Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability : II. Permeability changes to amethopterin and other folates in the drug-resistant mutant

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.     

Dihydrofolate Reductase Activity in Strains of Streptococcus faecium var. durans Resistant to Methasquin and Amethopterin

Resistance to the antifolates methasquin and amethopterin has been studied in new strains of Streptococcus faecium var. durans. Two methasquin-resistant strains (SF/MQ, SF/MQ(T)) and an amethopterin-resistant strain (SF/AM) were selected independently from the wild-type S. faecium var. durans (SF/O). SF/MQ(T) is a thymine auxotroph. Total dihydrofolate reductase activity was elevated in each of the resistant strains. The greatest increase (36-fold) was observed in extracts of SF/AM. The methasquin-resistant strains, SF/MQ and SF/MQ(T), had 29-fold and 8-fold, respectively, more dihydrofolate reductase activity than the parental strain. Total dihydrofolate reductase activity of SF/O was separable by gel filtration into two components: a folate reductase (11%) and a specific dihydrofolate reductase (89%). Folate reductase activity was associated with 88% of the total dihydrofolate reductase activity of SF/MQ(T), with specific dihydrofolate reductase activity accounting for the remaining 12%. In SF/MQ and SF/AM, folate reductase activity was associated with 97% of the total dihydrofolate reductase activity. Studies of the inhibition by methasquin and amethopterin of partially purified folate reductase and specific dihydrofolate reductase of the mutant strains suggested that resistance was not accompanied by changes in the affinities of these enzymes for either antifolate.     

Resistance to Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. II. Permeability changes to amethopterin and other folates in the drug-resistant mutant.

the accumulation of amethopterin in a Pediococcus cerevisiae strain resistant to this analogue was about 30% of that in P. cerevisiae/PteGlu, the sensitive parent. The uptake in the resistant strain was strictly glucose dependent, whereas in the sensitive parent about 16% accumulation occurred in absence of glucose. The transport in both strains was inhibited by iodoacetate and KF. Amethopterin uptake exhibited saturation kinetics with an apparent Km of 5 muM in P. cerevisiae/AMr and 0.5 muM in P. cerevisiae/PteGlu. The apparent V was 0.2 nmol per min per mg cells (dry weight); the same for both strains. The optimum pH for the uptake of amethopterin by P. cerevisiae/AMr and P. cerevisiae/PteGlu was pH 6.0. Folate and methyltetrahydrofolate competitivity inhibited amethopterin uptake with apparent Ki values of 8 and 0.7 muM, respectively. The uptake of folate exhibited a slightly increased Km value as compared to that of the sensitive strain, whereas the uptake activity velocity was in the same range. Methyltetrahydrofolate accumulated up to about 60-fold higher intracellular concentration than that of the medium, which is a markedly lower accumulation from that in the sensitive strain. The uptake was glucose dependent and inhibited by iodoacetate and KF. The pH optimum for methyltetrahydrofolate uptake in the resistant strain was the same as that in the sensitive parent (pH 5.7--6). In contrast to the increase in the apparent Km value for amethopterin in the resistant strain, the affinity of the carrier for methyltetrahydrofolate was apparently unchanged, whereas the V value was about 16 times lower than that in the sensitive strain. The Ki for amethopterin when added to increasing concentrations of methyltetrahydrofolate was 5.2 muM, a value about the same as that of the Km.     

Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following Mu-mediated transposition

The dihydrofolate reductase structural gene, folA, has been cloned into the multicopy vector pBR322 following the gene's enrichment by bacteriophage Mu-mediated transposition. Strains carrying the resultant plasmid, pJFMS, produce 25 to 30 times more dihydrofolate reductase than control strains. Consequently they are resistant to trimethoprim, an inhibitor of this enzyme. This elevation in enzyme production is due to an increase in the number of folA gene copies per cell. The higher yield of dihydrofolate reductase obtained will be extremely useful for purifying and characterising this trimethoprim-sensitive chromosomally derived enzyme. The plasmid will also be invaluable for studying the structure, function and regulation of dihydrofolate reductase.     

Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate

Interpretation of the ¹H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the ¹H-NMR spectra. Previous attempts to label the dihydrofolate reductase from over-producing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.     

A dihydrofolate reductase gene from Candida albicans: molecular cloning

The dihydrofolate reductase gene from Candida albicans has been cloned and partially characterized. A genomic bank from C. albicans strain 10127/5 was constructed in Escherichia coli and screened for trimethoprim resistance. A plasmid pMF1, carrying the resistance marker was isolated and characterized by restriction mapping and Southern blotting. Cells harbouring pMF1 were as sensitive as the parental cells to a wide spectrum of antibacterial agents, except for trimethoprim; the dihydrofolate reductase activity from these cells was trimethoprim resistant.     

The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388.

The R-factor R388 mediates the production of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different molecular weight and pH profile to the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatography. The purified R-factor enzyme was about 20 000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20 000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme suprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.     

Trimethoprim resistance in enterococci: microbiological and biochemical aspects

Synergy was found between sulphonamide and trimethoprim in ratios 1:1 and 20:1 against both trimethoprim-sensitive enterococci (17 strains) and trimethoprim-resistant enterococci (23 strains). Many of these strains were resistant to kanamycin, tetracycline, streptomycin and/or erythromycin. Resistance to kanamycin, but not to trimethoprim, was clearly associated with the presence of a plasmid of molecular weight 35-45 Md. Elimination of this plasmid in three out of four highly trimethoprim resistant strains brought about loss of resistance to both kanamycin and trimethoprim. Furthermore, it was possible to transfer trimethoprim resistance from three of five highly resistant strains, but not from three strains with low-grade resistance. It is concluded that resistance to trimethoprim in enterococci can be encoded on a plasmid, and that the gene responsible may be on a transposon. No significant differences were found between the specific activities of dihydrofolate reductase from trimethoprim-sensitive and -resistant strains. The enzyme from resistant strains was several orders of magnitude less susceptible to inhibition by trimethoprim than was the enzyme from sensitive strains.     

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Δyhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Δyhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.     

Dihydrofolate reductase and aminopterin resistance in Pneumococcus

Summary Wild-type pneumococci derived from Avery's strain R36A are sensitive to extracellular concentrations of the folate antimetabolite aminopterin exceeding 1.0x10^-6 M. Three classes of resistant strains are phenotypically distinguishable: amiB-r, amiA-r and amiD-r strains are resistant to low (1.5x10^-6 M), intermediate (0.5–4.0×10^-5 M) and high (4.5x10^-4 M) aminopterin levels respectively. The amiA and amiB regions are weakly linked, but linkage has not been established between either of these loci and the amiD region.Consistent with the maximum resistance conferred by mutations in the amiA locus, dihydrofolate (FH₂) reductase in cell-free extracts (CFE) of amiA-r strains has a two- to six-fold greater affinity for the substrate than dose the enzyme in wild-type CFE (Table 1); FH₂ reductase from amiA-r strains may also have reduced affinity for aminopterin. Specific activity of the enzyme is not affected by mutation in the amiA locus (Table 1) and its affinity for the cofactor (NADPH) is probably unaffected by mutation in this locus (Table 4). Dihydrofolate reductase activity in amiA5 CFE is considerably more thermolabile than that in wild-type CFE (Table 2).The enzyme in CFE of the high resistance strain amiD1 has the same affinity for the substrate, cofactor and antimetabolite as FH₂ reductase in wild-type CFE (Figs. 1–4, 8 and 9; Table 4). However, specific activity of the enzyme in amiD1 CFE is 11-fold higher than that in wild-type CFE (Table 1) and it is much more heat stable (Table 2).Some properties of FH₂ reductase in CFE of the high resistance recombinant strain amiA5amiD1 are intermediate between those in CFE of wild-type and amiD1.Preliminary results suggest that CFE of wild-type and amiA5 contain a factor, which is neither dialyzable nor heat sensitive, that has an inhibitory effect upon activity and stability of FH₂ reductase in amiD1 CFE (Tables 2 and 3).     

