Isolation and characterization of a cDNA for mitochondrial manganese superoxide dismutase (SOD-3) of maize and its relation to other manganese superoxide dismutases

Biochimica et Biophysica Acta, 951 (1988) 61-70 Elsevier

61

BBA91857

Isolation and characterization of a eDNA for mitochondrial manganese superoxide dismutase (SOD-3) of maize and its relation to other manganese superoxide dismutases

Joseph A. White and John G. Scandalios Department of Genetics, North Carolina State University Raleigh, NC (U. S.,4.) (Received 22 February 1988) (Revised manuscript received 11 May 1988)

Key words: Manganese superoxide dismutase; Superoxide dismutase; cDNA; Gene expression; Nucleotide sequence; Amino acid sequence; (Maize)

We describe the isolation of a eDNA clone for the nuclear-encoded manganese superoxide dismutase (SOD-3) of maize mitochondria. The eDNA, pSod3.1c, selects by hybridization an RNA which produces the SOD-3 precursor upon in vitro translation. The DNA sequence of pSod3.1c was determined from fragments subeloned in MI3. The amino-acid sequence deduced from the nucleotide sequence displays significant homology with the amino-acid sequences of prokaryotic and eukaryotic Mn-SODs, but displays greater homology with mammalian Mn-SOD than it does with yeast or bacterial Mn-SOD. A 31 amino-acid transit peptide also is encoded by the pSod3.1c clone. Analysis of poly(A) + RNA indicates that Sod3 mRNA is approx. 1250 nucleotides in length. The amount of Sod3 transcript in seedling leaves is increased by light.

Introduction Superoxide dismutases ( ' 0 2 - : 0 2 oxidoreductase; EC 1.15.1.1 (SOD)) catalyze the dismutation of the superoxide anion radical to molecular oxygen and hydrogen peroxide: "02- + "02 +2H + -. 02 +H202.

Superoxide can react with hydrogen peroxide through Fenton chemistry to produce the hydroxyl radical, one of the most potent oxidants known [1]. Through removal of the reactants of

Abbreviation: AMV, Avian myeloblastosis virus.

Correspondence: J.G. Scandalios, Department of Genetics, Box 7614, North Carolina State University, Raleigh, NC 27695-7614, U.S.A.

the Haber-Weiss reaction, SODs and catalases/ peroxidases have been implicated in the protection of cellular membranes and constituents from peroxidation and oxidative damage. The extreme reactivity of the hydroxyl radical and the impermeability of membranes to superoxide necessitate the presence of SOD at the site of superoxide formation [2]. Superoxide and hydrogen peroxide are generated in both plant [3] and animal [4] mitochondria, and in illuminated chloroplasts [5]. Thus, the essential role of SOD in these organelles is thought to be the removal of superoxide generated during electron transport, photoreduction, and other processes. The four SOD isozymes of maize are encoded by the four nonallelic nuclear genes: Sod1, Sod2, Sod3 and Sod4 [6]. The cytosolic isozymes, SOD-2 and SOD-4, and the chloroplast isozyme, SOD-l, are copper- and zinc-containing homodimeric enzymes. The mitochondrial isozyme, SOD-3, is a

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

e,2 manganese-containing homotetrameric enzymc [7,8]. SOD-3 has been shown to be synthesized as a precursor (preSOD-3) and is translocated into isolated maize mitochondria [9]. Comparison of complete amino-acid sequence data for manganese (Mn)-SODs [10,11] and one iron (Fe)-SOD [12] demonstrates that the MnSODs in eukaryotic mitochondria and the MnSODs and Fe-SODs of prokaryotes have significant homology. Sequence data from C u / Z n - S O D s indicate that these proteins are distinctly different from the Mn- and Fe-SODs. and that they have no homology with the latter proteins [13]. X-ray crystalographic data for T. thermophilus Mn-SOD [14] and Fe-SODs of P. oealis [15] and E. coil [16] indicate that these proteins are structurally related [17]. Alignment of the Fe-SOD sequence with Mn-SOD sequences indicates that these proteins have identical metal-binding residues [12]. Homology among Mn-SODs has been used to support the endosymbiotic hypothesis of mitochondrial origin [18]. With sequence data presented by Stallings et al. [14]. we have synthesized an oligonucleotide which we have used to isolate a eDNA for the maize manganese SOD. Materials and Methods

