Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in chlamydomonas reinhardi

Cell, Vol. 32. 99-l

07, January

1983,

Copyright

0 1983

by MIT

Regulation of Light-Harvesting Chlorophyll-Binding Protein (LHCP) mRNA Accumulation during the Cell Cycle in Chlamydomonas reinhardi Hurley S. Shepherd,* Gerard Ledoigt+ Stephen H. Howell* Biology Department, C-01 6 University of California, San Diego La Jolla, California 92093

and

Summary Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyllprotein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the lightdark cycle, is required for accumulation of this RNA. Introduction Chlamydomonas reinhardi and other unicellular algae can be entrained to divide synchronously by exposing cultures to alternating light and dark illumination cycles. C. reinhardi cultures subjected to 12 hr light-l 2 hr dark illumination cycles will divide synchronously 14-l 6 hr after the beginning of the light phase (2-4 hr into the dark phase). In addition to cell division, many other events occur synchronously, including nuclear DNA synthesis (Chiang and Sueoka, 1967). development of the capacity to form gametes (Schmeisser et al., 19731, the synthesis of various stage-specific proteins (Howell et al., 1977). the accumulation of components of the chloroplast (Armstrong et al., 1971, Beck and Levine, 1974; lwanij et al., 1975) and the accumulation of stage-specific RNAs (Ares and Howell, 1982). Chlorophyll accumulation is one of the most obvious stage-specific events that takes place in synchronized cells. Cultures “green” during the mid-light phase as cells accumulate chlorophyll (Schor et al., 1970). Chlorophyll is located in the internal or “thylakoid” membranes of the chloroplast and is noncovalently * Present

address:

Botany

Department,

University

rence, Kansas 66044. + Present address: Laboratoire de Cytophysiologie. Gif-sur-Yvette. France. * To whom correspondence should be addressed.

of Kansas, CNRS.

Law91190

bound in complexes called chlorophyll-protein complexes (see review by Thornber, 1975). The bulk of the chlorophyll is called light-harvesting chlorophyll (LHC) and functions to absorb light and channel that light energy into the photochemical components of the photosynthetic apparatus. Much of the LHC (both chlorophyll a and b) is organized into chlorophyllprotein complex II (CPII), and the apoproteins of this complex (or complexes) are called light-harvesting chlorophyll a/b proteins (LHCPs). In C. reinhardi and in higher plants, chlorophyll and the LHCPs are synthesized in different cellular compartments (Hoober, 1970; Chua and Gillham, 1977). Chlorophyll is synthesized in the chloroplast, and the LHCPs are synthesized in the cytoplasm and imported into the chloroplast (Schmidt et al., 1981). The regulation of chlorophyll and LHCP synthesis in C. reinhardi has been most intensively studied during greening in the mutant y-l, which requires light for chlorophyll synthesis. In the studies of the y-7 mutant, it has been found that light at wavelengths that photoconvert protochlorophyll to chlorophyll stimulates chlorophyll and LHCP accumulation (Hoober and Stegeman, 1973). It is not yet understood at what level-transcriptional, translational, posttranslational or other-light influences the accumulation of LHCP in C. reinhardi. On one hand, it has been shown that actinomycin D blocks the light-induced accumulation of LHCPs, suggesting that light may stimulate transcription of genes encoding LHCPs (Eytan and Ohad, 1972). On the other hand, Hoober and Stegeman (1976) showed that the accumulation of RNA encoding LHCPs was stimulated by heat, as well as light, because when heat-treated cells were placed in light, they synthesized LHCPs even in the presence of actinomycin D. Nonetheless, heat-treated cells did not synthesize LHCPs in the dark, indicating that some step other than the accumulation of LHCP mRNA must limit LHCP synthesis in the dark. In higher plants, the light regulation of LHCP synthesis is just as striking as in C. reinhardi and also appears to be controlled at two or more levels. In barley (Apel, 1979) and in the duckweek Lemna (Tobin, 19811, conversion of the pigment phytochrome by red light is correlated with the accumulation of mRNA encoding LHCPs. However, even in the presence of accumulated LHCP mRNA, LHCPs do not accumulate in the chloroplasts of higher plants in the absence of continuous white light (Ape1 and Kloppstech, 1980; Cuming and Bennett, 1981). It has been argued that the absence of LHCP accumulation in light-limiting conditions may be due.not to a failure to synthesize LHCPs, but to the inability of chloroplasts to maintain stably LHCPs in the absence of chlorophyll synthesis (Cuming and Bennett, 1981). The close tie between chlorophyll synthesis and LHCP accumulation is also observed in mutants defective in chlorophyll synthesis. For example, barley mutants defective

