The mitochondrial ATP synthase is the major enzyme responsible for the aerobic synthesis of ATP. The ATP synthase is composed of a water-soluble portion, the F-ATPase (EC 3.6.1.34), and a membrane portion, F. The F-ATPase has a subunit composition of with an overall mass of 360,000 Da(1, 2) . The recent determination of the high resolution (2.8 Å) structure of the bovine F-ATPase (3) and prior biochemical and mutagenesis studies (for review, see (4) ) indicate that the -subunits, together with small contributions from the -subunits, compose the active sites of the enzyme.

The binding change hypothesis for ATP synthesis proposed by Boyer et al.(5, 6, 7) suggests that the energy-requiring step for ATP synthesis is the release of newly synthesized ATP and not the phosphorylation step. The hypothesis proposes that three catalytic sites in F participate in the synthesis of ATP by a mechanism that involves conformational changes mediating changes in affinity of the active sites for ATP. The binding change hypothesis is supported by a number of biochemical studies (for review, see (6) ) and recently by the high resolution crystal structure of the protein (3) . The crystal structure indicates that the three active sites are not identical: one site is occupied by ADP (), one site by AMP-PNP ()(), and one site is vacant (). The differences in , , and are also demonstrated by large conformational differences in the active-site domain of each -subunit, including the structure and location of the P-loop motifs.

The P-loop motif (8, 9) is present in many nucleotide-binding proteins (9, 10) . The primary sequence of the P-loop in the -subunit of F is Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr. Crystallographic studies indicate that the backbone structure of the P-loop is the same in many nucleotide-binding proteins: p21, elongation factor Tu, myosin, RecA, adenylate kinase, and the ATPase(3, 11, 12, 13, 14, 15) . In p21, the P-loop has extensive hydrogen bonding with the -, -, and -phosphates of Mg-GMP-PNP(12) . Biochemical studies of mutants in Escherichia coli and yeast F indicate that the P-loop is critical for catalysis (16, 17, 18, 19) . Furthermore, recent studies in this laboratory indicated that the primary structural constraints of the P-loop in the -subunit of the yeast ATPase correspond very well with the known structure of p21(19) . As revealed by the crystal structure of F, the P-loop is in a dramatically different conformation in as compared with and , which suggests that the conformation of the P-loop may explain the vacancy of for nucleotides. As such, changes in the position, geometry, and structure of the P-loop of the mitochondrial ATPase may, in part, be responsible for changes in the biochemistry of the active site during the catalytic reaction cycle.

This study was initiated to identify residues in the -subunit that interact with the P-loop. Intragenic suppressors were isolated in strains with mutation codons coding for residues in, and immediately adjacent to, the P-loop of the -subunit of F. The primary structural constraints on these residues were postulated to be determined by steric interactions with other residues in the enzyme (19) . This study has identified critical interacting residues in the -subunit that appear to be important conformational links to the P-loop in the active site of the enzyme. These interactions may be important in the catalytic mechanism for the transition from the high to the low affinity conformation, or they may be important for modulating the activity of the enzyme to suit the needs of the particular organism.

MATERIALS AND METHODS

Strains and Growth Media

The following media were used for the growth of the yeast strains: YPD (1% yeast extract, 2% peptone, 2% glucose), YPG (1% yeast extract, 2% peptone, 3% glycerol), and SD (0.67% yeast nitrogen base without amino acids, 2% glucose). SD medium was supplemented with appropriate auxotrophic requirements at 20 mg/liter.

Isolation of Intragenic Suppressors

Natural Selection Method

Mutagenesis with Ethyl Methanesulfonate

Polymerase Chain Reaction Random Mutagenesis

Site-directed Mutagenesis

Construction of the Val-198 Mutants with L390F

Miscellaneous Procedures

Figure 3: Interaction pathway proposed for suppressor P353L. The bovine numbering system was used in Fig. 3Fig. 4Fig. 5since the figures are derived from the coordinates of the crystal structure of the bovine F-ATPase(3) . A, P353L (Pro-320) is proposed to disrupt the hydrogen bond of Asp-348 (Asp-315) with Arg-370 (Asp-337), which disrupts the hydrogen bond of Arg-370 with the P-loop main chain carbonyl. This structure is shown in the conformation with bound Mg-ADP. This structure does not differ much from the conformation. B, shown is the region corresponding to A in the conformation. The regions were superimposed using QUANTA Version 4.1.1 (29) as described under ``Materials and Methods.'' The perspective shown is the same as that in A. The relative positions of the P-loop, Pro-353, Asp-348, and Arg-370 are quite different in the conformation as compared with the conformation, with shifts of 10 Å.

