Plant Physiol. (1986) 81, 527-5320032-0889/86/81/0527/06/$0 1.00/0

Specific Polysome Immunoadsorption to Purify anAmmonium-Inducible Glutamate Dehydrogenase mRNA fromChlorella sorokiniana and Synthesis of Full LengthDouble-Stranded cDNA from the Purified mRNA1

Received for publication October 16, 1985 and in revised form February 9, 1986

NEWELL F. BASCOMB2, KATHERINE J. TURNER3, AND ROBERT R. SCHMIDT*Department ofMicrobiology and Cell Science, 1059 McCarty Hall, University ofFlorida,Gainesville, Florida 32611

ABSTRACT

A specific polysome immunoadsorption procedure, employing solublerabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linkedto an insoluble cellulose matrix, was used to immunoselect polysomestranslating mRNA for a chloroplastic ammonium-inducible NADP-GDHin fully induced cells of Chlorella sorokiniana. The immunoselectedpolysomes were dissociated, and the NADP-GDH mRNA was recoveredby oligo (dT)cellulose chromatography. The translatable NADP-GDHmRNA was estimated to be 0.07 and 90% of the total polysomalpoly(A)+RNA before and after immunoselection of the polysomes, re-spectively. The immunoadsorption procedure resulted in an 83% recoveryand 1,291-fold purification of translatable NADP-GDH mRNA. In vitrotranslation of the immunoselected poly(A)'RNA yielded a single radio-active protein (on sodium dodecyl sufate polyacrylamide gels) with amolecular weight of 58,500, i.e. size of the putative precursor-protein ofthe NADP-GDH subunit in the holoenzyme in fully induced cells. Thepurified NADP-GDH mRNA was used for synthesis of a high proportionof nearly full-length single-stranded cDNA and double-stranded cDNAmolecules.

A primary objective in this 'laboratory is to elucidate thedifferent types ofmolecular mechanisms that regulate the expres-sion of genes coding for enzymes in eukaryotic cells. Recentresearch has been focused on the expression of the gene(s) codingfor a chloroplast-localized (15) NADP-GDH4 that is inducibleby ammonium in Chlorella sorokiniana cells (12, 20). By use ofimmunochemical and biochemical procedures (27), the NADP-GDH has been shown to be synthesized as a precursor proteinon cytosolic polysomes (15), to undergo continuous synthesis

'Supported by United States Public Health Service Grant GM 29733from the National Institutes of Health; Florida Agriculture ExperimentStation Journal Series, No. 681 1.

2Present address: Experiment Station 402/4241, E.I. DuPont De-Nemours & Co., Wilmington, DE 19898.

'Present address: Genetics Institute, 87 Cambridge Park Dr., Cam-bridge, MA 02140.

4Abbreviations: NADP-GDH, nicotinamide adenine dinucleotidephosphate-specific glutamate dehydrogenase; IgG, immunoglobulin G;ss and ds cDNA, single-stranded and double-stranded complementaryDNA; PAB, para-aminobenzyl; CNBr, cyanogen bromide.

and rapid degradation in both uninduced and induced cells (1,23), and to undergo rapid inactivation after removal of ammo-nium from fully-induced cells (1). Because of these post-transla-tional processes, the catalytic activity or amount of antigen ofthis enzyme cannot be used as a reliable measure of the level ofexpression of its structural gene in cell cycle experiments (7, 16,20, 21, 26) nor in experiments in which changes are made in thenitrogen nutritional status of the cells (1, 6).A more direct approach to assess changes in expression of the

NADP-GDH gene would be to measure the levels or rates ofaccumulation of NADP-GDH mRNA sequences by hybridiza-tion with a radioactive cDNA probe. However, two problemswere encountered in the isolation of the NADP-GDH mRNAfor use in cDNA synthesis. The NADP-GDH translatable mRNAwas shown to be only 0.074% (23) of the total-cellular translat-able poly(A)+RNAs in fully induced cells and to have a size (22)in common (i.e. 2-3 kilobase) with many other cellular eukar-yotic mRNAs. Therefore, isolation procedures that take advan-tage of the high abundance (2) or of the unique size (3) of amRNA did not seem to be useful for purification of the NADP-GDH mRNA.

