comparison of the restriction endonuclease …

Copyright 0 1982 by the Genetics Society of America

COMPARISON OF THE RESTRICTION ENDONUCLEASE DIGESTION PATTERNS OF MITOCHONDRIAL DNA FROM

NORMAL AND MALE STERILE CYTOPLASMS OF ZEA MAYS L.

KATHLEEN S. BORCK AND VIRGINIA WALBOT

Department of Biological Sciences, Stanford University, Stanford, California 94305-2493

Manuscript received December 22, 1981 Revised copy accepted May 28, 1982

ABSTRACT

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, Sal1 and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization be- havior.-The total Sal1 digest and the larger Pst I fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.

ITOCHONDRIA are strictly maternally inherited in maize (PRING and NI LEVINGS 1978; CONDE, PRING and LEVINGS 1979). Hence changes in organelle DNA organization will be stabilized by uniparental inheritance because recombination between organellar genomes of different lines is not possible. Understanding the phylogenetic relationship among diverging organellar genomes allows assessment of when the last common maternal ancestor for that line may have existed (BROWN 1980). Mitochondrial DNA (mtDNA) appears to diverge rapidly in most animal and fungal species (UPHOLT and DAWID 1977; PRUNELL et al. 1977; SANDERS et al. 1977; AVISE, LANSMAN and SHADE 1979; BI~OWN 1980; FERRIS, WILSON and BROWN 1981) but not all (SHAH and LANGLEY 1979). In those species in which mtDNA does diverge rapidly as measured by changes in restriction sites, quantitation of this divergence has proven to be a useful tool in examining the relationship of closely related species, populations or even individuals (AVISE, LANSMAN and SHADE 1979; BROWN 1980).

In maize the restriction patterns of plastid DNA from a variety of modern inbred lines of maize, ancient maize lines, and races of teosinte, a close relative of maize, are almost invariant (TIMOTHY et al. 1979; PRING and LEVINGS 1978; WALBOT, unpublished data). However, within maize and teosinte mtDNA shows great variability (TIMOTHY et al. 1979). Variation in mtDNA exists within modern inbred maize as well (LEVINGS and PRING 1976, 1977; LEVINGS et al. 1979).

Genetics 102: 109-128 September, 1982.

110 K. S. BORCK AND V. WALBOT

For breeding purposes maize cytoplasms have been classified into four categories: normal and three cytoplasmically male sterile types T, C and S. The male sterile cytoplasmic types are distinguished from one another on the basis of which dominant nuclear genes restore male fertility (BECKETT 1971). PRING and LEVINGS (1978) demonstrated that unique restriction fragment patterns were found in the mtDNA from N, T, C and S cytoplasmic types maintained in the same nuclear background. Initial quantitation of the differences in mtDNA restriction fragment patterns between N and each male sterile type was based on the number of shared Hind111 fragments in digests in which approximately 200 kb of the genome was resolved (PRING and LEVINGS 1978). In that study the number of shared fragments was quite high with N sharing 88% with C, 78% with S and 73% with T.

However, more recent estimates of the size of the mtDNA genome indicate that the minimum complexity based on summation of restriction fragments is more than 450 kb (WARD, ANDERSON and BENDICH 1981 and this report) or even larger, based on the frequency of recovery of specific cloned sequences from a genomic library (LONSDALE, THOMPSON and HODGE 1981). As the complexity of maize mtDNA is considerably greater than initially suggested, we have reex- amined the question of the relationship among the four cytoplasmic types. We also report the first list of the sizes of the EcoRI, PstI and Sal1 fragments of normal mtDNA and restriction fragment sizes for T, C and S for the latter two enzymes.

MATERIALS AND METHODS

Zsolation of mitochondria and purification of mitochondria1 DNA: The procedure of isolation of mitochondria was based on that of PRING and LEVINGS (1978) with modifications. Buffers used were as follows: buffer A [0.35 M sucrose, 10 mM Tris-HC1 (pH 8), 1 mM EGTA, 10 mM NazEDTA (pH 8). 1% polyvinylpyrrolidone (PVP, ave. MW 40,000), 0.1% bovine serum albumin (BSA), 3 mM p- mercaptoethanol]; buffer B [0.30 M sucrose, 10 mM Tris-HC1 (pH 7.5), 2 mM NazEDTA, 0.1% BSA]; buffer C [0.3 M sucrose, 50 mM Tris-HC1 (pH 7.5), 18 mM MgClz]; buffer D [0.3 M sucrose, 20 mM Tris- HCI (pH 7.5),25 mM NazEDTA]. All buffers were sterile and contained 50 pg/ml chloramphenicol.

Seeds of B37N, C, T or S maize, obtained from Pioneer Hi-Bred International, were germinated in vermiculite, and dark-grown for 8 days at 25'. Coleoptiles were harvested into ice, surface sterilized with 10% hypochlorite for 2 min and quickly rinsed with 1 liter of buffer A, followed by 2-3 liters of sterile water. Typically, 500 g of tissue were homogenized in a Waring Blendor, using a volume of buffer equal to 2.5 times the wet weight of coleoptiles. Homogenates were filtered through four layers of cheesecloth and centrifuged for 15 min at 1300 x g. The supernatant was then centrifuged at 13,000 X g for 15 min. Pellets were resuspended in one fourth the original volume of buffer B, and the centrifugation steps were repeated. Final mitochondrial pellets were resuspended in 15 ml of buffer C and digested with 50 pg/ml DNase I (Worthington Diagnostics) for 1 hr at 4'.

NazEDTA was added to a final concentration of 50 mM and mitochondria were applied to a sucrose cushion of 0.6 M sucrose, 50 mM NazEDTA. After centrifugation at 13,000 X g the mitochondria were resuspended with buffer D and pelleted again at 13,000 X g; this wash procedure was repeated twice more to remove adherent nuclear DNA and DNase.