Tissue and nucleic acid isolation. Inbred maize line W64A was the source of all tissues employed. Seeds were surface-sterilized in 1% NaHCIO3 for 10 min and imbibed in distilled H 2 0 for 24 h before planting on moistened germination paper. RNA was isolated from scutella after 5 days and from roots after 8 days. Endosperm RNA was isolated from kernels 18 days after pollination. Etiolated leaves were harvested from seedlings grown in darkness for 10 days, whereas 24-h greened leaves were harvested from seedlings grown 9 days in darkness and 24 h in continuous light. RNA was isolated from maize scutella, endosperm, leaves or roots by the methods of Hall et al. [19] or Chirgwin et al. [20]. Poly(A)- RNA was selected from total RNA by two cycles of binding and elution from oligo(dT)-cellulose [21]. Polysomes were isolated by the method of Skadsen and Scandalios [22].

eDNA .s'vnthesis. cl)NA was synthesized from poly(A)' RNA by the method of Gtibler and Hoffman [23], except that Maloney murme leukemia virus reverse transcriptase (Bethesda Research l,aboratories) was used (200 units/tLg). After second strand synthesis, the cDNA was extracted with phenol/chloroform, subjected to Sephadex G-100 chromatography (equilibrated ill 10 mM T r i s - H C l / l mM EDTA (pH 8)) and precipitated with ethanol. T4 DNA polymcrase was used to produce blunt-ended I)NA [24]. The eDNA was extracted and precipitated as described above. It was then treated with l:'coRl methylase [24]. extracted and precipitated. EcoRl linkers ( G G A A T T C ( ' ) were phosphorylated with T, polynucleotide kinase [24]. Phosphorylated linkers and eDNA were ligated at a 50:1 molar ratio. Ligatcd DNA was then digested with EcoRI. extracted with p h e n o l / c h l o r o f o r m and precipitated twice from 2.5 M ammonium acetate. Linkcr-ligated cDNA was ligated to ~ g l l l vector (Promega) at a 1:2 molar ratio. ('oncatameric DNA was packaged into lambda particles with Gigapak packaging extracts (Vector Cloning Systems). Phage particles were plated with E. coli strain Y 1 0 8 8 ( r - ) [25] at a density of 10000 plaques per 15 cm plate. Phage DNA was transferred to nitrocellulose filters [24]. Oligonucleotide .STnthesi.~ and hybridizations. Mixed heptadecamers corresponding to the conserved ('-terminal manganese-binding region of manganese SOD [14] were synthesized on an Applied Biosystems DNA synthesizer by the method of Matteucci and Caruthers [26]. The chemically synthesized probe was 5'-end labelled with [V32p]ATP ( > 7000 C i / m m o l , 1CN) and T~ polynucleotide kinasc [24]. The procedure yielded specific radioactivities of greater than 1-10 s cpm/p.g. Nitrocellulose filters containing phage DNA were washed for several hours in 1 M NaCI/50 mM Tris-HCI (pH 8)/1 mM EDTA/0.1% SDS at 4 2 ° C to remove debris. The filters were prehybridized for 8-12 h at 4 2 0 ( ` in 6 × SSC (1 × SSC = 3 M NaC1/0.3 M sodium citrate (pH 7)), 5 × Denhardt's solution (1 × = 0.02% each of bovine serum albumin, ficoll and polyvinylpyrolidone), 50 mM sodium phosphate (pH 7), 100 p,g/ml denatured salmon sperm DNA and 0.1% SDS.

