Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.     

H-2 Effects on cell-cell interactions in the response to single non-H-2 alloantigens

Mice expressing mutant H-2Kb alleles were tested for their ability to generate cytotoxic effector T-cells specific for the non-H-2 histocompatibility alloantigen H-4.2. Cytotoxic effectors specific for H-4.2 are preferentially restricted by the Kb allele. Mutant Kb alleles were observed to differentially regulate the magnitude of the H-4.2-specific cytotoxic effector response. Mice expressing the Kbm5, Kbm6, Kbm7, and Kbm9 alleles generated cytotoxic T-cells to the same level as mice expressing the wild-type Kb allele. Kbm8 and Kbm11 responders generated intermediate levels of effectors, whereas Kbm1, Kbm3, and Kbm10 responders did not generate detectable levels of cytotoxic effectors. Kbm4 responders produced high levels of H-4.2-specific cytotoxic effectors that were variably reactive with wild-type Kb antigens with no H-4.2. The ability to generate H-4.2-specific effectors generally correlated with (1) the ability of mutant Kb molecules to present H-4.2 to wild-type Kb-restricted effectors, and (2) the position of the respective amino acid interchanges on the Kb molecule. Mutations that altered the amino acid sequence in the vicinity of the disulfide bond in the C1 domain had the greatest deleterious effects on Kb-controlled responsiveness to H-4.2. The only exception was the Kbm11 intermediate responder, which differs from Kbm3 in both responsiveness and in a single amino acid interchange. Therefore, the amino acid sequence in the vicinity of the disulfide bond in the C1 domain plays a prominent role in determining the H-4.2-specific immune response potential. These observations are the first to clearly demonstrate association between particular MHC gene product, amino acid sequences and immune responsiveness.     

Clonal analysis of H-2Kb + TNP recognition by T cells with the use of H-2Kbm mutants and H-2Kb-specific monoclonal antibodies

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.     

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.     

Changes in the coding sequence of the H-2Dk gene of metastatic cells that might account for immunogenic abnormality of its encoded antigen.

In previous studies we have demonstrated that metastatic cells, derived from T-10 fibrosarcoma, express an immunogenically abnormal H-2Dk glycoprotein which is involved in manifesting their metastatic phenotype. In the present study we show that these cells contain a remarkably high level of H-2Dk specific mRNA. Moreover, by cloning cDNA of this gene and analyzing its nucleotide sequence, we found 4 single nucleotide changes. Two of them did not change the encoded amino acids, whereas the others resulted in two amino acid substitutions in the alpha-2 domain of the protein product that might account for its immunogenic abnormality.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships

Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.     

The DNA sequence of the H-2kb gene: evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibilty antigens

We have determined the DNA sequence of the H-2Kb gene of the C57B1/10 mouse. Comparison of this sequence with that of the allelic H-2Kd shows surprisingly that the exons have accumulated more mutations than their introns. Moreover, many of these changes in the exons are clustered in short regions or hot spots. Additional comparison of these sequences with the H-2Ld and H-2Db sequences shows that, in several cases, the altered sequence generated at the hot spot is identical to the corresponding region of a non-allelic H-2 gene. The clustered changes are responsible for 60% of the amino acid differences between the H-2Kb and H-2Kd genes and suggest that micro-gene conversion events occurring within the exons and involving only tens of nucleotides are an important mechanism for the generation of polymorphic differences between natural H-2 alleles.     

Cross-reactive recognition of mouse cells expressing the bm3 and bm11 mutations within H-2Kb by H-2Kb-restricted herpes simplex virus-specific cytotoxic T lymphocytes