Cell 100

in chlorophyll synthesis accumulate neither CPll nor its apoproteins, the LHCPs (Thornber and Highkin, 1974; Machold et al., 1979). Hence, in higher plants, there seem to be at least two light-controlled steps in LHCP accumulation -one regulated by phytochrome conversion and the other by chlorophyll synthesis or accumulation. We have studied the regulation of LHCP synthesis during the cell cycle in C. reinhardi. We have examined whether the stage-specific accumulation of LHCP and chlorophyll observed during the cell cycle is correlated with a similar accumulation pattern of LHCP mRNA. A point at issue is whether the pattern of LHCP synthesis is truly a cell cycle event or is actually an event directly controlled by the light-dark illumination cycle used to synchronize cells. We have found that LHCP RNA is accumulated in a wave-like pattern during the cell cycle and that light is required for the accumulation.

A

B

Results The Proteins of CPU Delepelaire and Chua (1981) extensively examined the properties of LHCPs of the CPII complex of C. reinhardi. They observed that when thylakoid (inner chloroplast) membranes were solubilized and subjected to electrophoresis in the cold, the CPII complex migrated as a broad chlorophyll-containing band of about 29 kd (under specified gel conditions). On high resolution gels the complex separated into five closely spaced, pigmented bands designated CPII, through CPII,. When the CPII complex was isolated from gels and dissociated by heating, they found that it was composed of at least six polypeptides that could be separated on one-dimensional SDS-polyacrylamide gels. Unfortunately, insolubility of the polypeptides composing the CPII complex has prevented further analysis by two-dimensional gels because the prcteins do not readily focus in the first dimension of these gels. In view of the difficulty of resolving the proteins and ascribing them to specific subcomplexes, we have adopted the nomenclature of Chua (described in Chua and Gillham, 1977) and have numbered in descending order the bands obtained upon one-dimensional gel separation of the proteins from whole thylakoid membrane preparations. The bands which Delepelaire and Chua (1981) have shown to constitute the set of CPII polypeptides between 33 kd and 25 kd and are designated 10, 11, 14, 15, 16 and 17 (Figure 1, lane A). We have obtained an antiserum (provided by E. M. Tobin, UCLA) against purified CPII complex from Lemna gibba which cross-reacts with C. reinhardi chloroplast membrane proteins. The specificity of the cross-reaction was visualized by a filter blot procedure (Towbin et al., 1979) whereby chloroplast membrane

Figure 1. Separation by SDS-Polyacrylamide

of C. reinhardi Chloroplast Gel Electrophoresis

Membrane

Polyacrylamide gel is 11.2%. Coomassie-blue-stained A) are numbered according to Chua and Gillham components of the CPII complex as designated by Chua (1981), are proteins: 10 = 33 kd; 11 = 30 kd; = 27 kd: 16 = 25.5 kd; 17 = 25 kd. Proteins antiserum against CPII proteins of Lemna (lane 6) filter blotting (Towbin et al., 1979). Membrane proteins to nitrocellulose paper and reacted with antiserum, body is visualized with ‘251-labeled protein A.

Proteins

proteins (lane (1977). LHCPs. Delepelaire and 14 = 28 kd: 15 cross-react with as visualized by are transferred and bound anti-

proteins from C. reinhardi cells were transferred to nitrocellulose filters and incubated with antiserum, and the bound antibody was detected by ‘251-labeled A protein. As shown in Figure 1, lane B, the antiserum reacts most strongly with two members (proteins 16 and 17) of the rapidly migrating set of CPII proteins and weakly with protein 1 1. It is interesting that proteins 11, 16 and 17 which most strongly react with the antiserum to Lemna proteins, are also immunologically related to each other. Chua and Blomberg (1979) showed that a specific antiserum against protein 11 or C. reinhardi cross-reacts with proteins 16 and 17. To determine the relationship between members of the set of CPII proteins which react with the antiserum,