Mitochondria were lysed with 1% sarkosyl and digested with proteinase K (200 pg/ml) for 1 hr at 37'. The DNA was purified by phenol extraction and ethanol precipitation followed by centrifugation in 51.5% (w/w) CsCl (density adjusted to 1.62 g/cm3) containing 10 mM Tris-HC1 (pH 7.5). 1 mM Na2EDTA and 50 pg/ml ethidium bromide in a Beckman type 65 rotor for 48-60 hr at 40,500 rpm, 25'. The mitochondrial DNA (mtDNA) band was recovered with a bent Pasteur pipet. After removal of ethidium bromide with isopropyl alcohol, the mtDNA was dialyzed against 10 mM Tris- HCl, 1 mM NazEDTA, pH 8 (TE buffer) and then ethanol precipitated. The purity of mtDNA preparation was checked routinely by determination of the buoyant density in neutral CsCl as

MAIZE MITOCHONDRIAL DNA 111

described by SHAW and LEVINGS (1974) relative to Micrococcus luteus DNA (U = 1.731 g/cm3) in a model E Analytical Ultracentrifuge.

Restriction analysis by gel electrophoresis: All restriction enzymes except HindIII and EcoRI were purchased from New England BioLabs or Bethesda Research Labs and were used as directed by the supplier. HindIII was kindly provided by J. KINNAIRD and M. KEIGHREN, and EcoRI by M. SMITH. Mitochondrial DNA was digested with restriction enzymes, ethanol precipitated with yeast tRNA as carrier, and then resuspend4 in TE buffer before electrophoresis in 0.8%, 1.0% or 1.5% agarose (SeaKem ME grade). Electrophoresis buffer was 40 mM Tris-HC1.20 mM Na acetate, 18 mM NaCI, 2 mM NaZEDTA, pH 8.2 (TEA buffer). Molecular weight standards were: native lambda (21857,

lambda C1857 restricted witb SaII, SmaI, XhoI, EcoRI, HindIII and EcoR1:HindIII double digest (PHILLIPSEN, KRAMER and DAVIS 1978) and adenovirus 2 restricted with SmaI. Gels were electrophoresed for a total of 60-FA hr at 0.5-1 V/cm, but removed at 20 and 44 hr, stained with ethidium bromide and photographed on Ilford FP4 film. Negatives were scanned with a Joyce-Loebel densitometer. Gels were soaked in 0.25 N HCl for 30 min (WAHL, STERN and STARK 1979), then in 1.5 M NaC1, 0.5 M NaOH for 1 hr, then in 3 M NaCl, 0.3 M Tris-HC1, pH 7, for 1 hr and the DNA transferred to nitrocellulose (SCHLEICHER and SCHUELL, BA 85) by the method of SOUTHERN (1975).

All mobilities of restriction fragments were calibrated from densitometric scans of negatives relative to EcoRI fragment 5 or HindIII fragment 3 of lambda digests run as standards. The molecular weight vs. mobility of standards was plotted and a best fit curve determined by the least squares method; molecular weights of mtDNA restriction fragments were computed from this standard curve (SOUTHERN 1979). All data were analyzed on a Southwest Technical Products mini- computer. Areas under peaks were determined using a Numonics digitizer.

Construction of a mtDNA library: Purified mtDNA was partially restricted with EcoRI and cloned by in vitro packaging according to the method described by STERNBERG, TIEMEIER and ENQUIST (1977) and BLATTNER et al. (1978). The vector was lambda Charon 4A (BLATTNER et al. 1977) and the host, E. coli strain DP50 supF. T4 ligase was provided by W. BARNES. Approximately 93% of the resultant viable phage contained inserts; recombinant phage were purified and propagated in DP50 supF. All phage were further purified on CsCl gradients before DNA extraction. These experiments involving recombinant DNA were carried out under approved P1-HV2 conditions.

Preparation of probes and hybridizatioo methods: DNA from each phage preparation was restricted with EcoRI and fractionated on 0.8% agarose gels. Individual cloned mtDNA fragments were cut from the gel and electroeluted; they were then concentrated by ethanol precipitation. After resuspension in TE buffer, the DNA was nick translated by the method of RIGBY et al. (1977).

Nick-translated probes (approximately 5 x lo5 cpm) were hybridized to Southern transfers in 0.5 M NaCl, 0.1 M Na2HP04, 6 mM Na2EDTA, 1% sarkosyl, pH 7 containing 50 pg/ml denatured salmon sperm DNA for 16-20 hr at 65'. Filters were washed in 1x Denhardt (DENHARDT 1966) solution (0.02% BSA, 0.02% Ficoll, 0.02% PVP 360) in 2 x SSC (1X SSC is 0.15 M NaCl, 0.015 M Na3 citrate) for 1 hr, followed by two 45-min washes in 0.2X SSC, 0.1% sodium dodecyl sulfate (SDS) at 50'. Filters were then quickly rinsed with 1 liter of 0 . 2 ~ SSC, 0.1% SDS and then with 0 . 2 ~ SSC. After drying, filters were exposed to Kodak Ortho G X-ray film in cassettes with Lanex intensifying screens at -70'.

RESULTS

Restriction of normal mtDNA: The complexity of the restriction enzyme digestion profile of normal maize mtDNA can be seen in Figure 1 and the accompanying Table 1 where the molecular weight estimates are given for each visible band. Lengthy electrophoresis at low voltage of B37N (normal) mtDNA restricted by EcoRI has resolved more than 70 discrete fragments ranging in size from 13.9 kb to less than 1.4 kb. The enzyme PstI yields 59 fragments varying in size from 30.1 kb to 1.04 kb. Digestion with SaII produces 42 fragments ranging from 37.1 kb to 1.04 kb; this enzyme produces the fewest number of cuts of any restriction enzyme tried so far.