Figure 4: Interaction pathway proposed for suppressor T237I. Thr-237 (Ser-203) interacts with Lys-209 (Lys-175) in the conformation (A), but not the conformation (B). Mutation T237I is proposed to alter the conformation of the P-loop via changes in the position or conformation of helix B. As described for Fig. 3, the regions were superimposed in the and conformers, and identical perspectives are shown in A and B. Also shown is bound Mg-ADP in the conformation (A).

Figure 5: Interaction pathway proposed for suppressor L390F. The relative positions of Leu-390 (Ile-357) and Ala-181 (Ala-147) in the (A) and (B) conformations are shown. Leu-390 is located in -strand 9 of the nucleotide-binding domain. The crystal structure indicates that Leu-390 interacts with Ala-181. The mechanism of suppression is suggested to occur through the interactions of L390F with Ala-181, which distorts strand 3 and alters the conformation of the P-loop. These interactions are not largely different in the (A), , and (B) conformations despite large conformational changes in the region. As described for Fig. 3, the regions were superimposed in the and conformers, and identical perspectives are shown in A and B. Also shown is bound Mg-ADP in the conformation (A).

E. coli cells were transformed by electroporation as described (Bio-Rad). Yeast cells were transformed by electroporation (30) and by the LiAc/polyethylene glycol method(31) .

All DNA sequencing analysis was performed using dideoxynucleotides (32) , Sequenase Version 2 (Amersham Corp.), and S-dATP. Autoradiography was performed with Kodak XAR-5 film.

RESULTS AND DISCUSSION

The primary structural constraints of residues in the -subunit of the yeast mitochondrial ATPase P-loop suggested that this P-loop has the same geometry as those of other nucleotide-binding proteins, such as p21(19) . Comparative analysis of the structure of the p21 P-loop with GMP-PNP bound (12) with that of bovine (3) indicated that these structures were nearly superimposable, with a root mean square deviation of 0.41 Å. In addition, the nucleotide and the Mg ion are nearly superimposed in the structures of p21 and . These results suggest that the extensive hydrogen bonding pattern of the p21 P-loop with the nucleotide (12) is also present in the bovine F-ATPase. Thus, small changes in the conformation of the P-loop may disturb the hydrogen bonding and thereby alter the chemistry of the active site.

The initial goal of this study was the isolation of intragenic suppressors of mutations at or adjacent to the P-loop, specifically at residues 192 and 194 and residue 198. ()These residues, as are all residues in the P-loop, are completely conserved between -subunits of all F-type ATPases in the SWISS-PROT data base. ()Residues 192, 194, and 198 were postulated to interact sterically with other regions of the ATPase and thus may be important for the conformational changes observed in the catalytic sites during the reaction cycle(19) . The steric interactions need not be limited to the most stable structure of the enzyme, but may occur during the reaction cycle, as in the transition state of the enzyme. Analysis of the crystal structure of bovine F indicates that residues 192 and 194 do not interact with any residues outside the P-loop, while residue 198 may interact with the adenine ring of the bound nucleotide(3) .

Table 1shows growth phenotypes of yeast with various replacements at positions 192, 194, and 198 in the -subunit of the ATPase. The results described in Table 1suggest that the size of the amino acid side chain is an important determining factor in the function of the enzyme. Particularly, position 198 appears to require a side chain that is about the size of Val. If the side chain is too small, such as Ala or Ser, or too large, such as Met or Lys, the enzyme is defective, and with Gly at this position, the enzyme is inactive. The crystal structure of bovine F indicates that the side chain of Val-198 is 3.8 Å from the adenine ring of the nucleotide. Thus, Val-198 may serve to position the nucleotide or to stabilize the binding of the nucleotide. In comparison, Tyr-378 is also 3.8 Å from the adenine ring and serves as a major determinant in stabilizing nucleotide binding(33) . As such, V198S would have a diminished ability to act in this manner, and suppression of V198S would need to compensate for this loss.

The requirements at positions 192 and 194 are not so apparent. However, a large number of replacements at these positions give dysfunctional phenotypes (temperature or cold sensitivity or slow growth), suggesting that the residues are not involved directly in the catalytic mechanism. Instead, these residues may serve structural roles or roles in the conformational coupling of the enzyme.