,

Regulation 101

of LHCP mRNA

Accumulation

we excised the bands from the gels, labeled the proteins in vitro with “%iodine, digested the labeled protein with trypsin and subjected the digest to twodimensional fingerprint analysis (Figure 2). The analysis clearly shows that fast migrating LHCP pair, polypeptides 16-17 (Figures 2A and 26) are closely related; in fact, their fingerprint patterns are nearly indistinguishable. Polypeptides 1 l-l 2 (Figure 2C) are a doublet band, and the members of this doublet are not easily separated. Polypeptide 12 is not considered to be a component of CPII (Delepelaire and Chua, 1981). The pattern of peptides from the doublet is distinguishable from those of polypeptides 16-l 7, although one peptide, which migrates most rapidly in the electrophoretic direction, may be shared among all groups analyzed. Taken together these results indicate that the major LHCPs which react with the antiserum (polypeptides 11, 16 and 17) may be a closely related family of proteins. However, we do not know whether members of this family are different gene products or the same gene product, processed in different ways, to give a variety of electrophoretic forms. Furthermore, we do not know whether each electrophoretic band is a distinct protein species or a collection of closely related proteins. In higher plants, LHCPs are synthesized as larger precursor proteins and processed upon importation into the chloroplast (for example see Schmidt et al., 1981). When poly(A) RNA from C. reinhardi is translated from cell cycle stages(L6) in which active LHCP synthesis occurs, the typical constellation of LHCPs is not seen among translation products (Figure 3, lane A). However, when the translation products are immunoprecipitated with the Lemna antiserum raised against CPII and are examined on a one-dimensional SDS gel, two prominent bands appear-one of 32 kd and another of 30 kd (Figure 3, lane 6). These two bands are prominent translation products from L6 RNA, but are not among the translation products of, for example, D6 RNA (data not shown). In an attempt to establish identity between the in vitro translation products and the in vivo membrane proteins and to assign precursor-product relationships between them, partial proteolytic digest patterns of the relevant polypeptides were compared. However, in this case, making unequivocal assignments between in vitro and in vivo products is difficult for at least three reasons. First, the various LHCPs are small and closely related to each other, as shown in Figure 2. Second, it is difficult to label proteins in vitro and in vivo with the same radiolabeled compound. In these experiments, translation products synthesized in vitro were labeled with %-methionine while membrane proteins were labeled in vivo with 35S-H,S04. Unfortunately, C. reinhardi cells do not efficiently incorporate methionine; however, most 35S-H2S04 incorporated in LHCPs is found in methionine residues, since

B

C

.

Figure roplast Figure

2. Tryptic Membrane 1

Fingerprint Map Comparison of Three major ChloProteins Reactive to the Antisera as Indicated in

Proteins in bands separated by SDS-polyacrylamide gel electrophoresis were radioiodinated in vitro, digested with trypsin and separated on thin-layer chromatography plates by electrophoresis (vertical direction) and chromatography (right to left) as described by Elder et al. (1977). (A) band 17; (B) band 16: (C) doublet band 1 l-1 2.

Cell 102

A

B

A

B

C

D

Figure 4. Partial Proteolysis Digestion Patterns of %-MethionineLabeled Products Synthesized in a Rabbit Reticulocyte Translation System and lmmunoprecipitated by CPII Antiserum and Chloroplast Membrane Proteins Labeled In Vivo in the Presence of %-H&O., Labeled proteins and translation products were separated by onedimensional SDS-polyacrylamide gel electrophoresis and were excised from gel and digested with increasing amounts of Staphylococcus aureus V-8 protease (0.1 pg. 0.5 pg. 3.0 pg left to right) according to the method of Cleveland et al. (1977). (A) 32 kd translation product; (8) chloroplast membrane protein 17; (C) 30 kd translation product; (D) chloroplast membrane protein 11

Figure 3. Identification Gel Electrophoreis

of LHCP Precursors

by SDS-Polyacrylamide

PolyfA) RNA (1.5 pg) from mid-light-phase cells (L6) was translated in a wheat germ protein synthesizing system (lane A). Immunoprecipitation of 32 kd and 30 kd translation products (indicated by arrows) with antisera to Lemna CPII proteins (lane B).

methionine is the major sulfur-containing amino acid in C. reinhardi LHCPs. Methionine is found in quantities of approximately 4 mole residues per mole of protein, while cysteine/2 is found only in quantities of -1 mole residue per mole of protein (Hoober and Stegeman, 1976). Third, the presence of a possible leader sequence on a precursor will generate additional and altered peptides from the leader and the leader-junction peptides, respectively. Despite these potential shortcomings, the partial proteolysis pattern generated by Staphylococcus aureus V-8 protease digestion (Figure 4) suggests that the 32 kd translation product (group A digests) and membrane protein 17 (group I3 digests) share characteristic peptides (indicated with arrows). Likewise, the 30 kd translation product (group C digests) shares characteristic peptides with proteins from the 11-l 2