63

The filters were hybridized for 8-12 h at 42°C to labelled oligonucleotides (15-10). 106 cpm/ml in 6 x SSC/1 x Denhardt's solution/50 mM sodium phosphate (pH 7)/100/~g/ml salmon DNA.'The filters were washed at 51°C in 3 M tetramethylammonium chloride, as described [27]. After washing, the filters were air-dried and exposed to X-ray film for 2-4 days at - 70 o C. Subcloning. Isolated h phage were obtained by standard procedures [28]. Phage DNA was obtained by treatment with formamide followed by ethanol precipitation [28]. h DNA containing cloned cDNA was digested with EcoRI. Similarly digested pUC12 DNA was treated with calf intestinal alkaline phosphatase [24]. Digested lambda and pUC12 DNAs were mixed and treated with T 4 DNA ligase. Plasma pDT1-5 [29] was digested with AoaI. The 1.1 kb fragment containing the E. coli Soda gene was ligated to similarly digested pUC12 vector DNA. E. coli JM83 was transformed with the ligated DNAs, and the resultant colonies were screened as described above. Plasmid DNAs were isolated by standard procedures [24]. Hybridization-selection. Sod3 mRNA was selected from poly(A) ÷ RNA as described by Ricciardi et al. [30] and translated in rabbit reticulocyte lysate [31]. Immunoprecipitation of SOD-3 was conducted according to White and Scandalios [9]. Radioactive polypeptides were separated by electrophoresis in 13.5% polyacrylamide gels [32]. The gel was treated for fluorography [33], dried and exposed to X-ray film. RNA analyses. RNA was electrophoresed in 1.2% agarose/formaldehyde gels [34] and transferred to nitrocellulose [35]. DNA fragments were

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labelled with [ot-32p]dCTP by the random oligonucleotide primer method [36]. Nitrocellulose filters were prehybridized at 42°C for 12-14 h in 6 x SSC/5 × Denhardt's solution/50 mM sodium phosphate/0.1% SDS/100 ~g per ml salmon DNA/50% formamide. The filters were hybridized in the same solution plus radioactive probe (approx. 1 • 106 cpm/ml) at 42°C for 12-14 h. The filters were sequentially washed in 5 x SSC at 23°C, 2 x SSC/0.1% SDS at 60°C, and 0.2 x SSC/0.1% SDS at 60 o C. The filters were air-dried and exposed to X-ray film. DNA sequence analysis. Plasmid DNAs were digested with appropriate endonucleases, separated in agarose gels, and isolated from gel slices by electroelution in an ISCO sample concentrator (Model 1750). DNA fragments were ligated into appropriately digested M13mpl8 or M13mpl9 replicative form DNA (Pharmacia). E. coli JM107 or JM109 was transfected with ligated DNA by the calcium chloride method. The DNA sequence was determined by the dideoxynucleotide chain termination method [37] with (a-35S)-labelled dATP substituted for [a-32p]dATP. Sequencing gels (7% acrylamide/8 M urea) were fixed in 10% methanol/acetic acid for 20-30 min, dried and exposed to X-ray film. Sequence alignment. The manganese SOD sequences of Escherichia coli [38], Tetrahymena thermophilus [11], Bacillus stearothermophilus [39], Saccharomyces cerevisiae [101, Mus musculus [40], Homo sapiens [41] and Zea mays (this work) were aligned by the Needleman and Wunsch [42] algorithm of the University of Wisconsin Genetic Computer Group. The gapweight was 1.0, and the gaplengthweight was 0.6. A modified symbol com-

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Fig. 1. Oligonucleotide probe. (A) The conserved Mn-binding region of the Mn-SOD amino-acid sequence from T. thermophilus [14]. (B) The deduced mRNA sequence coding for the Mn-binding region. (C) The complementary D N A sequence for the Mn-binding region which was synthesized and used to probe c D N A clones.