Cytotoxic T lymphocytes, generated in C57BL/6 mice in response to herpes simplex virus type 1 (HSV) and known to be restricted in their recognition of HSV-encoded antigen(s) in association with the class I H-2Kb gene product, were consistently found to contain a subpopulation that recognized and lysed uninfected, SV40-transformed cells that expressed the H-2Kbm3 and H-2Kbm11 mutant class I gene products on their cell surface. The mutant cell lines, designated Lgbm3SV and Kbm11SV, share a common amino acid substitution at position 77, with the bm3 mutation having an additional amino acid substitution at position 89. Cross-reactive lysis was observed only after in vivo priming with HSV, suggesting an important role for an antigen-dependent driving step in the expansion of these cross-reactive CTL. The phenotype of the cross-reactive effector population was further confirmed as a T lymphocyte by negative-selection techniques. Limiting dilution analysis of the frequency of cross-reactive CTL precursors suggested that cross-reactivity was mediated by a subpopulation of HSV-specific CTL, and this was confirmed by clonal analysis of the reactivity patterns of short-term, HSV-specific CTL clones. However, analysis of the specificity of the cross-reactive CTL population by cold-target inhibition of bulk culture-derived CTL, or by Spearman ranking analysis of limiting dilution-derived CTL, indicated that the specificity of the cross-reactive population for HSV-infected H-2b target cells and for uninfected bm3 or bm11 target cells was quite distinct. These findings suggested that the cross-reactive CTL population played little, if any, role in the HSV-specific CTL response as measured in vitro. The findings also suggested that the HSV-specific CTL clones able to mediate cross-reactive recognition of the bm3 and bm11 targets had a higher intrinsic avidity for the foreign target than for the inducing antigen.     

Characterization of the spontaneous mutant H-2Kbm29 indicates that gene conversion in H-2 occurs at a higher frequency than detected by skin grafting.

A spontaneous mutation of H-2Kb, Kbm29, was discovered among the progeny of F1 hybrid parents. Unlike other characterized spontaneous class I variants, this mutant was detected with the use of antibody, rather than tissue grafting. Although Kbm29 is serologically indistinguishable from the previously described mutant molecule Kbm3, it is identical to the parental Kb by skin grafting and CTL assays. A full length cDNA of Kbm29 was amplified by polymerase chain reaction with locus-specific primers, cloned, and sequenced. Two nucleotides were found to be mutated, resulting in a single amino acid change (Lys----Ala) at amino acid 89 of the mature glycoprotein. This is consistent with the observed serologic changes, as the same amino acid substitution is responsible for the serologic profile of Kbm3. The occurrence of a mutation which is not detectable by the methods normally used to screen for H-2 mutants provides evidence that the high spontaneous rate of structural mutation described for the Kb molecule is underestimated.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

Primary structure of the H-2Db alloantigen

The complete amino acid sequence of the CNBr fragment comprising residues 229–284 of the murine major histocompatibility complex antigen H-2D^b has been determined using radiochemical methodology. The sequence was determined by N-terminal sequence analysis of the intact CNBr fragment and by sequence determinations of peptides derived from this fragment by trypsin and staphylococcal V8 protease cleavage. In addition to the amino acid assignments for H-2D^b, it was possible to assign the linkage position of the third N-linked glycosyl unit to the asparagine at residue 256. Additional amino acid sequence assignments have also been made for three other CNBr fragments that span residues 99–138, 139–228, and 308–331 of the H-2D^b molecule. The total protein sequence information available (222 of 338 residues) agrees in every comparable position with the protein sequence derived from the cDNA clone (pH203) isolated by Reyes and co-workers (1982b), which strongly suggests that this clone encodes H-2D^b. Combination of the protein sequence with that deduced from the cDNA clone provides the complete H-2D^b protein sequence. Comparison of this sequence with other available protein sequence information for murine class I molecules has revealed protein sequences that may be unique to either K or D region molecules.Abbreviations used in this paper HPLC high performance liquid chromatography - V8 Staphylococcus aureus V8 protease - MHC major histocompatibility complex     

Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen.

Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.     