doublet (group D digests). These results suggest the 32 kd product(s) synthesized in vitro are a precursor of protein 17 (25 kd) and/or 16 (25.5 kd), and that the 30 kd translation product is a precursor of protein 11 (30 kd). That relationship is also reflected in the comparative reaction of these proteins to the CPU antiserum. Membrane proteins 16 and 17 react most intensely to the antiserum in the filter binding analysis (Figure 1, lane B), and the 32 kd protein synthesized in vitro is more efficiently immunoprecipitated by the antiserum. The difference between the apparent molecular weight of the 32 kd precursor and its suggested products (A = 7 kd) may not be as great as it appears. The migration of these insoluble membrane proteins varies under different gel conditions. Copy DNA Clones for the 32 kd Precursor To examine the regulation during the cell cycle of mRNAs encoding the LHCPs, we attempted to clone double-stranded cDNAs representing mRNA encoding the LHCP precursors. A cDNA clone for a LHCP precursor in peas has been reported by Broglie et al. (1981). but it does not strongly hybridize to C. reinhardi DNA (data not shown). In C. reinhardi we attempted to clone cDNA for LHCP mRNA by taking advantage of the anticipated stage-specific appearance of LHCP mRNA during the cell cycle. Since in vivo synthesis of CPII apoproteins is restricted to a stage during the middle of the light phase of the cell cycle, we prepared a cDNA library from polyadenylated RNA extracted from cells at that time. The library was screened by colony-filter hybridization (Grunstein and Wallis, 1979) to detect clones containing sequences which preferentially hybridize to 32P-labeled cDNA synthesized from L6 mRNA, as opposed to D2 mRNA.

Regulation 103

of LHCP mRNA Accumulation

Copy DNA clones showing a differential response, amounting to 48 of 768 clones, were further tested by dot-blot filter hybridization, as shown in Figure 5. In these hybridizations, plasmid DNA from selected clones was immobilized on filters and hybridized to 32P-labeled cDNA prepared from total cell polyadenylated RNA extracted at various cell cycle stages. Most all the clones represented cell-cycle-regulated RNAs which peak in accumulation at mid- to latelight phase (6-10 hr after the beginning of the light phase). To determine whether any of these clones did, indeed, contain sequences homologous to LHCP mRNA, they were analyzed by hybrid-release translation in which selected plasmids were immobilized on filters and hybridized to L6 polyadenylated RNA, then hybridized RNA was released and translated in vitro in a rabbit reticulocyte translation system. The observed translation product from the RNA released from pHS4, pHS7 and pHS16 is a protein of 32 kd (Figure 6, lanes A-C) that can be immunoprecipitated with antiserum again LHCP (Figure 6, lanes E-G). Therefore, these plasmids appear to contain sequences complementary to the mRNA encoding a 32 kd precursor to LHCP. Plasmid pHS16, which contains a 300 bp insert, was chosen for further study.

L2 L6 LlO D2 D6 D10

pHS1 pHS2 pHS4 pHS7 pHSl0 pHS14 pHSl5 pHSl6 pHS20 pHS21 pHS44 pBR322 Figure 5. Dot-Blot Hybridization of Selected labeled cDNA Prepared from RNA Extracted Cell Cycle Stages

Cell Cycle Accumulation of LHCP mRNA To examine the pattern of accumulation of the mRNA coding for LHCP during the cell cycle, we used 32Plabeled pHS16 as a hybridization probe to detect LHCP mRNA on a Northern filter blot. RNA from a constant number of mother cells was isolated from cells at different cell cycle stages and subjected to electrophoresis on methylmercurichydroxide-containing agarose gels. The RNA in gels was transferred to nitrocellulose paper and hybridized to pHS16 DNA. Plasmid pHS16 strongly hybridized to a single 1.2 kb RNA band in the L6 and LlO RNA preparation. As shown in Figure 7 (lanes L2-DlO), that RNA accumulates in a wave-like pattern in the cell cycle. The RNA abruptly appears in L6 RNA preparations, begins to decay in Ll 0 and cannot even be detected in later stage preparations. Since C. reinhardi cells are synchronized by exposing cultures to an alternating light-dark cycle, it was important to determine whether the stage-specific accumulation of chlorophyll and LHCP mRNA is a “cell cycle” or a light-controlled phenomenon. We found that accumulation of LHCP mRNA (RNA hybridizing to pHS16) requires light. Cells previously entrained by

ABCDE

Figure 6. Translation Bound Plasmid DNA cDNA Clones to 32Pfrom Cells at Various

Cell cycle stages indicated in hours during hght phase (D). Filters were loaded with recombinant indicated (2 pg/dot) and hybridized with 2 X prepared from 1 pg of poly(A) RNA from each cell

phase (L) or dark plasmid DNAs as 10” cpm of cDNA cycle stage.

