Molecular Structure of a Functional Rat Gene for Manganese-containing Superoxide Dismutase Ye-Shih Ho, Adriann J. Howard, and James D. Crapo Laboratory of Molecular Biology, Division of Allergy, Critical Care and Respiratory Medicine, Department of Medicine, Duke University Medical Center, Durham, North Carolina

The manganese-containing superoxide dismutase (MnSOD) constitutes one of the major cellular defense mechanisms against the toxic effects of superoxide radical. The development of tolerance in adult rats to lethal exposure of O, (100%) after pre-exposing them to a sublethal concentration of O, (85%) was found to be closely associated with the increased activity of this enzyme in the lungs. Further experiments have shown that the transcriptional rate of the gene coding for MnSOD in rat lungs is increased at day 3 of 85% O2 exposure. To elucidate the nature of this transcriptional activation during hyperoxic insults, we chose to first understand the structure of the rat MnSOD gene. Three overlapping rat genomic fragments were isolated, and the DNA sequence containing the whole MnSOD gene was completely determined. The rat MnSOD gene contains at least five exons and is located in one piece of 16.4-kb EcoRl genomic DNA fragment. However, Southern blot analysis of total rat genomic DNA probed with MnSOD cDNA revealed an additional hybridizing 8.6-kb EcoRl genomic fragment besides the 16.4-kb one. To clarify the origin of this unexpected hybridizing genomic fragment, three unique genomic sequences derived from the promoter, intron 2, and the 3' untranslated region of the genomic clones were used to rehybridize the same Southern blot filter and were found to only hybridize to the 16.4-kbbut not 8.5-kb EcoRl genomic fragment. These data suggest: (1) two MnSOD genes are present per haploid rat genome, and (2) all three cloned genomic fragments are derived from the MnSOD gene, which is located in the 16.4-kb EcoRl genomic fragment. This cloned rat MnSOD gene has been designated as rat MnSOD-I gene. By using the unique 3' untranslated sequence of the MnSOD-I gene as a probe, RNA blot analysis has shown that this gene is functional under normal physiologic conditions in rat lungs and its expression is further enhanced in response to hyperoxia. The transcription initiation site was also mapped to 68 bp upstream from the first AUG of this gene by both Sl nuclease and primer extension analyses. Moreover, the same initiation site was found to be utilized during the activation of transcription by hyperoxia. The MnSOD-I gene promoter contains neither a "CAATbox" nor a "TATA box." It is highly G+C rich, contains two copies of Spl binding motif, two copies of SV40 core enhancer, one copy of adenoviral enhancer, and one copy of AP-l binding motif. These features may offer a clue to the mechanisms by which this gene is regulated.

Exposure of mammals to hyperoxia can cause extensive pulmonary injury (1). This type of lung damage is often observed in humans receiving oxygen therapy for respiratory insufficiency, and has been extensively studied in rats as a model of pulmonary oxygen toxicity (2-4). Cellular oxygen toxicity is initiated by overproduction of highly reactive oxygen species, including superoxide radical (Oi), hydroxyl (Received in original form May 21,1990 and in revised form September 13, 1990) Address correspondence to: Dr. Ye-Shih Ho, Box 3177, Department of Medicine, Duke University Medical Center, Durham, NC 27710. Abbreviations: Copper/zinc-containing superoxide dismutase, CuZnSOD; hydrogen peroxide, HzOz; manganese-containing superoxide dismutase, MnSOD; superoxide radical, O2.; hydroxyl radical, OH'; nucleotides, nt; superoxide dismutase, SOD. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 278-286, 1991

radical (OH') and hydrogen peroxide (HzOz) (5-7). These oxygen species can cause denaturation of cellular macromolecules such as lipids and proteins, resulting in cell injury. In order to reduce the intracellular concentration of these toxic oxygen species, which are also generated at a lesser extent during normal metabolism, various antioxidant defense mechanisms are present in the cell and include many small molecular scavengers as well as a series of antioxidant enzymes (7). Superoxide dismutases, catalase, glutathione peroxidase, and glutathione reductase are among those most studied antioxidant enzymes. The superoxide dismutases are thought to be the first line of antioxidant defense by catalyzing the dismutation of two superoxide radicals to yield hydrogen peroxide and oxygen (20i + 2H+ -+ Hz0 2 + Oz) (8). The two intracellular types of superoxide dismutases in mammalian cells are a cytosolic, dimeric copper/zinccontaining enzyme (CuZnSOD) and a mitochondrial, tetra-