Experimental evolution of propanediol oxidoreductase in Escherichia coli comparative analysis of the wild-type and mutant enzymes

A model for the study of experimental evolution is provided by the novel metabolic system responsible for the progressive utilization of l-1,2-propanediol by mutants of Escherichia coli (strains 3 and 430). In these mutant strains, propanediol oxidoreductase, which serves as l-lactaldehyde reductase in fucose fermentation by wild-type cells, became a key enzyme for aerobic catabolism of propanediol. In the wild-type strain (strain 1), the enzyme is inducible only anaerobically; in strains 3 and 430, the enzyme is synthesized constitutively even in the presence of air. The propanediol oxidoreductase from all three strains was purified to homogeneity by the same procedure. The enzyme of strain 3 clearly differed from that of strain 1 in several respects: K_m and V in both directions of the reaction, energy of activation, thermal stability, pH optimum and substrate specificity. However, no difference in any of the above characteristics was found between the enzymes of strains 3 and 430. All three enzymes presented the same electrophoretic mobility. According to immunological data, all three strains differed in their intracellular enzyme level.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O₂ and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O₂ exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.     

Trimethoprim resistance plasmids of different origin encode different drug-resistant dihydrofolate reductases.

Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofolate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R751 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a 10- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.     

Isolation of a small DNA fragment carrying the gene for a dihydrofolate reductase from a trimethoprim resistance factor

Summary DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate reductase. The enzyme was found to be stable at 100°. Some aspects of the synthesis of dihydrofolate reductase are discussed.Dedicated to Professor Peter Karlson on the occasion of his 60th birthday     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

Biochemical and genetic basis of the response to 5-fluorouracil in Drosophila melanogaster

Mutant strains sensitive and resistant to the drug 5-fluorouracil (FU) have been isolated from the wild-type Pac strain of Drosophila melanogaster. The resistant strain, termed flu^r, is resistant to at least 0.0035%FU (2.7 × 10^–4 m) in the food media and exhibits cross-resistance to 5-fluorodeoxyuridine (FUdR) but not to 5-fluorouridine (FUR). The sensitive strain termed flu ^S , exhibits over 90% mortality on 0.0008% FU (6 × 10^–5 m). Genetic analysis indicates that the flu gene is located on the third chromosome, which agrees with results of previous workers. An analysis of the enzyme thymidylate synthetase from the selected sensitive and resistant strains indicates that the resistant strain enzyme possesses an elevated specific activity. Levels 4 times that of the sensitive strain were observed when the enzymes were assayed at 20 C. This increase is apparently not due to induction by FU in the food media. It is suggested that the enzyme thymidylate synthetase may be involved in the resistance process.     

The development of resistance to methotrexate in a mouse melanoma cell line

PG19T3 mouse melanoma cells were selected for resistance to methotrexate. Nine sub-lines that are resistant to concentrations of methotrexate ranging from 1.27×10^–7 M, to 1×10^–4M methotrexate were selected and characterised in terms of their content of dihydrofolate reductase activity and their chromosomes. The intracellular level of dihydrofolate reductase activity increases with increasing resistance such that at the highest level of resistance PG19T3:MTX_R ^{10–4 M} cells contain approximately 1,000 fold more enzyme activity than the parental PG19T3 cells. It is shown that the enhanced activity is due to an increase in the amount of the enzyme rather than any structural change to the enzyme in resistant cellls. Comparisons of pH activity profiles, profiles under different activating conditions and titrations with methotrexate suggest that the sensitive and resistant cells contain identical dihydrofolate reductases. Analysis of the chromosomes of resistant cells shows the presence of up to 5 large marker chromosomes which contain homogeneously staining regions after G-banding. These same regions stain intensely after C-banding and fluoresce brightly after staining with Hoechst 33258. The size of homogeneously staining regions increases throughout the process of selection. For one marker chromosome this increase may have been mediated via a ring chromosome.     

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells.

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levls of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W.F., Davidson, S.V., and Siminovitch, L. (1976) Somatic Cell Genet.2,245-261). By selecting for increased resistance form these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In the report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.