64

parison table was employed: AA EE IL l,V QQ VV

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A cDNA library synthesized from maize scutellar poly(A) ÷ RNA was constructed with the cloning vector ~ g t l l (see Materials and Methods). Recombinant phage were screened with an oligonucleotide probe complementary to the deduced nucleotide sequence of the conserved Cterminal manganese-binding region of Mn-SOD [14] (Fig. 1). The probe was a mixture of 32 heptadecamers. Three positive clones were identified from 22 000 phage recombinants: two of these were subcloned into the vector pUCI2 for further analysis. These clones were labelled pSod3.1c and pSod3.2c. Both cloned cDNAs hybridized to a poly(A)' RNA capable of synthesizing a polypeptide of identical molecular mass to that of immunochemically isolated preSOD-3 (27 kDa) (Fig. 2). The amount of polypeptide synthesized by the selected RNA (Fig. 2, lanes 3 and 4) reflects the difference

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Fig. 2. Hybridization-selection o f maize RNA. Fluorograph of a 13.5% SDS-l:x)lyacrylamide gel showing the radioactive polypeptides synthesized in vitro from hybridization-selected R N A (see Materials and Methods). In vitro translation products were immunoprecipitated with SOD-3 antisera. Lanes 1 4: polypeptides synthesized from R N A selected by pUC12 vector (1); E. coil M n - S O D gene subcloned in pUC12 (2): pSod3.1c (3); pSod3.2c (4); lanes 5 and 7, nonselected poly(A) ~ R N A in vitro translation product; 6, endogenous reticulocyte R N A synthesis: 8. nonselected, nonimmunoprecipitated in vitro translation products.

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Fig. 3. Restriction enzyme digestion map and sequencing strategy. Restriction enzyme digestion maps of pSod3.1c and pSod3.2c (Av = At,al, B = B a m H i , E = E c o R l , H = H i n d l l l . P = P s t l , Pv = P , u l l ) are shown. Narrow lines indicate non-translated regions (5'-end to the left) and the wide boxes represent vector sequences. The coding regions are medium-thickness lines (the dark region denotes the mature polypeptide coding region and the clear area denotes the transit peptide coding region). The length and direction of sequenced fragments are indicated by arrows. Note the difference in length of the 3' nontranslated regions of the two clones.

65

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TTGATCATGT~TC~CCTGTCT~TGG~TrGT~TACATTrCACTGAGATAGACTAATGCACGGC 8qO ~ 86O 870 880 890 900 910 CTGCCC~TllTGTTCGTCCTGCTTC£GTGCTACTCTGTCTCTGCI'CCTA~TCATGIn'ATGTTGAGCAAG 920 930 O J l O 950 96O 970 980 990 GTGATGC~Tr CCCACTCTrGTCTCCATI'AATAAMTC AGCTGAC£TICCC~TGTTTGCTTC-GMTr C Fig. 4. DNA and amino-acid sequences of the EcoRI fragment of pSod3.1c. The DNA .sequence and the deduced amino-acid sequence of the open reading frame are shown. The underlined amino-acid residue (Va_._ll)at position 32 denotes the mature (SOD-3) amino-terminus. Arrows indicate repeated elements. The two polyadenylation signals are underlined and the probe-binding region is boxed. (The first digit of each number is above the base to which it refers.)