Examination of the photograph and the corresponding densitometric tracings (Figure 1) illustrates that bands exist in nonstoichiometric amounts. The range in stoichiometry is 1-13X based on the area under the peaks of the densitometric

TABLE 1

Average molecular weights (9 and standard deviation (un-1) of restriction fragments of B37N mtDNA

Sal1 PstI - Fragment EcoRI X On-1 X 0n-1

1 13.90 37.01 2.80 30.11 2.28 2 12.70 32.80 2.11 27.26 1.52 3 12.54 27.98 1.39 24.54 1.82 4 10.40 26.73 1.40 23.27 1.25 5 10.23 25.40 1.28 22.39 1.29 6 10.12 21.50 0.84 20.49 1.26 7 9.75 19.99 0.79 18.19 0.56 8 9.31 19.01 0.72 17.08 0.29 9 9.21 18.04 0.66 16.35 0.46

10 8.99 16.53 0.57 14.85 0.51 11 8.66 15.90 0.57 13.99 0.41 12 7.74 14.31 0.45 13.42 0.31 13 6.95 13.91 0.41 11.50 0.30 14 6.72 12.92 0.51 10.66 0.24 15 6.48 12.63 0.54 9.39 0.20 16 6.25 12.37 0.47 9.04 0.20 17 5.92 10.99 0.33 8.56 0.20 18 5.41 10.69 0.36 8.10 0.22 19 5.25 9.38 0.38 7.77 0.21 20 5.15 9.01 0.30 7.33 0.20 21 5.02 8.55 0.28 7.13 0.18 22 4.83 8.28 0.30 6.49 0.19 23 4.73 7.92 0.27 6.38 0.19 24 4.68 7.39 0.22 5.96 0.17 25 4.52 7.15 0.21 5.61 0.24 26 4.41 6.88 0.20 5.23 0.16 27 4.29 6.47 0.18 5.07 0.18 28 4.14 5.90 0.16 4.96 0.20 29 3.98 5.55 0.15 4.59 0.19 30 3.87 5.23 0.13 4.49 0.18 31 3.80 5.15 0.12 4.40 0.18 32 3.68 4.84 0.15 4.28 0.18 33 3.64 4.56 0.17 4.18 0.13 34 3.55 4.17 0.13 4.10 0.14 35 3.52 3.44 0.22 4.02 0.06 36 3.46 3.35 0.10 3.85 0.04 37 3.38 3.06 0.02 3.69 0.04 38 3.08 2.46 0.04 3.58 0.06 39 3.00 2.11 0.03 3.49 0.07 40 2.97 1.63 - 3.30 0.10 41 2.91 1.12 - 3.24 0.09 42 2.78 1.04 - 3.12 0.09 43 2.72 2.96 0.02 44 2.65 x = 473.35 2.77 0.01 45 2.60 2.71 0.0 46 2.57 2.54 0.0 47 2.50 2.00 - 48 2.45 1.98 - 49 2.43 1.94 - 50 2.33 1.90 - 51 2.30 1.86 - 52 2.25 1.64 - 53 2.16 1.59 -

112


TABLE 1-Continued

Sal1 PstI - Fragment EcoRI X an-1 X bn-1

54 2.14 1.47 - 55 2.06 1.35 - 56 2.05 1.27 - 57 2.01 1.21 - 58 1.97 1.09 - 59 1.86 1.04 - 60 1.80 61 1.76 Z = 446.77 62 1.72 63 1.68 64 1.64 65 1.60 66 1.56 67 1.52 68 1.50 69 1.45 70 1.42

X = 308.75

Average molecular weight values and standard deviations were computed from densitometric scans of agarose gels. All values are in kilobases. For PstI and Sa11 digests, five different gels were compared; a single well resolved EcoRI digest was used for molecular weight assignment. Standard deviations were not calculated for the EcoRI digest nor for bands less than 2 kb.

scans. Such variability in band intensity has been observed with these additional enzymes: SmaI, SacI, XhoI, XbaI, PvuII and BamHI. WARD, ANDERSON and BENDICH (1981) and SPRUILL, LEVINCS and SEDEROFF (1980) have also reported differences in the stoichiometry of restriction fragments of maize mtDNA. In the latter case many of the high intensity bands they observed may have been caused by overlapping fragments that did not resolve on short gels. By using very long electrophoretic times, low voltages and gel concentrations appropriate for the size range of restriction fragments being measured (SOUTHERN 1979), we have been able to separate bands of quite similar electrophoretic mobility. Within those regions of the gel where bands are clearly resolved we still see considerable differences in stoichiometry. For example, in Figure 1 B, compare bands 22, 23, 25 and 27 and in Figure lC, bands 13, 15, 16 and 17. We have therefore considered the means by which such differences could have arisen.

It is unlikely that the observed lack of stoichiometry is caused by the presence of partial digestion products. Our patterns have been consistent with each preparation and with increasing amounts of enzyme and digestion time. In addition, the hybridization of cloned fragments to unique bands in the digest (to be discussed) has indicated restriction was complete.

We also considered the possibility that mtDNA might be contaminated with extra-mitochondrial DNA, such as bacterial, chloroplast or maize nuclear DNA. The mitochondrial preparations were spread for bacterial contamination and found to contain no greater than lo5 bacteria/ml. At this level, any bacteria co- purified with mitochondria would contribute 0.01% to the total mass of mtDNA extracted. This quantity of bacterial DNA would not contribute significantly to the restriction patterns. In an effort to determine the degree of contamination by chloroplast or nuclear DNA we have hybridized probes for these genomes to


70 68 62 56 50 40 28 17 9 3

C

FIGURE 1.-Restriction enzyme digestion patterns of B37N mtDNA. The terminal digestion products from EcoRI (A), SalI (B) and PstI (C) cleavage of B37N mtDNA were displayed by electrophoresis on 1.5%. 0.8% and 0.8% agarose gels, respectively. The top of the gels is on the right. For reference some of the fragment numbers are given above the gel tracks. Densitometric scans of SalI and PstI digests are also shown.

mtDNA bound to nitrocellulose filters. Nick-translated chloroplast DNA did not hybridize to preparations of mtDNA (data not shown). Similarly, a cloned sequence of soybean nuclear ribosomal DNA that hybridizes to maize nuclear DNA with high fidelity (E. ZIMMER, personal communication) was used as a probe for nuclear contamination. Although intense hybridization was observed with maize nuclear DNA, none was seen with maize mtDNA, either restricted or unrestricted (K. NEWTON, unpublished). We conclude that our preparations of mtDNA are not sufficiently contaminated with foreign DNA to account for the complexity and variability in stoichiometry of mtDNA restriction patterns.