Several laboratories have developed polysome immunoadsorp-tion procedures (4, 8, 9, 17, 19, 24) for isolation oflow abundancemRNAs. These procedures exploit the ability of antibodies,prepared against the native protein of the mRNA to be purified,to bind to the nascent polypeptide chains on the polysomes. Thesoluble antibody-polysome complexes are recovered by adsorp-tion to anti-antibody that has been covalently linked to aninsoluble cellulose matrix or Sepharose (4, 17, 24) or by adsorp-tion to Protein A Sepharose (8, 9, 19). By use of one of thesespecific polysome immunoadsorption procedures, Groner et al.(4) purified lysozyme mRNA (estimated to be 2-3% of the totaloviduct mRNAs), Kraus and Rosenberg (9) purified the mRNAcoding for the precursor of ornithine transcarbamoylase, theprecursor of the ,3-subunit of propionyl-CoA carboxylase, andcystathionine f3-synthase (0.20 0.02, and 0.015% of total hepaticmRNAs, respectively), Sul et al. (19) purified murine ATP-citratelyase (0.03% of total hepatic mRNAs), Korman et al. (8) purifiedthe mRNA from human lymphoblastoid cells that codes for theheavy chain of human histocompatibility antigen DR (0.01-0.05% of total cellular mRNAs), and Vodkin (24) purifiedmRNAs coding for seed lectin and Kunitz trypsin inhibitor ofsoybean.The objective of the research described in this paper was to

use the specific polysome immunoadsorption procedure ofSchutz et al. (17) to purify the NADP-GDH mRNA from fully

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Plant Physiol. Vol. 81, 1986

induced cells of C. sorokiniana for two purposes: (a) to comparethe mol wt of the NADP-GDH antigen synthesized in an in vitrotranslation system, programmed by the purified NADP-GDHmRNA, with the mol wt of the NADP-GDH putative precursor-protein that was determined (15) after in vitro translation of totalcellular poly(A)+RNA followed by immunoprecipitation ofNADP-GDH antigen, and (b) to synthesize full-length ds cDNAfrom the purified NADP-GDH mRNA in preparation for cDNAcloning. The results described herein show that in vitro transla-tion of the purified NADP-GDH mRNA yielded a single proteinwith the mol wt identical to the one reported earlier for theNADP-GDH putative precursor-protein, and that full-length dscDNA can be synthesized from the purified mRNA.

MATERIALS AND METHODSMaterials. Protein A-Sepharose CL-4B, CNBr-Sepharose-4B

were obtained from Pharmacia; deoxyribonucleoside triphos-phates, P-L Biochemicals, Inc.; PAB cellulose, Accurate Chemi-cal Corp.; avian myeloblastosis virus reverse transcriptase,lambda DNA Hind III restriction fragments, vanadyl ribonucleo-side complex, Klenow fragment DNA polymerase I, BethesdaResearch Laboratories; oligo(dT)12-18-cellulose, Collaborative Re-search, Inc.; sodium heparin, Ricker Laboratories, Inc.; calf-livertRNA, Boehringer Mannheim; [32P]dCTP, [35S]methionine,Amersham; Sarkosyl, Sigma Chemical Co.; SDS, highest purity,Gallard-Schlessinger Chemical Corp.; methylmercury hydroxide,Alfa Products, Ventron Corp. All other chemicals and materialswere of the highest grade available from Calbiochem Corp. or

Sigma Chemical Co. or as described earlier (23, 27).Purification of Rabbit Anti-NADP-GDH IgG by Antigen-

Affinity Chromatography. By the procedures ofYeung et aL (27),the NADP-GDH was purified from ammonium-induced cells ofChlorella sorokiniana, covalently coupled to CNBr-activatedSepharose 4B, and its subunits were cross-linked together tomake a stable reusable antigen-affinity column. This column was

used to purify monospecific anti-NADP-GDH IgG from rabbitantiserum (27).

Purification of Preimmune Rabbit IgG. The IgG fraction frompreimmune rabbit serum was purified by affinity chromatogra-phy on a column of Protein A-Sepharose CL-4B by a proceduredescribed by Miller and Stone ( 1).