The dysfunctional mutants listed in Table 1were used to isolate intragenic suppressors. Because suppressors may be allele-specific, attempts were made to isolate suppressors from each of the mutants listed as dysfunctional in Table 1. Of these mutants, only A192V, V194Y, V194M, and V198S had strong enough phenotypes to isolate suppressors and provided a reversion or suppressor rate that allowed their isolation. Intragenic suppressors were isolated by a combination of methods as described under ``Materials and Methods'' and as summarized in Table 2. The number and type of intragenic suppressors isolated were very limited. For mutations V194Y and V194M, only suppressors T237I and P353L were isolated, respectively. For V198S, L390F was the only suppressor mutation identified, whereas for A192V, only revertants were isolated. This low number of intragenic suppressors may be due to limitations imposed by the genetic code or due to a severe limitation on the number of replacements that will suppress these mutations.

Fig. 1shows the growth phenotypes of the original mutants, the mutants with a suppressor mutation, and the suppressor mutations in a wild-type background. Despite the fact that the suppressors were isolated at one temperature, all of the suppressor mutations complemented the defective growth phenotype at 18 and 30 °C, while only T237I was effective at 37 °C. These data indicate that the suppressor mutations do not generally provide an enzyme with completely wild-type properties.

Figure 1: Growth phenotypes of the mutants and suppressors. A shows the key to the plates shown in B-D. Cells were diluted in water, placed on YPG medium, and incubated at 18 °C (B), 30 °C(C), and 37 °C (D). The first row shows the cells with the wild-type (wt) and null mutations in ATP2. For the second through fourth rows, the first columns show the phenotypes of the initial mutants, the second columns show the phenotypes of the initial mutant with the suppressor mutation, and the third rows show the phenotypes of the suppressor mutation in a wild-type background. The fifth rows show the growth phenotypes of L390A and L390G in an otherwise wild-type background. One-letter amino acid abbreviations are used to indicate the amino acid encoded by the codon.

If the amino acid residue corresponding to the suppressor forms a critical interaction with another residue, it would be expected that mutagenesis of this residue in an otherwise wild-type background would give a defective phenotype. This was tested by separating the suppressor mutation from the original P-loop mutation (Fig. 1). Suppressor L390F was defective at all temperatures, indicating that Leu-390 is an important residue in the F-ATPase. In contrast, T237I and P353L showed only moderate effects on the growth phenotypes, indicating the replacements modify, but do not eliminate, the enzyme activity.

Another indicator of the critical importance of Leu-390 was demonstrated by site-directed mutagenesis of Leu-390 to Ala and Gly. Fig. 1shows that strains with L390A or L390G have a negative growth phenotype on glycerol medium. These results indicate that Leu-390 makes important contacts with at least one other residue. Identification of the P-loop suppressor mutant L390F indicates that this interaction can be transmitted to the P-loop in the active site of the enzyme.

The allelic specificity of suppressor L390F was tested since this specificity should provide some information on the mechanism of the suppressor mutation. Specifically, the suppressor should show allelic specificity if it acts by reversing the effect of the primary mutation. This is in contrast to a mechanism whereby a suppressor improves the overall state of the enzyme, thereby overcoming the dysfunctional defect. Fig. 2shows the growth phenotypes of the L390F mutation in the presence of 10 different residues at position 198. The results indicate that the suppressor mutation is allele-specific. At 30 °C, a nearly wild-type growth phenotype for L390F was observed with Cys, Lys, Met, or Thr at position 198. However, at all three temperatures, L390F suppressed only Cys or Thr at position 198. Gly-198 was nonfunctional with either Leu or Phe at position 390. These data support the previous conclusion that Val-198 makes critical steric interactions with another residue or with the bound nucleotide.

Figure 2: Allelic specificity of the Phe-390 suppressor. A shows the key to the plates shown in B-D. Cells were grown as described for Fig. 1. The first row 1 shows the cells with the wild-type (wt) and null mutations in ATP2. The second through fourth rows show the phenotype of the initial mutant, V198S, in an otherwise wild-type background, followed by cells with mutations at position 198 with the L390F suppressor mutation.