One possibility is that variation in band intensity might result from partial methylation of mtDNA, so that only some of the recognized sites would be cleaved by a particular endonuclease. Using isoschizomers that will distinguish between methylated and unmethylated DNA, we have established that mtDNA contains no detectable 5-methyldeoxycytidine (m5C) or P-methyldeoxyadenen- ine (m6A). B37N mtDNA was restricted with HpaII and MspI which recognize the tetranucleotide 5’-C-C-G-G-3’ (MA” and SMITH 1978). While MspI will cleave the sequence 5‘-C-m5C-G-G-3’ HpaII will not (WAALWIJK and FLAVELL 1978). There was absolutely no difference in the restriction profiles produced by the two enzymes (data not shown). Likewise, endonucleases DpnI and Sau3A, which cut at tetranucleotides 5’-G-m6A-T-C-3’ and 5’-G-A-T-C-3’, respectively (as quoted by ROBERTS 1978), were used to investigate possible


adenosine modification. DpnI cleaves only methylated sequences and failed to cut mtDNA Sau3A recognizes the unmethylated tetranucleotide sequence and clearly yielded a limit digest (data not shown). It is our opinion that the heterogeneity observed in the restriction digest is not the result of partially methylated DNA. Similarly, the lack of methylation in mtDNA has been reported in wheat by BONEN, HUH and GRAY (1980) and in yeast, Neurospora, rat and calf (GROOT and KROON 1979).

Properties of normal mtDNA: Table 1 presents band numbers and molecular weights computed from densitometric scans of normal mtDNA restricted with either SalI, PstI or EcoRI. Disregarding nonstoichiometry, the sum of the molecular weights of fragments yields minimum molecular weight estimates of 473,447 and 319 kb for SalI, Pst I and EcoRI, respectively. The molecular weight obtained from an EcoRI digest is undoubtedly an underestimate because of loss of numerous very small fragments not resolved in this gel system. Because we feel confident we recovered all fragments from both SalI and PstI digests, we can reasonably set a minimum molecular weight based on restriction fragment analysis for maize mtDNA as 450-475 kb. The sum of fragment molecular weights reported for SalI and Pst I are not statistically different; the apparent

22 -

7.6- 5.9- 4.8- 4

A B C FIGURE Z.-Hybridization of unique and repetitive DNA probes to restricted mtDNA. B37N

mtDNA was digested with EcoRI and electrophoresed on a gel composed of 0.8% agarose at the top and 1.5% at the bottom (panel B). Clone AZmtB37NR 26-2 (3.8 kb) was hybridized to a Southern blot and the result is shown in panel A. Clone AZmB37NR 12-2 (3.6 kb) was also hybridized to a blot of the same digest as shown in panel C. Molecular weight (kb) is shown at the left.


25-kb discrepancy is caused by the inaccuracy of assigning molecular weights to fragments above 20 kb. Fragments present in higher stoichiometry may represent repetitive sequences or subsets of amplified mtDNA. The total complexity adding in the putative multiple copies of some sequences is > 2000 kb.

Hybridization tests indicate that the majority of fragments visualized on a gel

24 -

15.3-

12.1-

7.6- I) Y

3.4-

A B C FIGURE 3.-PstI cleavage patterns of B37N, T, C and S mtDNA, left to right, respectively, (A) and

hybridization of cloned fragments (B, C). PstI digests of the four cytoplasmic types electrophoresed concurrently on a 0.8% agarose gel are shown in panel A. This gel was transferred to nitrocellulose and hybridized to cloned EcoRI fragment hZmtB37NR 12-1 as described in MATERIALS AND METHODS. The autoradiograph (B) was exposed for 7 days. Several bands hybridize in all four tracks as discussed in the text. The same filter was then washed free of probe and rehybridized to fragment AZmtB37NR 26-1. Panel C shows an 8-day exposure; one different band in each track hybridizes.


represent unique sequence information. Twenty cloned EcoRI fragments prepared from a library of clones were hybridized back to an EcoRI digest of B37N mtDNA. Seventeen cloned fragments representing 88 kb each hybridized to a different single restriction fragment in a genomic EcoRI digest; in each case the hybridized fragment had a molecular weight identical to the cloned probe (Figure 2A shows an example of this hybridization pattern). These 17 cloned fragments were present in stoichiometry from 1-13X in the genomic digest indicating that fragments present in high as well as low yield can contain sequence information present on a single restriction fragment.

Three cloned fragments hybridized to multiple bands in a genomic EcoRI digest indicating repetitive DNA. Fragment hZmtB37NR 22-1 of 8.7 kb hybridized to two bands of 8.7 kb and 9.2 kb; fragment XZmtB37NR 12-2 of 3.6 kb hybridized to three bands of 2.05 kb, 3.6 kb and 6.3 kb (Figure 2C); fragment XZmtB37NR 16-1 of 12.5 kb hybridized to three bands of 4.0 kb, 6.5 kb and 12.5 kb. The cloned fragments representing repetitive DNA segments were each of intermediate stoichiometry. However, they also hybridized to genomic fragments of lower and higher stoichiometry indicating that similar sequences can be present on fragments with different representations in a genomic digest. The repetitive sequences detected in these hybridizations represented approximately 20% of the mass of fragments hybridized, suggesting that approximately 80% of the visualized fragments represent sequence information present on only a single restriction fragment size class.

Comparison of mtDNA from normal and male sterile lines: mtDNA was prepared from normal B37 and from T, C and S male sterile cytoplasmic types

N T C S

I -

A

I -

I

6 FIGURE 4.-Schematic diagrams of Sal1 (A) and PstI (B) digests of B37N. T, C and S mtDNA.

Diagrams were drawn from enlarged photographs of Sa11 and PstI digests of all four cytoplasmic types run in parallel on 0.8% agarose gels. Fragments are numbered consecutively from the top of the gel. For reference, a dot is placed beside every tenth fragment of the N digest. Thicker lines represent more intense bands on the gel. Molecular weight (kbp) is noted beside the drawings. Bands common to mtDNA of male sterile types only are marked with arrows.


TABLE 2A

Molecular weights (in kilobasesj of Sal1 fragments of B37N, T, C and S mtDNA with notation of shared restriction fragments*

Fragment N T C S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

X MW

34.61 31.59. 27.91 26.57 25.64 22.07 20.75 19.84- 18.91 17.340 16.74 15.140 14.610 13.81 13.62' 13.22- 11.582 11.360 10.12 9.55 9.052 8.84 8.40 7.74 7.49 7.15. 6.720 6.08 5.702 5.26' 5.15' 5.02 4.84. 4.18- 3.44. 3.35 3.06 2.46' 2.11' 1.63 1.12 1.04-

484.8

31.16. 27.91 25.95 24.63 21.50

19.65- 18.820 17.410 16.670 15.210 14.73 13.64' 13.150 12.4Z4,' 11.52 11.300 10.47' 10.34 9.414 8.80 8.38- 7.845 7.74. 7.50- 7.43 7.15. 6.984 6.70- 6.50

5.44 5.02 4.820 4.18. 3.88 3.45. 2.50' 2.11' 1.65 1.40 1.04.