Purification and Coupling of Sheep Anti-Rabbit IgG to PABCellulose. A yearling, 300 kg wethered male sheep was immu-nized by intramuscular injections (2-5 mg) of Protein A-purifiedpreimmune rabbit IgG in Freunds complete or incomplete ad-juvant. A partially purified IgG fraction was obtained from sheepantiserum by (NH4)2SO4 fractionation as described by Palmiteretal. (13).An affinity column was prepared by covalent attachment of

Protein A-purified preimmune rabbit IgG to CNBr-Sepharose4B exactly as described by Shapiro etal.(18). The partiallypurified sheep anti-rabbit IgG preparation was added to theaffinity column in phosphate buffered saline (0.01 m sodiumphosphate, 0.015 m NaCl [pH 7.2] at 22C). The column waswashed extensively with phosphate buffered saline to remove

unbound nonspecific IgG. The purified sheep anti-rabbit IgGwas eluted with 0.1 m glycine (pH 2.8) at22°C, and immediatelydialyzed against phosphate buffered saline. The affinity purifiedsheep anti-rabbit IgG was covalently attached to PAR-celluloseby use of a diazotization reaction exactly as described by Schutzetal. (17).

Isolation of Intact Total Cellular Polysomes. The proceduredescribed byTurner et aL. (23) was used to isolate total cellular,undegraded polysomes from asynchronous cells of C. soroki-niana that had been cultured in continuous light in 30 mmammonium medium (6, 21) for at least 10 h. Approximately 20A260 units of polysomes were obtained per g of broken cells in

the present study. Before cellular polysomes were used in theimmunoadsorption procedure, their integrity or intactness was

examined by sucrose density gradient sedimentation as describedearlier (23).

Specific Immunoadsorption of NADP-GDH Polysomes. TheNADP-GDH specific polysomes were selected from total cellularpolysomes by use of the immunoadsorption procedure describedby Schutz et al. (17) with only slight modifications. Total cellularpolysomes that had been stored in liquid N2 were thawed on iceand diluted to a concentration of 20 A260 units/ml with 25 mMTris-HCl, 25 mm KCI, 10 mM MgCl2, 5 mm EGTA, 200 units/ml heparin at pH 7.4, and then centrifuged at l0,OOOg for 10min. For every 20 A260 units/ml of total cellular polysomes, 0.2gg of affinity-purified anti-rabbit NADP-GDH IgG was added,and this mixture was incubated on ice for 2 h with occasionalmixing. The sheep anti-rabbit IgG cellulose was added to give a

final concentration of 500 gg per 20 A260 units/ml of cellularpolysomes. This mixture was allowed to incubate on ice for1 h,and the cellulose was resuspended every 5 min. After the 1 hincubation, the mixture was diluted with 5 ml of 10 mm Hepes,5mM MgCl2, 200 units/ml heparin at pH 7.4, and centrifugedfor 5 min at 5,000g. The nonspecific unadsorbed polysomes were

removed, from the NADP-GDH polysomes bound to the anti-antibody cellulose, by a series of five additional centrifugationwashes with 5 ml each of the same buffer. The wash step, withbuffer containing1% Triton and1% deoxycholate, used bySchutz et al. (17) was omitted in the present procedure.

IMMUNOADSORPTION OF SPECIFIC POLYSOMES

NADP- GDH

POLYSOME

TOTAL CELLULAR POLYSOMES RABBIT ANTI -NADP-GDH IgG

CH3COOH

60S SUBUNIT (I

40S SUBUNIT Ci

NADP-GHmMRNAI -

OLIGO (dT)CELLULOSECHROMATOGRAPHY

RIBOSOMALRNA 4-B PROTEINS

PURIFIED NADP-GDH mRNA

ds cDNA SYNTHESIS

cDNA CLONING

FIG. 1. Diagram of the steps involved in specific immunoadsorptionof polysomes, engaged in translation of the mRNA coding for an am-

monium inducible NADP-GDH in C. sorokiniana, and further purifi-cation of the mRNA by use of oligo(dT)cellulose chromatography. Thediagram was modified and redrawn from Schutz et al. (17).

