modifications. Seven to ten days before ‘5S-labeling, cells (at the stage of cap formation) were exposed to a 12 hr light/12 hr dark regime at 2O’C. After this treatment, the cells were kept under constant light and temperature conditions. At defined time intervals after the last dark period, samples of 20 cells were incubated in 2.5 ml of artificial sea water medium containing I .85 MBq
1141
1142
XIAO-PINGYANGand EG~N J.
of [35S]methionine for 2 hr in the light. The cells were then incubated in 2 ml of artificial sea water that contained 0.1% casamino acid for 30min. Isolation of the labeled cellular contents was performed according to Hartwig ef al. (1985). After fluorography the developed films were scanned with a LKB Ultro-Scan XL-laser densitometer (Pharmacia-LKB, Freiburg, Fed. Rep. Germany). isolation of total cellular RNA and poiy(A) + RNA
Total cellular RNA was prepared by the method of Li-Weber and Schweieer (1985) with slight modifications. Poly(A) + RNA was i&la&d b; oligo(dTj-cellulose affinity chromatography (Maniatis el al., 1982). In vitro translation of Poly(A)+ RNA
The quality of the poly(A)+ RNA preparations was determined by in vitro translation in a commercially available rabbit reticulocyte lysate system (Pelham and Jackson, 1976). Construction of the cDNA libraries
The cDNA was synthesized using reverse transcriptase and E. coli ribonuclease H (Gubler and Hoffman, l983), and cloned in the phage vectors lgtl0 and lgtll (Huynh et al., 1985).
Ah Timethrs]
DE GROOT
DNA labeling and hybridization
A commercially available random primer system (Amersham) was used for labeling the probes. Molecular weight standards for electrophoresis were end-labeled using the Klenow fragment of E. coli DNA polymerase and cl[32P]dNTP (Maniatis et al., 1982). Phage plaques were transferred to nitrocellulose filters and hybridized in situ as described in Maniatis et al. (1982). DNA was transferred from agarose gel to nitrocellulose filter or Hybond-N membranes (Amersham Buchler, Braunschweig, Fed. Rep. Germany) as described by Southern (1975). The filters were prehybridized in 5 x Denhardt’s solution (0. I % polyvinylpirrolidon. 0.1% bovine serum albumin and 0.1% Ficoll 400), 4 x SET solution (0.6 M NaCl, 0.12 M Tris-HCl, pH 8.0, 4 mM EDTA), at 42°C (H/IAc and HSC genes were used as probes) or at 68°C (cDNA of Acetabularia was used as a probe). Hybridization was carried out in 20ml of hybridization buffer (1 x Denhardt, 5 x SET, 0.5% SDS) at 42 or 68”C, for 16-24 hr. In each hybridization, 2-4 x 105cpm/ml of the labeled DNA probe were used. After hvbridization the filters were washed twice (30 min each) in 5do ml of 2 x SET, 0.2% SDS at 42 or 68°C and finally in 500 ml of 0.2 x SET, 0.2% SDS for 30min at room temperature.
I AAJ
Time 0
3
0
6
3
192 15 6 9 ::
18 21
24 27 / I
1.5
Distance
1
20
1
I
2.5
of migration
(cm1
Fig. 2. Densitometric tracing of the individual lanes shown in Fig. 1. The molecular weight range from 160 to 224 kDa (distance 2.5-1.5 cm) is displayed. The vertical arrow indicates the position of P200.
r,
L.5
1
5
1
5.5
Distance of migration (cm 1 Fig. 4. Densitometric tracing of the lanes shown in Fig. 3. The molecular weight range from 110 to 150 kDa (distance 5.7-3.9 cm) is displayed. The vertical arrow indicates the position of P130.
Two clock proteins hr
3
0
6
1143
in Acetabularia
9
12
15
18
21
24
MW
94
68
45
Fig. 1. Fluorogram of proteins prepared from the chloroplast fraction (supernatant). Twenty cells were labeled with [ZsS]methionine for 2 hr at different times after the last dark period. After lysis of the chloroplasts and centrifugation at lO,OOOg, the proteins from the supernatant fraction were separated by SDS-polyacrylamide gel electrophoresis. The proteins with a molecular weight of 200,000 (P200), which is synthesized with a circadian rhythm, is marked with an arrow. The following ‘%-labeled molecular weight markers were run in the first line to the left: myosin heavy chain (200,000); phosphorylase B (94,000); bovine serum albumin (68,000); and ovalbumin (45,000).
hr
0
3
6
9
12
15
21
24
21
MW x lo-
200
-
130
94
-
68
-
45
-
Fig. 3. Fluorogram of proteins prepared from the pellet of the chloroplast fraction. The protein with a molecular weight of 130,000 (P130), which is synthesized with a rhythm of about 12 hr, is indicated. The lane to the left represents “C-labeled molecular weight markers as in Fig. 1.