20.64~

5.974,5

463.1

31.80. 30.54 27.560 25.34 21.39 20.644 19.46 18.73. 17.34- 16.81 15.21 14.73 14.50 14.06 13.240

11.846 12.504*6

11.60' 11.37- 9.414 9.20 9.04' 8.75 8.36- 7.70. 7.48 7.35 7.13 6.954 6.670 5.9g4s6 5.92 5.68' 5.26' 5.14' 5.02 4.820 4.15 3.60 3.45 2.156 1.65 1.50 1.456 1.35 1.04.

494.9

31.37- 27.39. 24.49 21.50 20.85 19.65 18.82 17.73 17.26. 16.74. 16.18 15.21 14.67 14.39 13.74 13.240 12.505.6 11.846 11.30- 11.08 10.57' 9.68 8.84 8.43 7.865 7.78- 7.50- 7.20 6.75 6.18

5.63

5.19 S 2 5.10 5.00- 4.92 4.81 4.15 3.45. 2.156 1.65 1.456 1.30 1.25 1.04

5.985*6

5.25 S 1-1

489.0

in the same nuclear background. The mtDNA was restricted and electrophoresed in parallel tracks on the same gel. PstI digests are illustrated in Figure 3. For clarity, however, schematic diagrams have been drawn of both the Sal1

TABLE 2B

Molecular weights (in kilobases) of PstI fragments of B37N, T, C and S mtDNA with notation of shared restriction fragments

Fragment N T c S

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

X MW

29.382*3 26.85 24.86 23.41 22.53l 20.85 18.77 17.42l 16.39 15.110 14.18 13.57 11.7F~~ 10.81' 9.56l 9.19 8.75 8.24- 7.91 7.49 7.29. 6.65 6.5Z3 6.10 5.700

5.17l 5.06. 4 .63~

4.45. 4.40 9

423 4.191,2 4.05233 3 m 2 . 3

3.73 3.61 3.5Z2 3.25

5.342.3

4.59'

413.4

32.90 26.85 25.03 23.41 22.67l 17.51' 16.97 15.11- 13.694s5 13.23 11.914*5 11.554.5 10.33 9.62l 9.19. 8.75 8.21. 7.91 7.8Z4 7.29 7.06 6.61 6.48 6.42 5.67 5.46 5.16l 5.05. 4.91 4.65. 4.46. 4.38 4.26. 4.22' 4.16 4.01 3.93 3.84 3.69. 3.62 3.4743 3.20'

404.7

29.382s6 27.05 25.03 23.72 22.82 16.646 15.11 14.306 13.694s6 12.104-6 11.73'

10.8g2 9.22 8.80. 8.24. 7.98- 7.82' 7.29. 6.700 6.586 5.77 5.67- 5.362s6 5.216 5.06 4.650 4.612 4.43 4.38- 4.23. 4.18'

11.554*6

4.042*6 3 . d " 3.71 3.62 3.52' 3.484s6 3.20'

375.6

29.383s6 26.85 25.03 23.720 20.44 19.85 16.646 15.18- 14.43 14.306

13.17 13.695s6

12.20~.~ 11.645*6 11.09 10.97 10.01 9.280 8.80- 8.29- 7.96 7.29. 6.70- 6.616 6.533 6.01 S-1 5.72.

5.216 5.09 4.67- 4.53 4.46 4.380 4.26- 4.013~~~ 3.903,6 3.71 3.62 3.485*6 3.37

5.34336

421.9 ~ ~

Fragments shared in common between individual cytoplasmic types were determined by match- ing bands on a photograph or densitometric tracing of a gel containing samples of each cytoplasmic type in parallel tracks. Superscript numbers refer to those bands considered to have the same mobility in different cytoplasmic types: = fragment shared by all cytoplasms, 1 = fragment shared by N and T, 2 = fragment shared by N and C, 3 = fragment shared by N and S, 4 = fragment shared by T and C, 5 = fragment shared by T and S, 6 = fragment shared by C and S. Assignment of band identity in the table was made on the basis of comparison of several gel patterns; 1.5% or 1.0% gels were used to examine lower molecular weight fragments and 0.8% gels were used for higher molecular weight fragments. Table 2A was calculated from a single 0.8% gel of PstI fragments and Table 2B from a single 0.8% gel of Sa11 fragments. The S cytoplasm contains two linear DNA Fragments called S-1 and S-2 not found in the other cytoplasmic types (LEVINGS et al. 1980; THOMPSON, KEMBLE and FLAVELL 1980). These molecules and their restriction fragments are noted alongside the S column in the table.

119


(Figure 4A) and Pst I (Figure 4B) digests; molecular weights of all SalI fragments and PstI fragments greater than 3.2 kb are given in Table 2.

Measurements of total complexity from Sal I and PstI restriction fragments do not show that any obvious deletions or additions have occurred in the DNA of male steriles. The sum molecular weight of T is 16 kb less than N in SalI digests but C and S are actually larger. In PstI digests, mtDNA from male sterile cytoplasms are slightly smaller but this may not be significant. Only extensive hybridization experiments to compare sequence homology would reveal significant alterations in the DNA. However, there are two sets of bands common only to mtDNA of male steriles, those at 12.5 kb and 5.98 kb in Sal1 digests and those at 13.7 kb and 12.1 kb in PstI digests (Figure 4A,B). It is not known if the bands in SalI digests contain the same sequence(s) as those in PstI digests. In addition, the S cytoplasm has been reported to contain two linear “plasmids” (PRINC et al. 1977) which yield the fragments indicated in Table 2 (THOMPSON, KEMBLE and FLAVELL 1980).