Ho, Howard, and Crapo: Structure of a Rat Manganous Superoxide Dismutase Gene

meric manganese-contammg enzyme (MnSOD) (9-11). These two enzymes, though, exhibiting different molecular weights and different amino acid sequences, catalyze the same reaction. The hydrogen peroxide generated by the dismutation reaction is further scavenged by catalase and glutathione peroxidase (7). Previous studies have shown that augmentation of intracellular content of antioxidant enzymes by treating cells or animals with liposome-encapsulated superoxide dismutase and/or catalase can render the recipient cells or animals less susceptible to oxygen-mediated injury (12, 13). It has also been shown that tolerance of adult rats to 100% O2 can be induced by exposing them to a sublethal dose of O2 (85 %) for 5 to 7 d (4). Interestingly, this tolerance to hyperoxia is apparently associated with an increase in the activities of all the major pulmonary antioxidant enzymes as mentioned above. While the importance of these antioxidant enzymes in limiting the lung injury upon exposure to hyperoxia has been defined, there is little or no information regarding the molecular mechanisms that are involved in modulating the expression of these enzymes in rat lungs. Toward this end, our laboratory initiated studies to isolate cDNAs coding for various rat antioxidant enzymes (14-17). With the availability ofthese cDNA clones, we were able to demonstrate a dramatic increase of MnSOD mRNA in rat lungs at days 3 and 5 of 85% O2 exposure. I Furthermore, the increased MnSOD messages have been found.to result in part from the transcriptional activation of the rat MnSOD gene. I To further elucidate the nature of activation of the MnSOD gene transcription during hyperoxic insults, we chose to first understand the structure of the rat MnSOD gene. In this report, we describe the presence of two MnSOD genes per haploid rat genome; the sequence and structure of one ofthe MnSOD genes which is actively transcribed under normal physiologic conditions and is further activated in response to hyperoxia; and the nature of the controlling sequences regulating the expression of this gene.

Materials and Methods Recombinant DNA Techniques All the cloning and subcloning procedures were performed according to the methods described by Maniatis and associates (18). Rat lung RNAs were isolated using guanidine isothiocyanate as described by Chirgwin and colleagues (19). DNA and RNA blot analyses were performed according to the procedures described by Southern (20) and Thomas (21), respectively. Oxygen Exposure of Rats Adult male Sprague-Dawley rats weighing 200 to 250 g were used for exposures to 85 % oxygen and for controls exposed only to room air. Oxygen exposures were conducted in polystyrene chambers. The oxygen concentration varied less than 2 %, and CO2 concentration was maintained less than 0.5 % by providing seven to eight complete gas changes per hour. During the exposure, food and water ad libitum were provided, and the animals were kept in a 12-hour-on, 12hour-off cycle at all times. I

Ho, Y.-S., M. S. Dey, and 1. D. Crapo. Manuscript in preparation.

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Isolation of Genomic Clones A female Sprague-Dawley rat genomic DNA library, constructed in vector ACharon 4A with Hae III partially digested genomic DNA (Clontech Laboratories, Inc., Palo Alto, CA), was screened with the entire rat MnSOD cDNA-l insert according to the procedures described by Benton and Davis (22). Two positively hybridized clones, clones 2 and 25, were isolated and further characterized by restriction digestion analysis and DNA sequencing. The 5' end genomic DNA fragment of clone 25 (nucleotide residues -190 to +924) was again used to screen the same genomic library by which clone 44 was isolated. Sequence Analysis of Cloned Genomic DNAs Restriction DNA fragments derived from each genomic clone were isolated and subcloned into convenient sites of plasmid pKS or pSK (Stratagene, La Jolla, CA). Singlestranded plasmid DNAs were generated after infecting those bacteria harboring resultant recombinant plasmids with helper M13 phage VCS according to the procedures described by Levinson and associates (23). Nucleotide sequences of inserted DNAs were determined by Sanger's dideoxy chain-termination method using Sequenase (United States Biochemical Corporation, Cleveland, OH) or T7 DNA polymerase (Pharmacia LKB Biotechnology, Piscataway, NJ) with dITP reaction mixes to minimize the band compressions associated with high G+C content of the DNAs. A series of deletion subcloneswas generated by exolll/ mung bean nuclease method to allow performing further sequencing reactions from the cloning junction (24). Several synthetic oligonucleotides complementary to defined sequences of genomic DNA were also used to perform sequencing reactions. Figure 1 shows the entire sequencing strategy. All the nucleotide sequences in each of the exons have previously been determined on both strands as a cDNA form (MnSOD cDNA-l). Therefore, approximately 60% of the exons were only sequenced on one strand. Nucleotide sequences of approximately 60 % of the introns and 50 % of the DNA upstream from the first exon were determined on both strands. Based on our experience, the accuracy of sequence determination by collectingsequencing data from one strand of the DNA is approximately 99.5 %. Sl Nuclease Mapping and Primer Extension Experiments Two antisense, synthetic oligonucleotides with sequences 5' CACGTAGGlCGCGTGGTGCTT 3' and 5' TGCACGCCGCCCGACACAACATTGCTGAGG 3' complementary to nucleotide residues +337 to +357 and +61 to +90, respectively, of the genomic sequence were used in Sl nuclease mapping experiments. Each of these oligonucleotides was initially 5' end-labeled with [/,-32P]ATP and polynucleotide Kinase and then annealed to a sense, single-stranded plasmid containing nucleotide residues -190 to +924 of the genomic sequence, followed by synthesis of the complementary strand of DNA using the Klenow fragment of Escherichia coli DNA polymerase I. Each resultant double-stranded DNA fragment was digested with restriction enzyme EcoRI at the cloning site and then purified after separation on a polyacrylamide-urea gel. Fifty micrograms of total RNAs

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isolated from the lungs of rats exposed to air or 85% O2 for 3 d were used in SI nuclease mapping experiments. SI nuclease mapping was performed according to the procedures described by Greene and Struhl (25). The second oligonucleotide used in SI nuclease mapping experiments was also labeled at the 5' end with 32p and used in primer extension experiments. Fifty micrograms of total lung RNAs were used in primer extension experiments following the methods described by Kingston (26).