XIAO-PING YANG and EC~N J. DE GREAT
1144
Base
pair
315 227
-
181-
Fig.
5.
Alkaline
1
gel
2
electrophoresis of ds-cDNA from A. cltyfonii. Lane pBR329%HaeII/TaqI digest; lane 2: 32P-labeled ds-cDNA.
3
4
5
6
7
8
9
1: DNA
10
11
markers,
12
KbD
3.1 9.4
6.6
-
4.4
-
2.3
-
1.4
-
0.9 0.6
-
0.3
-
2.0
1.1
-
-
Fig. 6. Estimate EcoRI digestion
of the size of cDNA inserts of positive clones from the A. cliftonii cDNA library after by hybridization with 32P-labeled A. clifronii ss-cDNA. The position of the lgtl0 arms are marked (arrows).
Two clock proteins
1
2
Kbp
23.1 -
9.4
-
6.7
-
4.4
-
in Acetabularia
Kb P
23.1
9.4
-
6.7
-
-
2.2 -
2.2
1.8 -
1.8
1.2 -
1.2
1.0 -
1.0
-
0.6
-
0.5
-
Fig. 7. DNA of a positive clone from the A. clifronii cDNA library was restricted with JZcoRI and hybridized with ‘ZP-labeled Hj3Ac (lane 2).
1145
Fig. 8. DNA of a positive clone from the A. mediterranea cDNA library was restricted with EcoRI and hybridized with 3ZP-labeled HSC (lane 2).
XIAO-PING YANG and EGON J. DE GROUT
1146
Kbp
9.4
-
67-
Fig, 9. DNA
4.4
-
2.2
-
1.8
-
1.2
-
1.0
-
0.6
-
0.5
-
of 4 positive
clones from the A. clzftonii cDNA library were restricted hybridized with “P-labeled HSC (lanes 2-5).
with EcoRI
and
Two clock proteins in Acetabularia
DNA sequencing The 400 bp EcoRI fragment containing the sequence homologous to HBAc - was isolated, subcloned into plasmid pUC 1%and sequenced using the dideoxynucleotidd chaintermination method (Sanger et al., 1977; Chen and Seeburg, 1985).
RESULTS IdentiJication of proteins which are synthesized with circadian or semicircadian rhythm in A. cliftonii A. cftftonii cells, in the stage of cap formation, were labeled in uivo with [35S]methionine as described in Materials and Methods. The chloroplasts were lysed by several cycles of freezing and thawing and the sample was separated by centrifugation into two parts: supernatant (mainly chloroplast stroma fraction) and pellet (mainly chloroplast membrane fraction). In all experiments, the incorporation of [35S]methionine at different times exhibited relatively constant rates both in the supernatant and in the pellet. The electrophoretic analysis revealed that the supernatant was selectively enriched in many high molecular protein bands, while the pellet was depleted of them (Figs 1 and 3). Kinetics of methionine incorporation in the different protein bands revealed the presence of a band (P200) in the supernatant whose synthesis oscillated with a circadian rhythm. Its molecular weight was about 200 kDa and its synthesis reached a maximum 3 hr after the last dark period (Figs 1 and 2). A second protein (Pl30) was identified in the pellet whose synthesis also oscillated rhythmically. The protein has a molecular weight of 130 kDa and it exhibited maximum rate of [35S]methionine incorporation 6-9 hr after the last dark period (Figs 3 and 4). Poly(A)+ RNA and cDNA ,from A. chftonii and A. mediterranea
Approximately 1IO-150 pg of total cellular RNA were obtained from 1 g wet weight of A. clifonii and A. mediterranea. This corresponds to about 225 cells with a length of 2.5-3.5 cm. The yield of poly(A)+ RNA was in the range of 1.9-2.2% of the total RNA. The double-stranded cDNAs (ds-cDNA) were synthesized from the poly(A)+ RNAs. In different experiments, the percentage of poly(A) + RNA transcribed into first strand cDNA was 13.4-26.2%. The percentage of first strand cDNA transcribed into second strand cDNA was 61-92%. Alkaline agarose gel electrophoresis of small aliquots under denaturing condition revealed that ds-cDNA size range was from 0.13 to 6.5 kb (Fig. 5). Characterization of the cDNA libraries of A. cliftonii and A. mediterranea
Titration of recombinants and plaque hybridization with ss-cDNA as a probe indicated that the percentage of recombinants was 65% in the A. cltftonii cDNA library and 56% in the A. mediterranea cDNA library. The A. cliftonii library contained a total of 3.75 x lo5 clones. The A. mediterranea library contained a total of 5 x lo5 clones. The insert size of cDNA fragments ranges from 0.1 to 2.18 kb in the A. clifronii cDNA library (Fig. 6). In the case of