Interaction Pathways for P353L

The hydrogen-bonding pattern of Asp-348 with Arg-370 and of Arg-370 with Ala-192 is not present in the conformation (Fig. 3B). In the and conformations, Arg-370 is 3.0 Å from the carbonyl oxygen of Ala-192, while this distance increases to 4 Å in the conformation. This shift of 1 Å is sufficient to break the hydrogen bond between Arg-370 and Ala-192. More dramatically, Asp-315 is 12.6 Å from Arg-370 in the conformation, while they hydrogen-bond in the conformation. Thus, these interactions are specific for the conformations with nucleotide bound to the active site and may be important for stabilizing the and conformations. This hypothesis is supported by the fact that Pro-353 is in a catch region that interacts with the -subunit in the conformation (3) . Furthermore, Pro-353, Arg-370, and Asp-348 are conserved in all 56 sequences of F-type ATPases in the SWISS-PROT data base, suggesting that these residues are critical.

Mutagenesis of the residue that corresponds to Asp-348 in E. coli to Val (D301V) resulted in an enzyme that was defective in assembly of the ATPase complex(34) . Therefore, this residue is important, minimally, for forming a stable structure of the ATPase. There have been no reports on mutagenesis of residues corresponding to Pro-353 or Arg-370, but it is of interest to determine their roles in the structure and function of the ATPase. The identification of a suppressor mutation in the catch region that suppresses a mutation in the P-loop demonstrates genetically that the two regions are conformationally coupled. Further studies are required to determine conclusively if Pro-353, Arg-370, and Asp-348 are critical in the conformational coupling cascade during the catalytic cycle.

Interaction Pathways for T237I

Lys-209 is not a well conserved residue, but the size of the residue appears to be important (cf.Fig. 6). In E. coli, for example, the corresponding residue is Ile. This indicates that the charged group is not critical, but the steric interactions may be important. Replacement of Lys-209 with Val in yeast results in an enzyme that has a 3-fold increase in the K for ATP and GTP and a 3-fold decrease in the k for ATP binding. However, there was no change in the V for ATP hydrolysis as compared with the wild-type enzyme. ()This single change to a residue that is similar to that found in the E. coli enzyme significantly modified the kinetics of the yeast enzyme. Possibly, Ile at this position in the E. coli enzyme can account for some of the biochemical differences observed between the E. coli and mitochondrial enzymes(35, 36, 37) .

Figure 6: Corresponding residues from a number of species and enzymes (see Footnote 3) that are proposed to interact. The residues that correspond to Lys-209 and Thr-237 are shown in A, and residues that correspond to Ala-181, Leu-390, and Ser-161 are shown in B. Sequences were aligned using MaxHom (28) and the SWISS-PROT data base. The primary sequences of the -subunit of the ATPase were ordered into mitochondrial, bacterial, blue-green algae (BG), and chloroplast sequences, as shown. There were only partial sequence data available for sequence 27, and the space indicates that the sequence is not known. The dot in sequence 34 (A) indicates that the MaxHom program did not align this residue with the data base sequences.

The interaction of Thr-237 with Lys-209 is proposed to be important for modulating the activity of the enzyme. The importance is supported by the following data. First, T237I suppresses a P-loop mutation and thus must be able to alter the conformation of the active site. Since the P-loop forms multiple hydrogen bonds with the nucleotide, changes in the conformation of the P-loop have the potential of altering the hydrogen-bonding network. Second, T237I in an otherwise wild-type background is defective at 18 and 37 °C, but not at 30 °C as compared with the wild-type strain (Fig. 2). This indicates that the T237I interaction in the wild-type background modifies, but does not eliminate, the activity of the enzyme. Finally, mutagenesis of Lys-209 to Val has significant effects on the biochemistry of the ATPase. The variations of the residues at these two positions are proposed to modify the activity of the enzyme for the needs of the organism.

Interaction Pathways for L390F

Fig. 6indicates that residues corresponding to Ala-181 and Leu-390 in other species or enzymes may, in part, define biochemical differences between these enzymes. The putative interacting residues can be placed into two groups: the mitochondrial or bacterial and the blue-green algae or chloroplast ATPases. The exact identity of the corresponding residues is variable, but they are generally limited to hydrophobic interactions, such as Ile with Ala, Ala with Ile, or Gln with Met. For blue-green algae or chloroplast ATPases, the pair is limited to Arg (or Lys in one case) and Met. It is not clear how an Arg/Met pair could fit in the same space as the Ala/Ile pair in the structure of the bovine enzyme. However, Asp or Glu at position 161 is always coincident with Arg at position 181, and the carboxylate may form a salt bridge with the guanidinium group of Arg. These differences in primary sequence may provide unique properties to the chloroplast and blue-green algae enzymes(38, 39) . Mutagenic and biochemical studies on these residues in the yeast or bacterial enzyme should provide insight into the function of this putative salt bridge.