FGH

of RNA

Hybridized

and Released

Eight micrograms of poly(A) RNA from L6 cells was pg of filter-bound plasmid DNA. RNA released from pHS7 (lane B), pHS16 (lane C) and from pBR322 translated in a rabbit reticulocyte protein synthesizing lation products from pHS4 (lane E), pHS7 (lane F). and pBR322 (lane H) were immunoprecipitated with rum. Arrows indicate position of 32 kd band.

from

Filter-

hybridized to 4 pHS4 (lane A). (lane D) was system. TranspHSl6 (lane G) anti-CPII antise-

Cdl 104

.

4

.

Figure 7. Northern Filter Blot of ‘*P-Labeled pHS16 Hybridized RNA Extracted from Cells at Different Cell Cycle Stages during 12 hr Light-l 2 hr Dark Illumination Cycle

to the

Total RNA from 2 X 1 O6 mother cells was separated on methylmercurichydroxide-containing agarose gels, transferred to nitrocellulose filters and hybridized to labeled probe (4 X 1 O6 CPM). (Lanes L2’Ll 0’) RNA from 2 X 10’ mother cells kept in the dark during the normal light phase: (lanes D2’-DlO’) RNA from cells kept in the light during the normal dark phase. Arrows indicate prominent RNA species of 1.2 kb. Dots indicate RNA sizes determined relative to the migration of the 1631 base and 516 base Hinf I fragments of pBR322.

light-dark cycles and then kept in the dark during the normal light phase did not accumulate LHCP-specific mRNA (Figure 7, lanes L2*-LIO’). We also found that the decay of LHCP mRNA at the end of the light phase was not keyed to the onset of the dark period. Cells kept in the light following the end of the normal light phase lose their accumulated RNA and accumulate little, if any, more LHCP mRNA during a period corresponding to the normal dark phase (Figure 7, lanes D2*-DiO*). Thus it appears that light during the light phase is required for LHCP mRNA accumulation, but some other internal feedback mechanism, independent of the ongoing illumination condition, signals LHCP mRNA decay. Discussion These studies indicate that LHCP mRNA accumulates in a wave-like pattern during the cell cycle similar to the pattern of chlorophyll and LHCP synthesis, suggesting that LHCP synthesis during the cell cycle is governed largely by the availability of LHCP mRNA. Light is an important regulator of LHCP mRNA accumulation in C. reinhardi and is required for LHCP mRNA accumulation. In higher plants, continuous white light is required for accumulation of chlorophyll or LHCP; however, continuous light is not required for the accumulation of LHCP mRNA. Instead, pulses of

appropriate wavelengths of light to photoconvert phytochrome to active form stimulate LHCP mRNA accumulation (Ape1 and Kloppstech, 1980; Cuming and Bennett, 1981; Tobin, 1981). On the basis of what is found in higher plants, one might have predicted that LHCP mRNA in synchronous C. reinhardi would accumulate during the “darkened” light phase and not be translated; however, this was not so. It was also unexpected to find that the decay of LHCP mRNA, at the end of the normal light phase, did not result from a transition from light to dark. LHCP mRNA disappeared on schedule even in continuous light. This suggests that some internal feedback mechanism reverses the accumulation of the LHCP mRNA. In contrast to the dependence of LHCP mRNA accumulation on light, we have examined the effects of light on other stage-specific messages. We have found, for example, that the pattern of tubulin mRNA accumulation which occurs as a wave during the early part of the dark phase is unaffected by ongoing illumination conditions (Ares and Howell, 1982). The same is true for several other dark-phase messages. The lightdark regulation of chloroplast components appears to extend to the chloroplast genome of C. reinhardi, too. Matsuda and Surzycki (1980) have shown that at least one specific region of the chloroplast genome is transcribed more heavily at the mid-light-phase stage than any other time in the cell cycle. These experiments demonstrate the importance of the cell cycle and illumination conditions to the synthesis of various chloroplast components. It is interesting to change viewpoints and ask if the synthesis of these chloroplast components regulates, in turn, the cell cycle. Howell et al. (1975) found that various drugs and inhibitors which block chloroplast functions (photosynthesis) or the accumulation of chloroplast components (such as chloroplast protein synthesis) prevent C. reinhardi cells from dividing. Cells escape the effects of these agents, usually after the mid-light phase of the light-dark cycle. Cells show similar behavior in response to dark. Hence it appears that in order to divide, cells may rely on the acquisition of some chloroplast function or product during the light phase of the cell cycle. Spudich and Sager (1980) found that if previously synchronized cells were maintained in the dark, they would arrest at a stage comparable to the time of early light phase. It is possible that cells must acquire chloroplast components such as LHCP in order to progress further through the cell cycle. Experimental