1147
A. mediterranea, the size of the inserted cDNAs was up to 5.5 kb (Fig. 8). The detection of several full length SSU sequences in both cDNA libraries confirmed the quality of the two cDNA libraries. Twenty-eight of 2 x IO4plaques in the A. cliftonii cDNA library and 29 of 1.8 x lo4 plaques in the A. mediterranea cDNA library contained sequences homologous to the SSU cDNA (Schneider, 1989). Several of these DNAs were sequenced. Three clones from the A. cliftonii and 4 clones from the A. mediterranea libraries contained the complete sequence of the SSU gene together with the transit peptide (Schneider et al., 1989). Two further gene probes, the human /I-actinrelated pseudogene (Moos and Gallwitz, 1982, 1983) and the heat-shock gene from Chlamydomonas (Grimm et al., 1989) were used to screen for homologous sequences in the constructed cDNA libraries. When H/IAc- was used as a probe 1 positive clone was found in each of the two libraries. The positive clone from the A. mediterranea library was not further analyzed. The size of the cDNA fragment of the positive clone from A. cltftonii was about 400 bp (Fig. 7). The sequencing data showed that this positive clone consists of 383 bp (Fig. 10). The homology of this sequence to the HP AC - gene was 49%. One HSC positive clone with a size of 5500 bp was found in the A. mediterranea cDNA library (Fig. 8). Three HSC positive clones were found in the A. clifronii library. The sizes of these inserts were 900, 700 and 500 bp (Fig. 9).
DISCUSSION Detection of clock proteins in A. cliftonii
Previous studies on the mechanism of the circadian clock show that unicellular organisms play an important role. Among these organisms the unicellular and uninuclear marine green alga Acetabularia has been the most valuable. A protein (P230) has been detected in Acetabularia whose synthesis oscillated with a period length of about 24 hr under endogenous conditions (Hartwig et al., 1985). This result is in accordance with the coupled translation-membrane model that predicts the existence of one or a few essential clock proteins (Schweiger and Schweiger, 1977). Moreover, it has been shown that the circadian rhythm of the synthesis of this protein in Acetabularia was retained in the absence of the nucleus (Hartwig et al., 1986). The results presented here demonstrated that in Acetabularia cliftonii, a high molecular weight protein (P200), is synthesized rhythmically, in contrast to the behaviour of almost all other proteins. It is remarkable that a high molecular weight protein plays an important role in the function of the circadian clock in at least two different species of Dasycladaceae. Recently published results demonstrated the occurrence of an essential clock protein in the unicellular green alga Chlorella (Walla et al., 1989). Further evidence for the existence of a protein whose synthesis oscillates in a circadian manner comes from investigations on Chlamydomonas reinhardii. In this alga, a protein from the soluble cell fraction with a molecular weight of about 64,OOODa is synthesized
1148
XIAO-PING YANG and EG~N J. 10 ITT’lTCCTGA
AATTCCTTTT
20 60
ACAATTGGTT 90
ACAGATTTCT
30 AACGGGCTAT
50 TGAATTGAAT
DE GROOT
70 GATTTTAGTT
100 CATTAATTTT
110 ATATTTTTTC
130 140 150 TAAATCAGGA TAACACTTTA ATTTGGATAA
40 TTCCTKTTT 80 TATTTGTTGG 120 CCCATGAATA 160 CATTTTAATT
170 180 190 200 TGGACTATTT TAATTTAGAC TTTTGAAGTT GGTACAAATT 210 AAACACCGGTTAAGTGTTTA
220
230 240 AGTGTTTTAC AGTATGTTAT
250 260 270 280 AAACTTAGGA CAGAATTATT TTAATGGTTT GTTTGTTTGT 290 300 TTTGGCTGGC ATGTTGTTGG
310 320 TTAGAAAACA GATGATGATA
330 GTTAATTTTGTTTCGAAATG
350 GGGTGTAATAATTCAGATCT
340
360
370 380 TAATTCAGAG AGAAAAAAAA AAG
Fig. 10. DNA sequence of the EcoRI-fragment of A. clifroniiobtained after hybridization with (Hfi AC-) as a probe.