66

in length of the cloned fragments in pSod3.1c and pSod3.2c, 1037 and 229 bp. respectively (Fig. 3). Of the bands visible in the preSOD-3 immunoprecipitated from poly(A) ~ RNA products (Fig. 2, lanes 5 and 7), only the 27 kDa band is produced by the hybridization-selected RNA. The E. coil Mn-SOD gene, SodA, did not hybridize to any maize RNA sequence which produced preSOD-3 upon in vitro translation (Fig. 2, lane 2). This negative control demonstrates the specific hybridization of Sod3 mRNA to the homologous Sod3 eDNA, but not to the heterologous E. coil gene. The lack of proteins in lanes 1 and 2 indicates that unselected poly(A) ~ RNA had been sufficiently removed. Thus, we can rigorously state that the cDNAs specifically hybridize to Sod3 mRNA. The sequence of pSod3.1c was determined (Fig. 4) from fragments subcloned in M13 mpl8 or mpl9 (Fig. 3). The probe-binding region is between basepairs 592-608. Two polyadenylation signals are located in the 3'-nontranslated region at positions 767 and 954. The deduced amino-acid sequence of pSod3.1c has regions of significant homology to other manganese SODs [10] (Fig. 6). Several amino acids reported as conserved by Marres et al. [10] were not found in the maize sequence: keu-146, Asn-198 and Arg-208 (which correspond to amino acids 180, 232 and 242 in Fig. 6). The N-terminal sequence of SOD-3 was determined by sequential Edman degradation: Val-Thr-Thr-Val-Ala-Leu-Pro-Asp-keu-Serl 0-TyrAsp- Phe-Gly-Ala- Leu-Glu-Pro-Ala-Ile20-Ser-Gly -Glu-lle-Met-Arg-keu-His28. The mature aminoterminus begins at codon 32 of the deduced amino-acid sequence. The only difference observed between this sequence and the deduced amino-acid sequence shown in Fig. 4 is the replacement of Thr (codon ACA) for Ala (codon GCA) at position 36 of the deduced sequence. The putative initiator codon is the only ATG codon in the DNA sequence 5' to the mature amino-terminal codon. This ATG is in frame with the remaining coding sequence and its flanking sequences follow Kozak's rules for the sequences surrounding an initiator codon [43]. To study the level of Sod3 mRNA in various maize tissues, the Sod3 cDNA was hybridized to poly(A)" RNA from line W64A (Fig. 5A). Scutel-

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Fig. 5. RNA hybridization. Autoradiograph.,, of RNA transfer~ hybridized to a2P-labelled pSe,d3.1c EuoR! fragment containing the Sod3 eDNA. (A) Lanes: 1, RNA markers: 2, poly(A) RNA from W64A scutella; 3.-.6. poly(A) " RNA:, from scutella (3), liquid endosperm (4), etiolated leaves (5), 24-h greened leaves (6): 7, total RNA from roots. Positions of the RNA markers are given in nucleotides. (B) Lanes: 1. polysome-bound RNA isolated from etiolated leaves: 2. polysome-bound RNA isolated from 24-h greened leaves: 3, poly(A)" RNA isolated from etiolated leaves: 4, poly(A)" RNA isolated from 24-h greened leaves. Note that there is an increased amount of hybridization to greened-leaf poly(A)* RNA.

lar poly(A) RNA shows no hybridization, as expected (Fig. 5A, lane 2). Sod3 hybridized to an RNA of approx. 1250 nucleotides in each of the remaining RNA preparations. Surprisingly, much more hybridization was seen in 24-h greened leaf poly(A) ÷ RNA than in etiolated leaf poly(A) ~ RNA (Fig. 5A, lanes 5 and 6): this was confirmed by analysis of both polysome-bound RNA and poly(A)* RNA from etiolated and light-grown leaves (Fig. 5B). However, no difference in hybridization of Sod3 to polysome-bound RNA from etiolated and greened leaves (Fig. 5B, lanes 1 and 2) was seen. On a per microgram basis, scutellar poly(A) + RNA displayed the greatest amount of hybridization (Fig. 5A, lane 3). The Sod3 mRNA has a broad size range of 1050-1450 nucleotides. The significance of this range is discussed below. Di~ussion

In this communication, we have reported the isolation of a cDNA clone for maize manganese SOD (SOD-3). The pSod3.1c plasmid contains a 1037 bp inserted EcoRl fragment. The cDNA