It is immediately evident that the overall restriction patterns of the four cytoplasmic types vary considerably, but that some fragments have the same relative mobility in every track. To quantify the fraction of the genome contained in similar size restriction fragments, and hence to gain some insight into the fraction of the genome that has not undergone deletion, rearrangement or restriction site divergence, we have denoted the shared fragments in Table 2. Fragments of apparently identical molecular weight were determined after comparison of multiple gels in which limit digests of all four mtDNA types were electrophoresed in parallel tracks of a gel in which numerous molecular weight standards were also included. The molecular weight assignment was made using a computer program in which the molecular weights from a single gel pattern were calculated and fitted to a curve using the least squares method (SOUTHERN 1979).

As a further check on the homology of fragments of the same apparent molecular weight, cloned EcoRI fragments of N mtDNA represented by fragments of identical molecular weight in T, C and S mtDNA were hybridized to SOUTHERN (1975) transfers of the PstI or SalI restriction digests of N, T, C and S mtDNA. For example, we hybridized a cloned EcoRI fragment of 6.0 kb, hZmtB37NR 12-1, to a SOUTHERN transfer containing all four cytoplasmic types restricted with PstI. The probe hybridized strongly with a 27-kb fragment in all digests (Figure 3B). Bands of 8.8 and 3.7 kb, as well as other bands, have also hybridized to some extent. This is caused by the contamination of the 6.0 kb probe with fragment hZmtB37NR 12-2 (3.6 kb) which co-purified on a gel. This fragment hybridizes primarily with the 8.8- and 3.7-kb fragments of a PstI genomic digest, but it also hybridizes with additional fragments. Because clone hZmtB37NR 12-2 hybridizes with three fragments in an EcoRI genomic digest, we have concluded that the clone contains repetitive DNA. A 4.3-kb probe, ZmtB37NR 26-1 (Figure 3C), hybridized to band 15 of N (9.56 kb), band 14 of T (9.62 kb), band 24 of C (5.36 kb) and band 14 of S (11.64 kb). At least some of the sequence information of 26-1 has been retained in all of the cytoplasmic types, but it is found on different fragments in T, C and S compared with N.

Cloned EcoRI fragments totaling 40 kb have been hybridized to SOUTHERN transfers containing restricted mtDNA from N, C, T and S cytoplasmic types.


Where there were fragments of identical molecular weight (i.e., mobility) in all four types, the fragments were found to be homologous by hybridization to the probe. Therefore, it is likely that fragments of the same molecular weight contain the same information. Hybridization patterns were found to differ (as with clone 26-1) when there were no corresponding bands in all four cytoplasmic types. Fragments of identical molecular weight totaling 95 kb in PstI or SalI digests have been verified as containing some of the same sequence information in N, T, C and S by hybridization to cloned EcoRI probes; 95 kb represents about 20% of our estimated minimum molecular weight.

On the basis of identical mobility and molecular weight, 19 bands in the SalI digests and 18 in the PstI digests are shared in common among the four types. The sum molecular weight for common bands in the Sal I patterns is 247 kb and represents 50-55% of the total molecular weight recorded for each cytoplasmic type (Table 2). In the PstI digests, fragments shared in common total 175 kb or 42-47% of the fragments compared. Therefore, approximately 50% of the genome appears to be conserved in the same arrangement in all the cytoplasmic types, and thus about half may be rearranged or have lost or gained a restriction site. An additional point is that with only two exceptions (bands corresponding to N-3 and N-8 in SalI digests) each set of bands is of the same relative intensity in N, T, C and S. For example, in the PstI digests, bands corresponding to N-2 are bright, but those corresponding to N-15 are less intense (Figure 3). Whatever its basis, stoichiometric differences are conserved for the most part in each cytoplasmic type.

We have also calculated the fraction of shared fragments in each pairwise combination of mtDNA type as suggested by NEI and LI (1979). Their calculation to construct phylogenetic trees relies on the assumption that no rearrangements have occurred, a condition not met in maize mtDNA. However, as a large fraction of the genome is conserved and a large data set was available, the comparison is still of interest and significance as an index of similarity. In Table 3 the calculated fraction of shared restriction fragments and 95% confidence limits are shown. It is clear that the C cytoplasm type mtDNA is closely allied with the S type with 67% shared restriction fragments, 65% with N and 60% with T. The N and T cytoplasmic types are reasonably similar to each other with 59% shared fragments. The S mtDNA, however, is more diverged from both N and T as only 56% and 53% of the fragments are shared, respectively.

The position of C as most similar to the other three cytoplasmic types is reinforced by noting the number of unique fragments in each cytoplasmic type (Table 4). C mtDNA has only 13 of 85 unique fragments in the Sal1 and PstI digests combined whereas the others have 20 or more unique fragments. Although it is interesting to note that only 28.6 kb of the PstI digest of C mtDNA is found in unique fragments, the mass involved is a poor measure of restriction site divergence because creation of large fragments caused by restriction site loss can greatly bias the result.

DISCUSSION

Properties of maize m t D N A : By optimizing resolution on agarose gels, we have been able to assign a number and molecular weight to fragments of maize mtDNA resulting from cleavage by SalI, PstI and EcoRI restriction enzymes.

122 K. S . BORCK AND V. WALBOT

TABLE 3

Analysis of shared fragments in Pstl and Sal1 digests of N, T, C and S maize mitochondrial DNA

No. of shared fragments Total frag- Pairwise compar- ments com- Fraction of shared frag-

ison PstI digest Sa11 digest pared ments' Variance of (F)*

N, T 23 26 166 0.590 k 0.004 0.002 N, C 27 27 167 0.647 k 0.002 0.002 N, S 23 22 169 0.532 f 0.004 0.002 T, C 24 27 169 0.604 1- 0.004 0.002 T, S 22 26 171 0.561 & 0.004 0.002 c, s 30 27 171 0.667 k 0.004 0.002

"The fraction of shared fragments was calculated as described by NEI and LI (1979) using the equation F = 2nx,/(n, + ny) in which F = the fraction of shared fragments, nxy = the number of shared fragments, n, = the number of fragments analyzed in one cytoplasmic type, and n, = the number of fragments analyzed in the other cytoplasmic type. The 95% confidence interval of the Fraction (F) was calculated as described by SOKAL and ROHLF (1969).