The BM12 mutation and autoantibodies to dsDNA in NZB.H-2bm12 mice

Molecular and genetic tools have been used to shed light on the genes that contribute to susceptibility to murine lupus and the mechanisms that lead to immunopathology. The MHC genes and their products have been consistently shown to contribute toward the development of disease. To understand the contribution of MHC-class II genes, our laboratory had derived two inbred strains of mice, NZB.H-2bm12 and NZB.H-2b. These new colonies of mice were studied and compared in the 10th generation backcross; inbreeding was serially followed by H-2 typing, responses to beef/porcine insulin, and the presence of the B6 Ig allotype, IgG2ab. Of great interest is the finding that NZB.H-2bm12, in contrast to NZB.H-2b or NZB (H-2d), mice develop high titer autoantibodies to dsDNA. This result is unique because NZB (H-2d) mice, unliked NZB x NZW (NZB/W F1) or NZB x SWR (SNF1) hybrids do not develop autoantibodies to dsDNA, even after immunization. NZB mice, in contrast, are characterized only by autoantibodies to ssDNA. Our observation is also striking because the gene conversion that resulted in the I-A beta bm12 mutation occurred at amino acid residues 68, 71, and 72 of I-E beta b. Recently the contribution of NZW to accelerated autoimmunity in the NZB x NZW F1 hybrid has also been linked to H-2 and a single amino acid change at amino acid 72 of I-E beta. Thus, amino acid residue 72 may be a hot spot for disorders of immune regulation when superimposed on the appropriate genetic background. NZB mice expressing the I-Abm12 mutation will allow specific dissection of the requirements for autoantibody production to dsDNA uncomplicated by heterozygosity.     

Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells.     

The molecular basis of sulfonylurea herbicide resistance in tobacco

The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide-resistant mutants, C3 and S4-Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro-Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4-Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4-Hra mutant includes a Pro-Ala substitution at amino acid 196 and a Trp-Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.     

Structural basis of Kbm8 alloreactivity. Amino acid substitutions on the beta-pleated floor of the antigen recognition site

We have analyzed the functional significance of the four amino acid differences between the parental H-2Kb and mutant H-2Kbm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2Kb variants was analyzed using monoclonal antibodies which bind to well-defined H-2Kb epitopes. No alterations introduced into the conformational structure of H-2Kb by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2Kb anti-H-2Kbm8, H-2Kbm8 anti-H-2Kb, or third party anti-H-2Kb. The alloreactivity between H-2Kb and H-2Kbm8 is dominated by the amino acid substitution at residue 24 (Glu----Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr----Phe). The H-2Kbm8 anti-Kb bulk CTL reacted with both variant H-2Kbm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2Kbm8 anti-Kb CTL clones indicated the complexity of the self Kbm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met----Ile) and/or 30 (Asp----Asn) did not impact significantly on the alloantigenic properties of Kbm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2Kb and H-2Kbm8.     

Biochemical studies on the H-2K antigens of the MHC mutantbm1

Biochemical analysis of the H-2K-gene product from the MHC mutant strainbml and from the C57BL/6 parent strain has been carried out in order to characterize the structural differences between parent and mutant K-gene products. Based on comparative tryptic peptide mapping of the cyanogen bromide fragments from these glycoproteins, two peptide differences were localized to the CN-Ia fragment. Partial amino-acid sequence analysis revealed two alterations in the primary structure of K^bml involving substitutions of tyrosine for arginine at position 155, and tyrosine for leucine at position 156. Both of these amino-acid replacements require a minimum of two nucleotide base changes at the nucleic acid level. These changes were the only alterations noted differentiating the K^bml and K^b glycoproteins. However, because our techniques allow us to analyze only 75 to 80 percent of the extra cellular portion of H-2K^b, it is possible there are other undetected changes. Nonetheless, the biochemical data are consistent with the hypothesis that the structural alterations noted in the K^bml mutant glycoprotein are directly related to the observed immunological specificity relative to the parent K^b molecule. Peptide comparisons of the K^b molecules of two C57BL/6 sublines and of the H-2^b lymphoblastoid cell line, EL-4, disclosed no difference.     

Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance

The amino acid substitutions in subunit 6 of the mitochondrial ATPase complex have been determined for 4 oligomycin resistant mutants of Saccharomyces cerevisiae. The data were obtained for each mutant by nucleotide sequence analysis of the mitochondrial oli2 gene. Amino acid substitutions conferring oligomycin resistance in subunit 6 are located in two conserved regions that are thought to form domains which span the inner mitochondrial membrane. The disposition of these amino acid substitutions is consistent with the view that these two membrane-spanning domains interact structurally and functionally with the DCCD-binding proteolipid subunit 9 in the Fo-sector.