Procedures

Growth of Cells A cell-wall-defective strain of C. reinhardi CW15 mating type + (provided by D. R. Davies) was grown photoautotrophically under asynchronous conditions (constant illumination) or synchronous conditions (12 hr light-l 2 hr dark alternating cycles). Synchronous cells were subject to at least three light-dark cycles before they were harvested for various experiments.

Regulation 105

of LHCP mRNA Accumulahon

RNA Extraction and In Vitro Translation RNA was extracted from CWl5’ cells according to a modification of a procedure by Dobberstein et al. (1977). In this procedure, l-l .5 liters of synchronous cells were harvested by centrifugation and resuspended in 10 ml of 100 mM Tris-HCI (pH 8.5). 400 mM LiCI, 10 mM EGTA, 5 mM EDTA. to which is added 200 ~1 of 4 mg/ml proteinase K (Sigma) and 1 ml of 20% SDS. The mixture was shaken at 4°C for 10 min and extracted with an equal volume of watersaturated phenol and CHC13 (1 :I ). The aqueous phase was extracted with phenol and CHC13 until the interface cleared, and nucleic acids were precipitated with 2 volumes of ethanol and collected by centrifugation. The precipitate was washed at room temperature with 2 M LiCl and dissolved in 10 mM Tris-HCI (pH 7.4). 1 mM EDTA. 0.1% sodium dodecyl sarcosinate. RNA was precipitated by adding sodium acetate (pH 5.0) to 0.1 M and 2 volumes of ethanol. RNA for hybrid-release translation was further purified through cesrum chloride according to the method of Glisin et al. (1974). RNA (-2 mg per pellet) was suspended in 2 ml of 100 mM Tris-HCI (pH 8.0), 2% sodium dodecyl sarcosinate. to which was added 1 g/ml solid cesium chloride. RNA solution was layered over a 1.2 ml cushion of 6 M cesium chloride in 100 mM EDTA (pH 7.5) and overlaid with 0.25 ml of 100 mM Tris-HCI (pH 8.0). 2% sodium dodecyl sarcosinate. Tubes containing the cesium chloride gradients were centrifuged in a SW50.1 rotor at 35,000 rpm for 18 hr at 20°C. The RNA-containing pellet was dissolved in 10 mM Tris-HCI (pH 8.0) containing 0.2% sodium dodecyl sarcosinate and phenol and CHC13. extracted and precipitated with ethanol. PolyfA) RNA was obtained by passing RNA through oligo(dT)cellulose as described by Weeks and Collis (1976). PolyfA) RNA was translated in a rabbit reticulocyte (Amersham) or wheat germ translation system (Weeks and Collis. 1976). Selected samples (10 ~1) were immunopreciprtated with 10 ~1 antiserum in 125 ~1 of TNT (50 mM Tris-HCI [pH 7.61. 100 mM NaCI. 0.1% Triton X-100, at 4°C overnight. Followmg incubation with antiserum, 60 pl of a suspension of heat-treated Staphylococcus aureus cells (Pansorbin, CalbiochemBehring) were added, and incubation was continued at room temperature for 0.5 hr. The cells with adsorbed antigen-antibody complexes were collected by centrifugation and washed five times with 0.5 ml aliquots of TNT. The washed cell pellet was suspended in 20 gl of TNT containing 2 M urea and 2% SDS and heated in a boiling-water bath. The cells were again pelleted, and the supernatant fluid containing the released translation product was prepared for one-dimensional polyacrylamide gel electrophoresis according to Laemmli (1970). Synthesis of cDNA and Construction of Recombinant cDNA Clones Copy DNA and double-stranded cDNA were prepared from poly(A) RNA according to the method of Wickens et al. (1978), with AMV reverse transcriptases from J. W. Beard and E. coli DNA polymerase I from Boehringer. In the preparation of “P-labeled cDNA for hybridization experiments, 50 PCi3’P-dCTP (-600 Ci/mmole from Amersham) was used as a radiolabel. Copy DNA and double-stranded cDNA were phenol-extracted, isolated by gel filtration centrifugation through Bio-Gel P60 (Bio-Rad) and concentrated by ethanol precipitation. Double-stranded cDNA for cloning was digested with Sl nuclease according to the method of Ullrich et al. (1977). phenolextracted and concentrated as described above. Double-stranded cDNA was tailed with about 10 to 12 deoxycytosine residues by use of terminal deoxynucleotidyl transferase (Miles) and was inserted into the Pst I site of pBR322 similarly tailed with deoxyguanosine residues according to the method of Otsuka (1981). Recombinant plasmids were used to transform CaCI,-treated E. coli C600-SF8 or HE101 as described by Bolivar and Backman (1979). Tetracycline-resistant, ampicillin-sensitive clones were selected for further testing. To detect transformants containing plasmids with inserted cDNA that hybridized to RNA accumulated in the light phase of synchronous C. reinhardi cells, 32P-labeled cDNA was prepared from poly(A) RNA from mid-light-phase (L6) and early dark-phase (D2) cells. The labeled cDNAs were used as probes in a colony-filter hybridization procedure similar to that of Grunstein and Wallis (1979). Colonies hybridizing