Although suppressor mutations of mutants in the P-loop of the E. coli ATPase have been reported(40, 41) , there are a number of important differences compared with this study. In the E. coli study, the initial mutations were in Gly-149 (Gly-190 in yeast). In the bovine enzyme, this residue has very unusual dihedral angles that only allow Gly at this position(3) . Assuming that the geometry of the P-loop is the same for the E. coli enzyme, then the geometry of the P-loop must be altered in the mutations at this residue. Suppressors of these mutations may be limited to those that can have a broad effect on the conformation of the P-loop. Furthermore, the primary structural constraints of the E. coli P-loop are different from those of yeast. Although there has not been an extensive examination of the P-loop constraints in E. coli, Ser can replace Gly-149, and mutant F has nearly normal activity(35, 36) . This is in contrast to the yeast enzyme, where the corresponding mutation, G190S, severely impairs F activity(19) . A third important difference is that the primary sequence of the -subunit from E. coli is more divergent from bovine than is yeast. The biochemical differences between the E. coli and mitochondrial enzymes must certainly be defined by the differences in their primary structures. However, primary structural differences may also change possible replacements that could suppress any given mutation, such as mutations in the P-loop mutations. For these reasons, the suppressors identified in the E. coli enzyme need not correspond to those identified in yeast.

The suppressors of mutations in the P-loop in the E. coli studies were G172D, S174F, D192V, and V198A. These residues are located at the beginning of -sheet 4, in -sheet 4, in helix C, and at the end of helix C, respectively. Interestingly, these suppressors are clustered and are in the region of the yeast P-loop suppressor T237I. The clustering of these suppressor mutations provides additional evidence that helix C and -sheet 4 affect the conformation of the P-loop and thus the biochemistry of the enzyme.

Although some residues may diverge because their importance is relatively minor in defining the biochemistry of an enzyme, there must be other residues that diverge due to the different requirements of the organism or organelle. Suppressor studies may be an important tool for the identification of residues that are important in determining the biochemistry of the enzyme. The suppressors identified in this study all originated from mutations that were in or near the P-loop of the mitochondrial ATPase. As such, they all are able to correct a defect located in this critical motif. Since two of the putative interacting pair of residues are not strictly conserved between species and enzymes, Thr-237/Lys-209 and Ala-181/Leu-390, it is suggested that this divergence can be responsible, in part, for their biochemical differences. Of course, there are five different subunits that compose the F ATPase, each with divergent residues. Any, or many, of these differences may contribute to the biochemical differences observed between enzymes. However, certainly not all of the residues that are divergent are important for modulating the kinetics of the enzyme, as is postulated for the interacting pairs Thr-237/Lys-209 and Ala-181/Leu-390.

The P-loop undergoes dramatic conformational changes in the or to the conformation ( (3) and Fig. 3Fig. 4Fig. 5) and possibly in the transition from the high to the low affinity site in the catalytic mechanism. The understanding of the conformational coupling pathway from the proton, to F, to F, to the catalytic site requires molecular details of essential residues that interact and trigger this transition. Suppressor studies may be an important tool to identify interacting residues, such as Asp-348 and Arg-370, that are key components in the conformational coupling pathway.

Summary

FOOTNOTES

*

This work was supported in part by Grant R01GM44412 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Mineral Metabolism Div., Loma Linda University, Loma Linda, CA 92357.

¶

Supported in part by a grant from the Autonomous National University of Mexico.

**

To whom correspondence should be addressed: Dept. of Biological Chemistry, Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 847-578-8606; Fax: 847-578-3240; muellerd{at}mis.finchcms.edu.

()

The abbreviations used are: AMP-PNP, adenosine 5`-(,-iminotriphosphate); GMP-PNP, guanosine 5`-(,-iminotriphosphate).

()

The corresponding residues of the -subunit of F-ATPase from bovine () heart are as follows: Ser-161 (Ser-127), Ala-181 (Ala-147), Ala-192 (Ala-158), Val-194 (Val-160), Val-198 (Val-162), Lys-209 (Lys-175), Thr-237 (Ser-203), Asp-348 (Asp-315), Pro-353 (Pro-320), Arg-370 (Arg-337), Tyr-378 (Tyr-345), and Leu-390 (Ile-357).