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Fig. 6. C o m p a r i s o n o f a m i n o - a c i d s e q u e n c e s f o r M n S O D o f m a i z e a n d o t h e r o r g a n i s m s : a l i g n m e n t o f m a n g a n e s e S O D s e q u e n c e s o f

E. coli [38], T. thermophilus [11], B. stearothermophilus [39], S. cereoisiae [10], M. musculus [40], H. sapiens [41] a n d Z. mays (this work). T h e a l i g n m e n t w a s g e n e r a t e d f r o m p a i r w i s e c o m p a r i s o n o f e a c h o f the seven s e q u e n c e s b y t h e N e e d l e m a n a n d W u n s c h [42] algorithm. Boxed sequences are regions of homology. The amino-terminal residues of each sequence are underlined.

specifically hybridized to Sod3 mRNA (Fig. 5), and its deduced amino-acid sequence has significant homology to other Mn-SODs (Fig. 6). The amino-acid sequence deduced from clone pSod3.1c consists of 235 residues and has a calculated molecular weight of 25525. The mature amino terminus is located 32 residues downstream from the putative initiator methionine (Fig. 4). The additional 31 codons code for a transit peptide of 3055 Da. This is slightly greater than the size of the preSOD-3 transit peptide (approx. 2000 Da)

reported by White and Scandalios [9]. The transit peptide has characteristics similar to other mitochondrial transit peptides [44], notably, the basic residues at positions 4, 9, 10, 21 and 30 (Fig. 4). However, this sequence also contains Pro residues at positions 15 and 22. It has been hypothesized that transit peptides can form a-helical structures [45]. The significance of these residues in the preSOD-3 transit peptide is being investigated. The remainder of the deduced amino-acid sequence has a calculated molecular mass of 22 469

68

Da, which compares favorably with the value (24000 Da) determined by analytical ultracentrifugation [7]. The deduced amino-acid sequence displays significant homology with the amino-acid sequences of Mn-SOD from other organisms (Fig. 6). The alignment differs from that of Marres et al. [10]. Rather than simply use their alignment, we realigned the sequences for two reasons: (1) the maize sequence is the first reported plant Mn-SOD sequence, and (2) by use of the Needleman and Wunsch [42] algorithm and a comparison table based on the chemical similarity of certain amino acids, we believe a better alignment has been obtained. From the alignment presented in Fig. 6, the percent homologies (identical residues) were derived (Table I). The minimal level of homology among these sequences is approx. 40%, i.e., that between yeast and E. coli, and between E. coli and Z. mays. The number of highly conserved residues, 50 (outlined in Fig. 6), corresponds to a homology of 26% of the shortest sequence in this comparison. The greatest homology shown is between the human and mouse sequences, as might be expected for these mammalian sequences. However, the three bacterial sequences appear to have significantly greater homology (approx. 60%) to one another than they do with the eukaryotic Mn-SOD sequences. The maize Mn-SOD sequence shows greatest homology (approx. 56%) with the mammalian sequences: thus, it appears to

be more closely related to these sequences than to the yeast or the bacterial sequences (at least 50%). Nevertheless. all of the mitochondrial Mn-SOI)s have significant homology with the prokaryotic Mn-SODs, and thus lend support to the endosymbiotic hypothesis of mitochondrial origin [18]. It is interesting to note that the E. coli and B. stearotherrnophilvLs" Mn-SOD enzymes are dimeric. whereas the T. thermophilus and eukaryotic MnSOD enzymes are tetrameric. Homology of the E. co// or the B. stearothermophilus sequence with any of the eukaryotic sequences is relatively low (less than 50%). The disparity between the N-terminal protein sequence data and our deduced amino-acid sequence has several possible explanations: (a) a reverse transcriptase copying error; (b) incorrect N-terminal sequence data; or (c) a second, previously undetected isozyme for SOD-3 in maize line W64A. We currently have no conclusive data indicating that a second isozyme for Mn-SOD exists in W64A. The error rate for AMV reverse transcriptase (1 per 1000 bases) is known to be quite high among polymerases [46,47]. Data for Maloney murine leukemia virus reverse transcriptase are unavailable, but AMV and Maloney murine leukemia virus reverse transcriptases perform equally well in DNA-sequencing reactions [48]. However, the possible substitution of A for G by Maloney murine leukemia virus reverse transcriptase in our