* Variance of the fraction ( F ) of shared fragments was calculated as described by LI (1981) using the equation

V(F) = [F(1 - F) - F2(1 - dF){1 + %(I - dF))]/fi

in which ii = (n, + n,)/2.

We have also established the lack of stoichiometry that is observed in these digests as a unique property of mtDNA itself and not caused by partial methylation of the DNA, incomplete digestion or contamination.

Based on SalI and PstI restriction digests we estimate a minimum molecular weight for maize mtDNA as 450-475 kb. This sum is in agreement with calculations of 500 kb based on reassociation kinetics (PALMER and THOMPSON 1980; WARD, ANDERSON and BENDICH 1981). Our sum is higher than the 277 kb estimated from BamHI restriction digests (SPRUILL, LEVINGS and SEDEROFF 1980) but is in agreement with the sum of SalI fragments of C mtDNA reported by WARD, ANDERSON and BENDICH (1981). Hybridization of cloned EcoRI fragments to EcoRI digests of normal mtDNA has indicated that a large proportion of the mitochondrial genome is composed of unique sequences, although our estimate of 80% is not as great as the 93% previously reported (SPRUILL, LEVINGS and SEDEROFF 1980).

Several types of repetitive sequences exist in maize mitochondrial DNA. LONSDALE, THOMPSON and HODGE (1981) reported the presence of a 26-kb sequence repeated on each side of a fragment hybridizing to the S-1 or S-2 molecules. The maize mitochondrial genome does not appear to contain a large inverted repetitive DNA element as is found in the maize chloroplast genome (BEDBROOK, KOLODNER and BOGORAD 1977). Dissociation of native, unsheared mtDNA followed by neutralization and electron microscopy (DAVIS, SIMON and DAVIDSON 1971) revealed only very short (ca. 100 bp) repeated sequences widely dispersed throughout the genome (data not shown). Furthermore, reassociation experiments have not detected a highly repeated component of maize mtDNA (WARD, ANDERSON and BENDICH 1981).

Within the mitochondrial genome different fragments exist in different relative stoichiometries, a phenomenon for which there is currently no explanation. Those restriction fragments that are common to all four cytoplasmic types


appear at equivalent levels of intensity, a fact that may have some implications regarding genome organization of mitochondrial DNA. Maize mtDNA is isolated as several circular molecules found with variable frequency (LEVINGS et al. 1979; BORCK and WALBOT, unpublished observations). Thus, the high stoichiometry of some bands could be caused by repetition of a sequence on a given molecule or molecules or the high ploidy of a molecule carrying that sequence. WARD, ANDERSON and BENDICH (1981) report that maize C cytoplasm mtDNA restriction fragments fall into a simple stoichiometric series of lX, 2X and 3X. However, we find band intensities vary from 1-13X. WARD, ANDERSON and BENDICH (1981) used a C cytoplasm in the single cross FR9 x FR37 nuclear background, and we have used the inbred B37 nuclear background. There is at least one cytoplasmically inherited difference in these two C cytoplasm sources, as we report that there are 46 SalI fragments and WARD, ANDERSON and BENDICH (1981) report 47 Sal I fragments. Although WARD, ANDERSON and BENDICH (1981) did not report the size of sizes of the SalI fragments, the overall pattern is remarkabIy similar to ours with the exception of the relative stoichiometry of some fragments. These differences in relative stoichiometry may result from differences in the nuclear backgrounds or tissue composition. We have noted a change in the relative intensity of mtDNA restriction fragments in a given cytoplasm as the nuclear background is changed (BORCK and WALBOT, unpublished data).

The proportion of mtDNA isolated as circular forms depends on the tissue used for isolation of mitochondria. mtDNA isolated from coleoptile tissue of various inbred lines contains only a small percentage of total mass in circular forms (LEVINGS et al. 1979; BORCK and WALBOT, unpublished data), whereas mtDNA from callus tissue culture of Black Mexican Sweet background can be 2040% circular forms (DALE 1981). These circular forms include 1.5-kb and 1.8- kt minicircular forms found in high concentration and numerous additional circular forms that form a ladder of discrete size classes when unrestricted mtDXA is electrophoresed to separate size classes. All of the €arger circular forms hybridize to the minicircular molecules suggesting that the larger circular forms inay represent a multimeric series based on amplification of the smaller circles. Alternatively, the larger circular forms may contain both unique information as well as copies of the smaller circular forms. Attempts at electropho-

TABLE 4

Number and molecular weight (in kilobases) of unique fragments in Sal1 and Pstl digests of N, T, C and S mtDNA

PstI unique fragments % of unique fragments (sum of PstI and So11 digest) Sol1 unique fragments

Cytoplasm - type No kb No. . kb No. kb

N 12 176.7 8 100.6 24 31 T 10 118.6 13 119.7 27 28 C 11 134.8 2 28.6 15 20 S 17 184.5 10 113.9 31 34

The number of unique rekction fragments in each digest was obtained by counting the number of restriction fragments in Tables 2A and 2B for each cytoplasmic type that does not match another restriction fragment, and the inolecular weight was summed far each class of unique restriction fragments.

124 K. S . BORCK AND V. WALBOT

retic separation of size classes of unrestricted mtDNA isolated from coleoptile tissue yields no discrete bands except minicircular forms (BORCK, unpublished data). We have detected circular forms of from 1.5 kb to 168 kb by electron microscopy of similar samples. However, these circular forms constitute only about 1% of the total mass of mtDNA (BORCK and WALBOT, unpublished data). Because the circular forms are so rare it is unlikely that they could be visualized by electrophoresis of unrestricted mtDNA samples. Such circular forms will, however, contribute to the restriction pattern. If the circular forms contain a repetitive DNA sequence element or form a multimeric series of a single sequence, the circular forms would generate a limited set of restriction fragments that would be visualized as high stoichiometry fragments even though the relative concentration of circular forms is low. Differences in the relative stoichiometry of fragments between different inbred lines could represent differences in the number of circular forms containing subsets of the mitochondrial genome. Within the B37 inbred line the relative stoichiometry of fragments in the N, T, C and S cytoplasms is nearly identical. This observation suggests that nuclear background rather than cytoplasmic type could determine the relative concentration of subsets of the mitochondrial genome.