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PURIFICATION OF AMMONIUM-INDUCIBLE GLUTAMATE DEHYDROGENASE mRNA

Table I. Purification ofC. sorokiniana NADP-GDH mRNA by Specific Polysome Immunoadsorption and Oligo(dT)-Cellulose ChromatographyThe data were obtained from a rabbit reticulocyte lysate mRNA dependent in vitro translation system as described by Turner et al. (23). The

radioactivity in NADP-GDH was obtained after immunoprecipitaton with anti-NADP-GDH IgG and SDS gel electrophoresis of the immunopre-cipitates (23). To calculate the fold-purification and percent of recovery of the NADP-GDH mRNA, the assumption was made that the NADP-GDH mRNA was translated in vitro with equal efficiency before and after purification from the total polysomal mRNAs.

Type of Radioactivity Incorporation NADP-GDH ofPoly(A)+RNA R

Into ttal protein Into NADPGDH Total Protein Synthesized Purification Recovery

Mg cpm/ug RNA % -fold %Total polysomal 2,300 420,000 290 0.07 1 100Immunoselected polysomal 2 270,000 244,000 90 1,291 83

The immunoadsorbed NADP-GDH polysomes were releasedfrom the anti-antibody cellulose by the addition of 2.5 ml of 20mM EDTA, 10 mm Hepes, and 100 units/ml heparin at pH 7.4.After 5 to 10 min, the anti-antibody cellulose was removed fromthe mixture by centrifugation for 5 min at 5000g. To dissociatethe polysomes and release the polysomal poly(A)+RNA (i.e.NADP-GDH mRNA), 125 Ml of 20% SDS and 20% Sarkosylwere added to the supernatant and incubated for 10 min. Aftercentrifugation, the supernatant was placed at 68°C for 2.5 min,and then cooled rapidly in an ethanol-ice bath. The polysomalpoly(A)+RNA was recovered by use of oligo(dT)-cellulose chro-matography (23). Based on an extinction coefficient for RNAequal to 1 A260 unit/24 mg RNA, 100 A260 units of total cellularpolysomes (from 5 g fresh weight of cells) yielded 2 ug ofimmunoselected polysomal poly(A)+RNA.To facilitate precipitation of the immunoselected poly

(A)+RNA, calf-liver tRNA was added to give a final concentra-tion of 5 MAg/ml. The RNA was precipitated by the additions of0.1 volume of 2.4 M ammonium acetate and 2.5 volumes ofredistilled ethanol at -20°C. After 6 h at -20°C, the precipitatedRNA was recovered by centrifugation for 1 h at 30,000 rpm ina SW 41 rotor at -5°C. The pellet of RNA was lyophilized todryness and stored in liquid N2.

Rabbit Reticulocyte Lysate mRNA-Dependent in Vitro Trans-lation System. The purification and recovery ofthe NADP-CGDHmRNA was estimated by use of the mRNA-dependent in vitrotranslation system of Pelham and Jackson (14) as modified byTurner et al. (23). The incorporation of [35S]methionine intoNADP-GDH antigen synthesized in vitro was measured afterimmunoprecipitation of enzyme antigen from the lysates (23).To estimate the amount ofcontamination ofthe immunoselectedNADP-GDH mRNA by other translatable mRNAs, the in vitrotranslation of the immunoselected poly(A)+RNA was allowed toproceed for 60 min, and the radioactive proteins that weresynthesized in vitro were subjected to SDS-PAGE as describedearlier (23, 27). The gels were fixed, dried, and autoradiogramsprepared.

Synthesis of NADP-GDH cDNA. Avian myeloblastosis virusreverse transcriptase was used to synthesize ss cDNA from 1 to10 MAg of immunoselected NADP-GDH mRNA as previouslydescribed (2, 25). The conditions for the reaction were 0.1 M

Tris-HCl (pH 8.3); 10 mM MgCl2; 140 mM KCI; 10 Mg oligo(dT)-cellulose; 2 mm methylmercury hydroxide; 30 mM jl-mercapto-ethanol; 1 mm vanadyl ribonucleoside complex; 1 mm of eachdeoxyribonucleoside triphosphate, [32P]dCTP (0.5Ci/mmol); 1

to 2 Mg immunoselected poly(A)+RNA; 40 units reverse tran-scriptase; total reaction volume, 50 ,l; incubation at 42°C. TheKlenow fragment of DNA polymerase I was used to catalyzesecond strand synthesis by the procedure of Wickens et al. (25).Alkaline agarose-gel electrophoresis was performed on the sscDNA and ds cDNA exactly as described by Maniatis et al. (10).The ds cDNA was not treated with Si nuclease before electro-phoresis. Mol wt standards were lambda DNA Hind III restric-

tion fragments. The positions of the radioactive cDNAs in thegels were determined by autoradiography.