TABLE I P E R C E N T H O M O L O G Y BETWEEN MnSOD S E Q U E N C E S F R O M O T H E R O R G A N I S M S Homologies (given as percent) between pairwise comparisons of the Mn-SOD sequences as aligned in Fig. 6 are listed in the upper-right portion of the table. These values were determined from the number of matches (listed in the lower-left portion of the table) divided by the length of the shorter sequence in the comparison. The lengths ~ of each sequence used in the comparisons are listed in the lower part of the table. Maize

Human

Maize Hum an Mouse Yeast E. coil B.st T. th.

109 112 103 85 88 95

55.6 181 102 88 97 97

Length

204

196

Mouse

Yeast

E. coil

B. st.

56.6 92.3 99 89 97 99

50.5 52.0 50.0 84 83 90

41.7 44.9 44.9 40.8 125 110

43.6 49.5 49.0 41.1 61.9 127

206

202

198

207

'~ The transit sequences of the maize, mouse and yeast sequences were omitted from the comparisons.

T. therm. 46.8 49.5 50.0 44.3 54.2 62.9 203

69

experiments, producing Thr instead of Ala at position 36 in the amino acid sequence, cannot be eliminated. The Sod3 mRNA appears to have a broad size range (nucleotides 1050-1450) (Fig. 5), and may terminate after at least two different poly(A) addition signals (nucleotide positions 767 and 954). Clone pSod3.2c has a 3' nontranslated region approx. 190 bp shorter than that of pSod3.1c (Fig. 3). Neither clone has a poly(dA) tail; however, the polyadenylation signal is found 33 and 15 bp upstream of the EcoRI site in clones pSod3.1c and pSod3.2c, respectively (Fig. 4). The minimum coding sequence for preSOD-3 is 708 bp. Depending on the poly(A) tail length, a large portion of the 5' leader for Sod3 mRNA may be absent from our clone. A repeated element (AGCG) is located in the 5' leader region from bp - 2 1 to -10, and is repeated three times. The probability for occurrence of this 4 bp sequence being repeated twice is 1.526-10 -5, or once in approx. 65 500 bp. The significance of this repeated sequence is not known. The Sod3 mRNA appears to be synthesized to a greater extent in 24-h greened leaves than in etiolated leaves of the same age. This may reflect a general increase in transcription in the growing seedling, or an increase in the transcription of nuclear-encoded mitochondrial proteins in greened leaves. A similar increase in NADP-malic enzyme mRNA in maize has been reported [49]. Since malic enzyme is compartmentalized in the chloroplast, the increase in mRNA of both these proteins in greened versus etiolated tissue probably represents an increase in the synthesis of nuclear-encoded organellar proteins. We are studying this further by quantitation of mRNA levels with the Sod3 cDNA. In conclusion, the isolation of a cDNA for maize manganese SOD will allow for the further study of the evolution of manganese SOD en-' zymes and the compartmentalization of the SOD-3 precursor as directed by its transit peptide.

Acknowledgements The authors thank Ron Cannon and Ron Skadsen for helpful suggestions and stimulating discussions, Lisa Brooks for help with protein sequence

alignment, Mark Hermodson for doing the Nterminal protein sequencing, Danielle Touati for providing the E. coli SodA gene, and Suzanne Quick for typing the manuscript. This work was supported by Research Grant No. GM-33817 from the U.S. National Institutes of Health to J.G.S. This is Paper No. 11292 of the Journal Series of the NC Agricultural Research Service, Raleigh, NC.

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