Comparison of N, T, C and S maize mtDNA: Previous studies of the extent to which the same mtDNA restriction fragments are present in normal cytoplasm and the T, C and S male sterile cytoplasm types of maize indicated a high proportion of shared restriction fragment sizes in the 200 kb of the genome analyzed (PRING and LEVINGS 1978). Utilizing higher resolution gel electrophoresis to separate a greater number of restriction fragments, we estimate that approximately 50% of the mtDNA mass is shared by all four cytoplasmic types. By analyzing the extent of shared restriction fragments in each pairwise combination of N, T, C and S utilizing the entire limit Sal1 digest and approximately 40 PstI fragments of each mtDNA type, we have detected closer relationships between some cytoplasmic types than between others.

The mitochondrial genome with the most homology to each of the other three is the C type mtDNA that shares 67% of fragments with S, 65% with N and 60% with T. Previous studies have emphasized the normal mtDNA patterns as the reference point for studying the changes in the mtDNA patterns of male sterile cytoplasm. However, it appears from our analysis that the C mtDNA may serve as a better reference genome for the interpretation of the changes resulting in the N, T and S cytoplasmic types.

The comparison of the fraction of shared fragments between pairwise com- binations of mtDNA types was performed as suggested by NEI and LI (1979). At the present time there is no physical map of maize mtDNA so it is not possible to verify that the linear order of restriction fragments has remained invariant in the four cytoplasmic types. It is possible that some rearrangement has occurred in the mtDNA of each cytoplasmic type. Hybridization results indicate that restriction site divergence has occurred and is probably the major contributor to the changes in fragmentation pattern observed in maize mtDNA (BORCK and WALBOT, unpublished data). Thus, the NEI and LI formulas can be used as an index of similarity for retention of homologous restriction fragments.

Rearrangement is known to occur in S mtDNA: In male sterile lines two linear DNAs designated S-1 and S-2 are found; these become inserted into the main mitochondrial genome in strains in which fertility has been restored by cyto-


plasmic reversion (LEVINGS et al. 1980). The S-1 and S-2 sequences were also detected by DNA hybridization with mtDNA of the N cytoplasmic type and they possibly share limited homology with mtDNA of C and T as well (THOMP- SON, KEMBLE and FLAVELL 1980). The role of these molecules in generating genomic rearrangement is not established. Our S cytoplasmic type was an unrestored strain in which the S-1 and S-2 molecules are in the unintegrated state and thus do not contribute to restriction fragment size variability by insertion into the main mitochondrial genome.

Our major observation, however, is that one of the male sterile cytoplasms, the C type, shares a significantly greater fraction of restriction fragments with the other three cytoplasmic types than does the normal mtDNA. Within the C cytoplasm group there is some restriction fragment heterogeneity, however, the percentage of shared fragments is reported to be greater than 95% for a single enzyme comparison (PRING, CONDE and LEVINGS 1980). We have only a single band difference (N = 46) in the SalI digest of C mtDNA compared to the report of WARD, ANDERSON and BENDICH (1981) in which 47 SalI fragments were found. Because they did not report the size of their SalI fragments we cannot determine the nature of their extra fragment. Although the C cytoplasm shares 67% of the fragments with S, 65% with N and 60% with T, there are large differences between C and each of the other cytoplasmic types compared to the few reported differences within the C cytoplasmic group.

The definition of N cytoplasm is purely empirical: N cytoplasms are those that have failed to segregate for male sterility in numerous crosses. Quite possibly certain N cytoplasms could result in cytoplasmic male sterility in some nuclear backgrounds. Cytoplasmic male sterility in T, C and S is noted only in the absence of nuclear restorer genes. The observation that male sterility or semifertility is a common result of wide crosses (DUVICK 1965) suggests that the concept of a normal cytoplasm is valid only within the context of a particular nuclear background.

The C cytoplasm was originally isolated from a Brazilian maize (BECKETT 1971). These C plants were shown to segregate for cytoplasmic male sterility in the maternal line when crossed by various inbred lines of maize (D. N. DUVICK, personal communication). This origin suggests that the original C plant contained a dominant nuclear restorer gene that was lost during repeated crosses to an inbred line lacking the restorer gene. The C type cytoplasm has been recovered in numerous breeding programs, probably from independent sources and nuclear restorers of C are common in modern inbred maize lines (D. N. DUVICK, personal communication).

It is premature to speculate on the evolutionary relationships among the four mtDNA types of maize, although with strict maternal inheritance and the assumption of a monophyletic origin of maize, one mtDNA type should be more closely allied with the progenitor species of modern domesticated maize than the others. Based on nuclear DNA hybridization studies, Mexican teosintes such as Balsas and Chalco appear to the closest to modern maize (HAKE and WALBOT 1980). A preliminary study of the restriction enzyme digestion patterns of mtDNA from many races of teosinte in which perhaps 200 kb of the genome could be examined indicated that considerable diversity exists within the teosintes (TIMOTHY et al. 1979). If C is the most ancestral mtDNA type, then we would predict that it would share greater homology with more distant relatives

126 K. S. BCIRCK AND V. WALBOT

of maize than do the N, T and S WtDNA types. We would also predict that the C type cytoplasm would exist in the teosintes and modern maize. This latter point has already been demonstrated in breeding programs (BECKETT 1971; D. N. DUVICK, personal communication). Further work with teosintes and other maize relatives will be required to evaluate the distribution of the C type mtDNA organization and to determine the possible derivation of the other cytoplasmic type during evolution. That the C type mtDNA differs considerably from N, T and S suggests that changes such as rearrangements, insertions and deletions rather than simply restriction site divergence may be involved in generating the different mtDNA organization patterns.

We thank E. H. COE, Jit. for introducing us to this problem. We also thank Pioneer Hi-Bred International, Inc. for seeds. The assistance of M-C. YAO with cloning experiments, D. HOISINCTON with computer analysis, W. NULTY for model E analyses, G. M. VEITH with photography, and S. MORAN with manuscript preparation is very gratefully acknowledged. We also appreciate the advice and help of Drs. K. NEW TO^ and E. A. ZIMMER. A portion of this project was carried out in the laboratory of J. R. S. FINCHAM University of Edinburgh, and we thank him for generously providing facilities. The project was supported by a grant from the National Institutes of Health.

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