strongly to L6 cDNA and not to D2 cDNA were selected. Plasmid DNA was prepared from selected transformants and isolated on ethidium bromide-cesium chloride gradients in a procedure described by Kahn et al. (1979). Plasmid DNA was labeled by nick translation with 32P-dCTP (Amersham, -600 Ci/mmole) by the procedure of Rigby et al. (1977). Labeled DNA was separated from nucleotides by centrifugal gel filtration through Bio-Gel P60 (Bio-Rad), extracted with phenol and precipitated with ethanol. Northern Filter Hybridization For Northern filter hybridization. poly(A) RNA (2 pg per lane) was subjected to electrophoresis on 1.5% agarose gels containing 10 mM methylmercurihydroxide (Alpha Products) according to Bailey and Davidson (1976). RNA was transferred to nitrocellulose paper (BA83, Schleicher and Schuell) and hybridized in dextran-sulfate-containing solutions to “P-labeled nick-translated probe DNA (usually 2 x 1 O6 cpm) according to the method of Wahl et al. (1979). Hybrid-Release Translation Hybrid-release translation was performed with a modification of a procedure described by Durica et al. (1980). Two dots from the dot filters described in the previous section were cut into pie-shaped pieces and incubated in an Eppendorf tube for 1 hr at 65°C in 100 ~1 of hybridization mixture containing 50% deionized formamide, 0.6 M NaCI, 0.1 M PIPES-NaOH (pH 6.4) plus 100 pg/ml polyadenylic acid (Calbiochem-Behring Corp.). PolytA) RNA (8 $g/tube) was incubated with filter-bound DNA in 50 pl of hybridization mixture. The temperature of the incubation mixture was regulated by a temperature programmer set to drop the temperature linearly over a range of 65°C to 45°C during a 3 hr period. Following incubation, filter pieces were washed at 50°C first with 100 mM PIPES-NaOH (pH 6.4). 200 mM NaCI. 0.5% SDS, then with the same buffer without SDS. The filter pieces were subsequently washed with 10 mM Tris-HCI (pH 7.5). 1 mM EDTA. and DNA was eluted by boiling the filters in 100 gl of Hz0 for 2 min. Eluted RNA was precipitated by adding 2 rrg calf-liver tRNA (Boehringer), potassium acetate (pH 5.0) to a final concentration of 0.2 M and 2 volumes of ethanol. Precipitated RNA was suspended in H20 and translated in a rabbit reticulocyte lysate system. Translation products were immunoprecipitated as described above. Chloroplast Membrane Preparation Chloroplast membranes were prepared from asynchronously grown C. reinhardi cells. ?S-methionine-labeled membranes were obtained from cells grown for 2 hr in sulfate-free high-salt medium (HSM) with 200 pCi ‘“S-H&O4 (New England Nuclear). Cells were harvested by centrifugation. washed with cold 25 mM HEPES (pH 8.0) and resuspended in the same buffer. Cells were broken by sonication for 30 set with a Sonifer Cell Disruptor (Branson Sonic Power Co.) with microtip (power setting 4). Sonicated cells were subject to centrifugation in a Sorvall SS-34 rotor, 5 min, 6000 rpm. 4°C. to remove intact cells and starch granules. Membranes in the supernatant fluid were sedimented for 1 hr in a Beckman Ti50 rotor, 40,000 rpm, 4°C. The green pellet was resuspended by sonication in 25 mM HEPES (pH 8.0) and collected again by sedimentation. Membranes were dissociated by resuspending the membranecontaining pellet in 10 mM Tris-HCI tpH 8.0). 2% SDS and 5% mercaptoethanol and heating m a boiling-water bath for 2 min. Samples containing 5-20 fig chlorophyll were subjected to electrophoresis on 11.2% polyacrylamide gels(acrylamide:bisacrylamide, 37.5:1) according to the method of Laemmli (1970). Peptide Mapping LHCPs were analyzed by tryptic fingerprint mapping. Chloroplast membrane proteins separated by polyacrylamide gel electrophoresis were radioiodinated and trypsinized in gel slices by the method of Elder et al. (1977) as modified by Zweig and Singer (1979). Peptide mapping was carried out on 10 x 10 cm cellulose-coated plastic thinlayer chromatography plates (Brinkmann Instruments) as described rn Elder et al. (1977).