RESULTS AND DISCUSSIONImmunoadsorption Procedure for Isolation of NADP-GDH

Polysomes. The four steps involved in the specific immunoad-sorption of NADP-GDH polysomes, from total cellular poly-somes isolated from Chlorella sorokiniana, are shown in Figure1. Crucial to the success of this procedure is the ability ofantibodies, prepared against the native enzyme or its subunits,to recognize the growing nascent polypeptide chains on intactpolysomes engaged in synthesis of the enzyme. Earlier in thislaboratory (5, 27), antibodies were prepared in rabbits againstthe purified NADP-GDH holoenzyme. The anti-NADP-GDHIgG was purified by antigen-affinity chromatography and shownto be monospecific by a number ofimmunochemical procedures(27). Moreover, the affinity-purified anti-NADP-GDH IgG waslabeled with 1251, and used in binding studies with intact totalcellular polysomes isolated from ammonium-induced cells of C.sorokiniana (23). The '25W-labeled anti-NADP-GDH IgG wasshown to bind to a specific class of intact polysomes (i.e. approx-imately 15 to 20 ribosomes/polysome), presumably by recogni-tion of the nascent polypeptide chains on the NADP-GDHpolysomes.

Percent Recovery of NADP-GDH Polysomes by Immunoad-sorption. In the present study, a mRNA dependent in vitrotranslation assay (14, 23) of poly(A)+RNA, isolated from totalcellular polysomes and from immunoselected polysomes, wasused to estimate the percent recovery of the NADP-GDH poly-somes by the immunoadsorption procedure. An 83% recoveryof the NADP-GDH polysomes was obtained (column 7, TableI). The NADP-GDH mRNA was shown to be only 0.07% of thetotal polysomal poly(A)+RNA (column 5, Table I) which isconsistent with its being reported to be only 0.074% of the totalcellular poly(A)+RNA in an earlier study (23). Thus, consideringthat the NADP-GDH polysomes belong to the low abundanceclass of cellular polysomes, the 83% recovery of NADP-GDHpolysomes is quite remarkable.

Estimation of Purity of Immunoselected NADP-GDH Poly-somes and Size of in Vitro Translation Product. As judged bythe amount of radioactivity incorporated into NADP-GDH an-tigen, during the in vitro translation of poly(A)+RNA isolatedfrom the immunoselected polysomes, the NADP-GDH mRNAwas estimated to be approximately 90% pure, with respect tocontamination by other translatable mRNAs (column 5, TableI). An 1,291-fold purification of the NADP-GDH mRNA wasachieved by the polysome immunoadsorption procedure (col-umn 6, Table I). To obtain another estimate of the purity of theimmunoselected NADP-GDH mRNA, a sample of the in vitrotranslation mixture was subjected to SDS slab-gel electrophoresisbefore immunoprecipitation of the NADP-GDH antigen, andthe position of the radioactive protein(s) in the gel was deter-mined by autoradiography. Only a single radioactive protein,with a mol wt of 58,500, was detected in the gel, containing the

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Plant Physiol. Vol. 81, 1986

I 2Mr x 10D kb

2 3.5

9..7v.S6.--1

..--92,, --4.3

--67 -2.2-2,1

-45

--0.59

FIG. 2. Autoradiogram of 35S-labeled protein(s), separated by SDS-PAGE, after synthesis in a rabbit reticulocyte lysate mRNA-dependentin vitro translation system programmed by C. sorokiniana poly(A)+RNAisolated from total cellular polysomes or from NADP-GDH specificpolysomes selected by immunoadsorption by the procedure shown inFigure 1. Lane 1, radioactive proteins synthesized under direction of 1.75Mg total cellular polysomal poly(A)+RNA in the in vitro translation systemof Pelham and Jackson (14) as modified by Turner et al. (23). Lane 2,radioactive protein (i.e. putative precursor-protein ofNADP-GDH sub-units) synthesized under the direction of 0.23 ug of immunoselectedpolysomal poly(A)+RNA. Ten MIA of the 50 M1 in vitro translation mixturewere added to the gel. The total cellular polysomes were isolated fromcells cultured for at least 10 h in 30 mm ammonium medium.