in a circadian rhythm under constant conditions (Weidmann and Schweiger, 1990). These results show that in different organisms a particular protein is synthesized with a circadian rhythm. This observation is in agreement with the coupled translation-membrane model, which predicts that such essential clock proteins are elements of the circadian clock. Further biochemical characterization is required to answer the question of whether the clock proteins identified in different organisms show similar biochemical features. After microsequencing of the isolated clockproteins and synthesis of the corresponding oligonucleotides, the next step would be to carry out hybridization studies in the corresponding cDNA libraries. Therefore, we constructed and characterized cDNA libraries of A. mediterranea and A. clifonii. Construction and characterization of’ cDNA libraries
The isolation of RNA from Acetabularia is complicated by the presence of large amounts of high molecular weight polyphosphates (Thilo et al., 1956; Niemeyer, 1977). Therefore, Baltimore (1966) suggested the use of LiCl for the precipitation of RNA. Components of the cell wall and of the vacuole also interfere with the isolation of total cellular RNA (Shoeman and Schweiger, 1982). This problem was solved by gently homogenizing the cells to prevent large amounts of contaminations being released from cell wall or vacuole. The number of clones that must be screened to ensure isolation of a particular sequence is given by the formula N = ln(1 -P)/ln(l
-n)
where N is the number of clones required; p is the desired probability of isolating the clone (usually set
at 99%); and n is the fractional proportion of the total mRNA population represented by a single mRNA species (the clone that is sought) (Jendrisak et al., 1987). Generally, not all mRNAs are present in the same proportion in cells. In the case of mouse hepatocytes, evidence has been presented for existence of two or three mRNA abundance classes (Ryffel and McCarthy, 1975; Young et al., 1976; Hostie and Bishop, 1976; Tse et al., 1978). A total clone bank of cDNA synthesized from mouse liver poly(A) + RNA of 4 x 10’ clones was obtained (Norgard et al., 1980). In case of a typical vertebrate cell with about l-3 x IO4different mRNA sequences, an initial library of lo6 recombinants ensures statistically that a very rare mRNA will be contained within that library (Norgard et al., 1980; Williams, 1981; Jendrisak et al., 1987). A comparison of the ratio of genome size to the size of the complete cDNA library in mouse hepatocyte, in a typical vertebrate cell and in the two species of Acetabularia, indicated that the Acetabularia libraries we have constructed of 3.75 x lo5 and 5 x lo5 clones, respectively, were large enough to contain sequences representing even the rarest mRNAs. Detection of‘fuil length cDNA genes demonstrated by the SSU sequence
To check the completeness of our libraries we screened them with several different genes. Genetic analysis indicated that the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase is located in the large single copy region of the chloroplast genome of most higher plants (Bedbrook et al., 1979). The gene for the small subunit (SSU) is present as a multigene family in the nuclear genome (Berry-Lowe et al., 1982).
Two clock proteins in Acerabularia
Several clones containing the DNA sequence corresponding to the SSU were found in the cDNA libraries of A. cliftonii and A. mediterranea. The complete sequence was located in 3 clones in A. clifionii and in 4 clones of A. mediterranea (Schneider et al., 1989). The occurrence of the full length cDNA genes demonstrated the high quality of the libraries in the sense that they contained full length cDNA copies. The fact that several different SSU clones were found in each of the two libraries indicates that a high diversity of mRNAs is represented in the libraries. The detection of clones containing sequences homologous to the H/I Ac gene and the HSC gene in both Acetabularia cDNA libraries is additional evidence of the high quality of the libraries. The construction of the two cDNA libraries of Acetabzdaria gives us the possibility to screen these libraries with oligonucleotides based on the protein sequences of essential clock proteins from Acetabuiaria. Furthermore, it is feasible to screen the libraries with oligonucleotides derived from clock proteins from different organisms. One example is the identified clock protein isolated from Chlorella (Walla, unpublished results). The sequence of another clock protein isolated from Chlamydomonas (Wiedmann, unpublished results) may also be used to construct oligonucleotides. Furthermore, these libraries are a prerequisite to search for sequences homologous to genes such as per or frq that play an essential role in the function of the circadian clock. We hope information derived from such hybridization studies will help us to further elucidate the biochemical mechanism of the circadian clock.