Results Isolation and Characterization of a Rat MnSOD Gene Two rat MnSOD eDNA clones (one full-length and the other truncated), containing different 3' untranslated sequences, have previously been isolated and characterized in our laboratory (15). The eDNA fragment of the full-length clone (designated as rat MnSOD eDNA-I) was used to screen a female Sprague-Dawley rat genomic library constructed in vector ;\Charon 4A. Three overlapping rat genomic DNA fragments were isolated and the nucleotide sequence of the DNA containing the whole MnSOD gene was completely determined (Figures 1 and 2). By comparing the genomic and the cDNA-l sequences, the rat MnSOD gene is divided into at least fiveexons interrupted by four introns (Figure 3). The nucleotide sequence of these five exons is identical to that of MnSOD eDNA-I, except nucleotide residue +4802 resembles that of the truncated cDNA clone (designated as rat MnSOD cDNA-2; Figure 2). This difference may result from the polymorphism of the MnSOD gene. As shown in Figure 2, the sequences at the 5' and 3' boundaries of each intron are in agreement with the consensus sequences proposed for donor and acceptor sites of introns (27). All introns are bound by the dinucleotide GT at the 5' end and AG at the 3' end. Organization of the Rat MnSOD Genes In order to understand the organization of the rat MnSOD gene(s), we prepared Southern blot filters containing Sprague-Dawley rat genomic DNA digested with various restriction enzymes and then probed with different regions of

Figure 1. Sequencing strategy for the rat MnSOD-I gene. The top lines show the overlapping recombinant phage clones. E* represents EcoRI restriction site artificially generated during the construction of the genomic library. The middle line shows restriction sites of the the rat MnSOD-I gene. B, H, and P represent restriction sites for enzymes BamHI, HindIII, and PstI, respectively. The open boxes indicate the exons. The bottom lines show the sequencing strategy. Arrows indicate the extents and directions of sequencing reactions of various subclones.

cDNA and genomic DNA fragments. Figure 4b shows that two EcoRl genomic DNA fragments of approximately 16.4 kb and 8.6 kb hybridized with the entire cDNA insert of clone MnSOD eDNA-I. Our cloning data indicated that the rat MnSOD gene was located in one piece of I6.4-kb EcoRl genomic DNA fragment (Figure 3), which corresponded to the same size EcoRl fragment observed in the Southern blot analysis. The hybridizing 8.6-kb EcoRl genomic DNA fragment was somewhat unexpected. A similar observation was also obtained from a Southern blot analysis of BamHI digested rat genomic DNA (Figure 4b). While the I4.0-kb hybridizing BamHI genomic DNA fragment was found to correspond to the size of DNA fragment predicted from the genomic clones, the second hybridizing 1O.0-kb BamHI genomic DNA fragment was unexpected. Furthermore, the sizes of hybridizing HindIII, Pstl, and Sac! digested genomic DNA fragments were identical to those predicted from the genomic clones, except there was an additional genomic DNA fragment in each of these enzyme digested genomic DNA hybridized with the cDNA-I fragment (Figure 4b, as marked by arrowheads). We interpreted these results to suggest that those additional, hybridizing restriction genomic fragments might result from a second rat MnSOD gene, rather than originating from restriction fragment length polymorphisms (RFLP) of the gene. It is less likely that polymorphisms would occur in a manner that would be found with five different restriction enzymes. This notion was substantiated by rehybridizing the same Southern blot filter with an unique probe of intron 2 (nucleotide residues +921 to +1206 obtained from genomic clone 25). As shown in Figure 4c, this probe only hybridized to the 16.4-kbEcoRl and I4.0-kb BamHI genomic DNA fragments but not to the 8.6-kb EcoRl and 1O.0-kb BamHI fragments. This intron probe also hybridized to other enzyme digested genomic DNA of predicted sizes. Furthermore, DNA fragments at the promoter and proximate 5'-flanking region of the gene (nucleotide residues -1107 to +21 isolated from genomic clone 44, as shown schematically in Figure 3) and at the 3' end of the gene (nucleotide residues +5862 to +6296 of the gene, isolated from MnSOD eDNA-I, as shown schematically in Figures

Ho, Howard, and Crapo: Structure of a Rat Manganous Superoxide Dismutase Gene

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Molecular structure of a functional rat gene for manganese-containing superoxide dismutase.

The manganese-containing superoxide dismutase (MnSOD) constitutes one of the major cellular defense mechanisms against the toxic effects of superoxide...
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