translation product(s) from the assay mixture programmed bythe immunoselected poly(A)+RNA (Fig. 2). Thus, although thein vitro translation assay (Table I) indicated that the immunose-lected NADP-GDH mRNA was only 90% pure, the absence ofother radioactive proteins in the SDS gel (Fig. 2), strongly suggeststhat the immunoselected NADP-GDH mRNA preparationmight not have been contaminated by other translatable mRNAspecies.The 58,500 D protein, synthesized in vitro in the present study,

is identical in size to the NADP-GDH antigen that Prunkard etal. (15) immunoprecipitated from in vitro translation reaction-mixtures programmed with total cellular poly(A)+RNA that was

FIG. 3. Autoradiogram after electrophoresis in an alkaline agarose gelof ss cDNA synthesized from immunoselected NADP-GDH mRNA byuse of avian myeloblastosis virus reverse transcriptase. The reaction wasprogrammed with 10 Mg of immunoselected polysomal poly(A)+RNA(i.e. NADP-GDH mRNA).

extracted from fully induced cells of C sorokiniana. Based ondata obtained from a number of experimental approaches, theseworkers concluded that the NADP-GDH mRNA codes for aprotein precursor (Mr = 58,500) which is synthesized in thecytosol and presumably is processed in the chloroplast to form asubunit with a mol wt of 53,000. A catalytically active NADP-GDH holoenzyme, composed ofidentical subunits (Mr = 53,000)was observed (15) to accumulate in the chloroplast of fullyinduced cells growing in 30 mm ammonium medium.

Synthesis of Single and Double-Stranded cDNAs from Im-munoselected NADP-GDH mRNA. Avian myeloblastosis virusreverse transcriptase was used to synthesize ss cDNAs from theimmunoselected NADP-GDH mRNA. When cDNA synthesis

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PURIFICATION OF AMMONIUM-INDUCIBLE GLUTAMATE DEHYDROGENASE mRNA

-23.5

-9.7-6.6

4.3....-2.2

2.1

-:0.59

FIG. 4. Autoradiogram after electrophoresis in an alkaline agarose gel

of ds cDNA synthesized from NADP-GDH ss cDNA by use of the

Klenow fragment of DNA polymerase The reaction conditions for ds

cDNA synthesis were identical to those described by Wickens et at. (25),

except the reaction was programmed by 3 gg of NADP-GDH ss cDNA

synthesized as described in Figure 3. Lanes 1, 2, and 3 contained 5, 3,

and ul of the ds cDNA reaction mixture after h incubation and then

dialysis to remove the radioactive substrate. The ds cDNA was not

treated with SI nuclease before alkaline gel electrophoresis.

was performed with gg of mRNA, the reaction ceased in 30

min, and the cDNA product was stable for at least 50 minthereafter (data not shown). For jzg of input mRNA, 0.3 usg ofss cDNA was synthesized. The NADP-GDH mRNA pro-

grammed the synthesis of ss cDNA of rather uniform size, in the

range of 2.1 to 3.3 kb (Fig. 3). By use of sucrose density gradient

centrifugation of denatured total cellular poly(A)'RNA, Turner

(22) estimated the translatable NADP-GDH mRNA to range in

size from less than 2 kb up to 3 kb. Therefore, the length of the

NADP-GDH mRNA appears to be in excess of the approxi-

mately 1.5 kb minimum-size mRNA required to code for a

precursor protein with a mol wt of 58,500. Since the size of the

ss cDNA, synthesized from the immunoselected NADP-GDH

mRNA, ranged approximately 2.1 to 3.3 kb (Fig. 3), it would

appear that many of the cDNA transcripts were in the size range

of the full-length NADP-GDH mRNA.