SUMMARY
Two proteins have been identified in the unicellular and uninucleate green alga Acetabularia clifronii, whose synthesis exhibit a pronounced endogenous rhythm. The first of these proteins has a molecular weight of 200,000 Da(P200) and is synthesized with a period of 24 hr. The second protein has a molecular weight of 130,000 Da(Pl30) and shows a semicircadian rhythm with a period length of 12 hr. To search for genes involved in the mechanism of the circadian clock, cDNA libraries from A. clzytonii and A. mediterranea were prepared. The cDNA of A. cl$tonii was cloned in the vector Igt 10, the cDNA of A. mediterranea was cloned in 1gt 11. The size of the cDNA inserts ranged up to 2.18 kb in the case of A. clifronii and up to 5.5 kb in the case of A. mediterranea. A total of 3.75 x lo5 clones was obtained from the original cDNA library of A. cliftonii. The A. mediterranea library yielded 5 x lo5 clones. Different genes were used as probes to characterize the cDNA libraries. The quality of both cDNA libraries was confirmed by the detection of full length SSU sequences. One clone of each of the two Acetabularia libraries was found to hybridize with the human /?-actin related pseudogene. The size of the insert of the positive clone identified in the A. mediterranea library was about 400 bp. The sequence homology of this clone to the human /I-actin-related pseudogene was 49%.
1149
One clone containing
sequences homologous
to
Chlamydomonas heat-shock gene was found in the library of A. mediterranea. Four clones were found in the library from A. cliftonii. The size of the insert from A. mediterranea was about 5.5 kb, and the size of the inserts from A. cliftonii were between 500 and
900 bp. Acknowledgemenrs-We wish to thank Dr Michael Leible and Dr Diedrick Menzel for invaluable advice and many fruitful discussions, and Professor Dieter Gallwitz and Professor Klaus Kloppstech for kindly supplying the HPAC- and HSC clones. The skilful technical assistance of Mrs Brigitte Seib and Mrs Ute Rath is appreciated. We also thank Mrs Erika Schindler for preparing the photographs and Dr Robert Shoeman for critical reading of the manuscript. This investigation was partly supported by the Deutsche Forschungsgemeinschaft.
REFERENCES
Aschoff J. (1965) Circadian Clocks. North-Holland, Amsterdam. Baltimore D. (1966) Purification and properties of poliovirus double-stranded ribonucleic acid. J. molec. Biol. 18, 421-428 Bargiello T. A. and Young M. W. (1984) Molecular genetics of a biological clock in Drosophila. Proc. natn. Acad. Sci. U.S.A. 81, 2142-2146. Bedbrook J. R., Coen D. M., Beaton A. R., Bogorad L. and Rich A. (1979) Location of the sinale aene for the large subunit ‘of ribulose-bisphosphate carboxylase on the maize chloroplast chromosome. J. biol. Chem. 254, 909-910. Berry-Lowe S. L., McKnight T. D., Shah D. M. and Meagher R. B. (1982) The nucleotide sequence, expression, and evolution of one member of a multigene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean. J. molec. appl. Gener. 1.483498. Biinning E. (1973) The Physiological Clock, Circadian Rhythms and Biological Chronometry. Springer, Berlin. Chen E. and Seeburg P. H. (1985) Supercoil sequencing: A fast and simple method for sequencing plasmid DNA. DNA 4, 165-170.
Edmunds L. N. Jr (1983) Chronobiology of the cellular and molecular levels: models and mechanisms of circadian time keeping. Am. J. Anat. 168, 389-431. Edmunds L. N. Jr (1988) Cellular and Molecular Bases of Biological Clocks. Springer, New York. Ehret C. F. and Trucco E. (1967) Molecular models for the circadian clock. I. The chronon concept. J. Theor. Biol. 15, 240-262.
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