Cdl 106

Antibody Binding Chloroplast membrane proteins of C. reinhardi reacting with antiserum to Lemna CPII proteins (provided by E. M. Tobin, UCLA) were detected by Western filter binding procedures. Chforoplast membrane proteins separated by SDS-polyacrylamide gel electrophoresis as described above were transferred electrophoretically to nitrocellulose paper CBA-83. Schleicher and Schuell) according to Towbin et al. (1979). Antiserum (50 pl) was incubated with 3 x 12 cm filter strips that had been preincubated in 0.9% NaCI. 10 mM Tris-HCI (pH 7.4) and 3% egg albumin (Sigma). Bound antibody was detected with lz51labeled protein A from Staphylococcus aureus (Pharmacia) according to the method of Renart et al. (1979). Acknowledgments We wish to thank Dr. Stephen Zweig for his assistance in the tryptic fingerprinting, Dr. Elaine Tobin for the Lemna antiserum and Dr. Ben Murray for his aid in Western blots. This work was supported by a grant from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

April 5. 1982;

revised

Ocotber

18. 1982

Apel. K. (1979). Phytochrome-induced appearance of mRNA activity for the apoprotein of the light-harvesting chlorophyll a/b protein of barley. Eur. J. Biochem. 97, 183-188. Apel, K. and Kloppstech, K. (1980). The effect of light on the biosynthesis of the light harvesting chlorophyll a/b protein. Planta 750, 426-430. Ares, M. and Howell, S. H. (1982). Cell cycle stage-specific mulatin of mRNAs encoding tubulin and other polypeptides mydomonas. Proc. Nat. Acad. Sci. USA 79, 5577-5581.

accuin Chla-

Armstrong, J. J., Surzycki. S. J., Mall. B. and Levine, Ft. P. (1971). Genetic transcription and translation specifying chloroplast components in Chlamydomonas reinhardi. Biochemistry 10, 692-701. Bailey, J. M. and Davidson, N. (1976). Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal. Biochem. 70, 75-85.

Bolivar. F. and Backman, K. (1979). Plasmids cloning vectors. Meth. Enzymol. 68, 245-268.

of the lightRNA activity

Delepelaire, P. and Chua, N.-H. (1981). Electrophoretic purification of chlorophyll a/b-protein complexes from Chlamydomonas reinhardtii and spinach and analysis of their polypeptide compositions. J. Biol. Chem. 256, 9300-9307. Dobberstein, B.. Blobel. G. and Chua, N.-H. (1977). In vitro syntheses and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase of Chlamydomonas reinhard;. Proc. Nat. Acad. Sci. USA 74, 1082-l 085. Durica. D. S.. Schloss, J. A. and Crain, W. R. (1980). Organization of actin gene sequences in the sea urchin: molecular cloning of an intron-containing DNA sequence coding for a cytoplasmic actin. Proc. Nat. Acad. Sci. USA 77, 5683-5687. Elder, J. H.. Pickett, R. A., Hampton, J. and Lerner. R. A. (1977). Radioiodination of proteins in single polyacrylamide gel slices. J. Biol. Chem. 252, 651 O-651 5. Eytan, G. and Ohad, I. (1972). Biogenesis of chloroplast VIII. Modulation of chloroplast lamellae composition induced by discontinuous illumination and inhibition protein synthesis during greening of Chlamydomonas mutant cells. J. Biol. Chem. 247, 122-l 29.

membranes and function of RNA and reinhard; y-l

Glisin. V.. Crkvenjakov, R. and Byus, C. (1974). Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 26332637.

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