()

The species and SWISS-PROT accession numbers are as follows: 1) Bos taurus (bovine), P00829[GenBank]; 2) Rattus norvegicus (rat), P10719[GenBank]; 3) Homo sapiens (human), P06576[GenBank]; 4) Drosophila melanogaster (fruit fly), Q05825[GenBank]; 5) Rhodospirillum rubrum, P05038[GenBank]; 6) Rhodopseudomonas blastica, P05440[GenBank]; 7) Schizosaccharomyces pombe (fission yeast), P22068[GenBank]; 8) Neurospora crassa, P23704[GenBank]; 9) Saccharomyces cerevisiae (bakers' yeast), P00830[GenBank]; 10) Chlamydomonas reinhardtii, P38482[GenBank]; 11) Daucus carota (carrot), P37399[GenBank]; 12) Hevea brasiliensis (Para rubber tree), P29685[GenBank]; 13) Nicotiana plumbaginifolia (leadwort-leafed tobacco), P17614[GenBank]; 14) Oryza sativa (rice), Q01859[GenBank]; 15) Zea mays (maize), P19023[GenBank]; 16) Chlorobium limicola, P35110[GenBank]; 17) Cytophaga lytica, P13357[GenBank]; 18) Bacteroides fragilis, P13356[GenBank]; 19) Lactobacillus casei, Q03234[GenBank]; 20) Bacillus firmus, P25075[GenBank]; 21) B. firmus, P22478[GenBank]; 22) Bacillus megaterium, P12698[GenBank]; 23) Bacillus subtilis, P37809[GenBank]; 24) thermophilic bacterium PS-3, P07677[GenBank]; 25) Bacillus caldotenax, P41009[GenBank]; 26) Mycoplasma gallisepticum, P33253[GenBank]; 27) Streptococcus downei (Streptococcus sobrinus), P21933[GenBank]; 28) Vibrio alginolyticus, P12986[GenBank]; 29) Escherichia coli, P00824[GenBank]; 30) Thiobacillus ferrooxidans (sulfur-metabolizing), P41168[GenBank]; 31) Pectinatus frisingensis, Q03235[GenBank]; 32) Propionigenium modestum, P29707[GenBank]; 33) Synechococcus sp. (strain PCC 6716), Q05373[GenBank]; 34) Anabaena sp. (strain PCC 7120), P06540[GenBank]; 35) Synechocystis sp. (strain PCC 6803), P26527[GenBank]; 36) Synechococcus sp. (strain PCC 6301), P07890[GenBank]; 37) Dictyota dichotoma (chloroplast), P30158[GenBank]; 38) Pylaiella littoralis (chloroplast), P26532[GenBank]; 39) Galdieria sulphuraria (cyanidium caldarium), Q08807[GenBank]; 40) Chlamydomonas reinhardtii (chloroplast), P06541[GenBank]; 41) Euglena gracilis (chloroplast), P31476[GenBank]; 42) Chlorella ellipsoidea (chloroplast), P32978[GenBank]; 43) Angiopteris lygodiifolia (turnip fern), P28250[GenBank]; 44) Marchantia polymorpha (liverwort; chloroplast), P06284[GenBank]; 45) Aegilops columnaris and Aegilops crassa, Q01396[GenBank]; 46) Triticum aestivum (wheat; chloroplast), P20858[GenBank]; 47) Hordeum vulgare (barley; chloroplast), P00828[GenBank]; 48) Oryza sativa (rice; chloroplast), P12085[GenBank]; 49) Zea mays (maize; chloroplast), P00827[GenBank]; 50) Isum sativum (garden pea; chloroplast), P05037[GenBank]; 51) Spinacia oleracea (spinach; chloroplast), P00825[GenBank]; 52) Cuscuta reflexa (Southern Asian dodder; chloroplast), P30399[GenBank]; 53) Ipomoea batatas (sweet potato; batate), P07137[GenBank]; 54) N. plumbaginifolia (leadwort-leafed tobacco; chloroplast) and Nicotiana bigelovii (Bigelov's tobacco; chloroplast), P26529[GenBank]; 55) Nicotiana tabacum (common tobacco; chloroplast), P00826[GenBank]; 56) Nicotiana rustica (Aztec tobacco; chloroplast), P26530[GenBank]; and 57) Nicotiana sp. (strain 92; tobacco; chloroplast), P26531[GenBank].

()

V. Bulygin, unpublished results.

ACKNOWLEDGEMENTS

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