Synthesis of the second strand of the cDNA was catalyzed bythe Klenow fragment of DNA polymerase I. Without priortreatment with nuclease, the ds cDNA preparation was sub-

jected to alkaline agarose gel electrophoresis. Thus, the first and

second strands remained covalently joined by the hairpin loop.Under denaturing conditions of the electrophoresis, the ds

cDNAs were observed to migrate at approximately 0.5 the rate

of the ss cDNAs, indicating that second-strand synthesis was

essentially complete (Fig. 4). The ds cDNAs, containing thehairpin loop, ranged in size from 4.2 to 6.6 kb.Cloning of these nearly full-length NADP-GDH cDNAs is

currently in progress. These cDNA clones will be used for se-quence analysis and as hybridization probes to study the molec-ular biology and expression of the NADP-GDH gene(s) andmRNA(s) in C. sorokiniana.

Acknowledgments-The authors appreciate the review of this manuscript by Dr.P. M. Achey, Dr. F. C. Davis, and Dr. K. T. Shanmugam. Thanks are also extendedto Mrs. Waltraud I. Dunn for her technical help and to Ms. Martina Championfor typing the manuscript.

LITERATURE CITED

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13. PALMITER RP, T OKA, RT SCHIMKE 1971 Modulations ofovalbumin synthesisby estradiol- 1 7# and antinomycin D as studied in explants of chick oviductculture. J Biol Chem 246: 724-737

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16. SCHMIDT RR, KJ TURNER, NF BASCOMB, CF THURSTON, JJ LYNCH, WTMOLIN, AT YEUNG 1982 Post-transcriptional regulation of expression ofthegene for an ammonium-inducible glutamate dehydrogenase during the cellcycle of the eukaryote Chlorella. In KS MCCARTY, JR, GM PADILLA, eds,Genetic Expression in the Cell Cycle. Academic Press, New York, pp 199-229

17. SCHUTZ G, K SHALOM, B GRONER, AE SIPPEL, DT KURTZ, P FEIGELSON 1977Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody. Nucleic Acids Res 4: 71-84

18. SHAPIRO DJ, JM TAYLOR, GS McKNIGHT, R PALACIOS, C GONZALEZ, MLKIELY, RT SCHIMKE 1974 Isolation of hen oviduct ovalbumin and rat liveralbumin polysomes by indirect immunoprecipitation. J Biol Chem 249:3665-3671

19. SUL HS, LS WISE, ML BROWN, CS RUBIN 1984 Cloning of cDNA sequencesfor murine ATP-citrate lyase. J Biol Chem 259: 1201-1205

20. TALLEY DJ, LH WHITE, RR SCHMIDT 1972 Evidence for NADH- and NADPH-specific isozymes of glutamate dehydrogenase and the continuous inducibil-ity ofthe NADPH-specific isozyme throughout the cell cycle ofthe eucaryoteChlorella. J Biol Chem 247: 7927-7935

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BASCOMB ET AL.

21. TURNER KJ, RM GRONOSTAJSKI, RR SCHMIDT 1978 Regulation of initial rateof induction of nicotinamide adenine dinucleotide phosphate-specific gluta-mate dehydrogenase during the cell cycle of synchronous Chlorella. J Bac-teriol 134: 1013-1019

22. TURNER KJ 1980 Evidence for post-transcriptional regulation of induction ofNADP-specific glutamate dehydrogenase by accumulation of its mRNA inuninduced synchronous Chlorella cells. Ph.D. dissertation. Virginia Poly-technic Institute and State University, Blacksburg

23. TURNER KJ, NF BASCOMB, JJ LYNCH, CF THURSTON, WT MOLIN, RRSCHMIDT 1981 Evidence for mRNA of an ammonium-inducible glutamatedehydrogenase, and synthesis, covalent-modification, and degradation ofenzyme subunits in uninduced Chlorella cells. J Bacteriol 146: 578-589

Plant Physiol. Vol. 81, 1986

24. VODKIN LO 1981 Isolation and characterization of messenger RNAs for seedlectin and Kunitz trypsin inhibitor in soybeans. Plant Physiol 68: 766-771

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