Purification and molecular cloning of succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex. Absence of a sequence motif of the putative E3 and/or E1 binding site

Full-length cDNA clones for succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex were isolated from rat heart cDNA libraries in lambda gt11. The cDNA clones were identified as those for rat succinyltransferase by the identity of their predicted amino acid sequence with the NH2-terminal amino acid sequence of rat succinyltransferase determined by protein chemical analysis and the known amino acid sequence of bovine succinyltransferase. The clone with the longest cDNA consisted of 2747 base pairs and coded for a leader peptide of 56 amino acid residues and a mature protein of 386 amino acid residues. The primary structure of rat succinyltransferase showed close similarity to Escherichia coli and Azotobacter vinelandii succinyltransferases, in the COOH-terminal part forming the lipoyl-binding domain and the NH2-terminal part forming the inner core-catalytic domain. However, the rat succinyltransferase did not contain a sequence motif that has been found as an E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168, Reed, L. J., and Hackert, M. L. (1990) J. Biol. Chem. 265, 8971-8974). The absence of this sequence was confirmed by direct sequencing of the polymerase chain reaction product of rat heart mRNA and by computer analysis. These results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site.     

Expression and assembly of mature apotransacylase (E2b) of bovine branched-chain alpha-keto acid dehydrogenase complex in Escherichia coli. Demonstration of transacylase activity and modification by lipoylation

A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex (Griffin, T. A., Lau, K. S., and Chuang, D. T. (1988) J. Biol. Chem. 263, 14008-14014) was used to construct a prokaryotic expression vector for recombinant mature E2b. The overexpression in Escherichia coli correlates with the presence near the 5'-terminus of the mature E2b coding region (nucleotides 20 to 28) of the sequence 5'-TCAAACT-CT-3'. It has been proposed that this sequence is involved in secondary mRNA recognition through interaction with the 5'-terminus of the bacterial 16 S rRNA. The mature E2b protein has transacylase activity when assayed with exogenous dihydrolipoamide and [1-14C] isovaleryl-CoA as substrates. However, the recombinant protein has no attached lipoic acid. This was established by the absence of radiolabel incorporation when transformed E. coli cells were grown in a medium containing DL-[2-3H]lipoic acid. The recombinant mature E2b protein was purified to greater than 95% purity in one step using Sepharose 4B column chromatography. The purified recombinant protein was shown to have a cubic 24-mer structure by electron microscopy and to possess a specific activity similar to that of the purified natural bovine E2b. The purified recombinant mature E2b was lipoylated in vitro in the presence of 2 mM ATP using a mitochondrial extract prepared from bovine liver. The above results provide the first evidence that the proper folding and assembly of mature bovine E2b is independent of the attachment of lipoyl moieties and that mammalian lipoylation activity is present in mitochondria.     

H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms

We analyzed the production of Torpedo marmorata acetylcholinesterase (AChE) in transfected COS cells. We report that the presence of an aspartic acid at position 397, homologous to that observed in other cholinesterases and related enzymes (Krejci, E., N. Duval, A. Chatonnet, P. Vincens, and J. Massoulié. 1991. Proc. Natl. Acad. Sci. USA. 88:6647-6651), is necessary for catalytic activity. The presence of an asparagine in the previously reported cDNA sequence (Sikorav, J.L., E. Krejci, and J. Massoulié. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1865-1873) was most likely due to a cloning error (codon AAC instead of GAC). We expressed the T and H subunits of Torpedo AChE, which differ in their COOH-terminal region and correspond respectively to the collagen-tailed asymmetric forms and to glycophosphatidylinositol-anchored dimers of Torpedo electric organs, as well as a truncated T subunit (T delta), lacking most of the COOH-terminal peptide. The transfected cells synthesized similar amounts of AChE immunoreactive protein at 37 degrees and 27 degrees C. However AChE activity was only produced at 27 degrees C and, even at this temperature, only a small proportion of the protein was active. We analyzed the molecular forms of active AChE produced at 27 degrees C. The H polypeptides generated glycophosphatidylinositol-anchored dimers, resembling the corresponding natural AChE form. The cells also released non-amphiphilic dimers G2na. The T polypeptides generated a series of active forms which are not produced in Torpedo electric organs: G1a, G2a, G4a, and G4na cellular forms and G2a and G4na secreted forms. The amphiphilic forms appeared to correspond to type II forms (Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:776-785; Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:786-794), which are abundant in the nervous tissue and muscles of higher vertebrates (Bon, S., T. L. Rosenberry, and J. Massoulié. 1991. Cell. Mol. Neurobiol. 11:157-172). The H and T catalytic subunits are thus sufficient to account for all types of known AChE forms. The truncated T delta subunit yielded only non-amphiphilic monomers, demonstrating the importance of the T COOH-terminal peptide in the formation of oligomers, and in the hydrophobic character of type II forms.     

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.     

Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants

Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f.     

Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli

A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH. 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5. The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R. W., Green, G. D. J., and Barrett, A.J. (1985) Biochem. J. 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate. However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH. Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein. These constructions were expressed in the E. coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins. Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system. Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity. These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity.     

Molecular cloning of a novel chaperone-like protein induced by rhabdovirus infection with sequence similarity to the bacterial extracellular solute-binding protein family 5

Previously we demonstrated that a novel stress protein is induced in fish cells by the infection of a fish rhabdovirus (Cho W. J., Cha, S. J., Do, J. W., Choi, J. Y., Lee, J. Y., Jeong, C. S., Cho, K. J., Choi, W. S., Kang, H. S., Kim, H. D., and Park, J. W. (1997) Biochem. Biophys. Res. Commun. 233, 316-319). In this paper, we present the molecular cloning and characterization of a gene encoding this protein named virus-inducible stress protein (VISP). The VISP was purified partially by immunoprecipitation using a monoclonal antibody against the VISP and further purified by the electroelution from a SDS-PAGE gel. The protein was subjected to internal protein sequencing, and the sequence of three peptides was determined. Degenerate oligonucleotides based on the three peptide sequences were used to screen a cDNA library from rhabdovirus-infected CHSE-214 fish cells, and a cDNA of a 2193-bp open reading frame encoding the VISP with 730 amino acid residues (M(r) = 79.84) was identified. Whereas the nucleotide sequence of VISP shows no similarity with other genes in the GenBank(TM), the amino acid sequence of the VISP has similarity with the bacterial extracellular solute-binding protein family 5 (SBP_bac_5) that is proposed to have chaperone activity. Thus, we explored whether the VISP also had chaperone-like activity. Purified recombinant VISP expressed in Escherichia coli promoted the functional folding of alpha-glucosidase after urea denaturation and also prevented thermal aggregation of alcohol dehydrogenase. These results suggest that the VISP has amino acid sequence similarity with SBP_bac_5 and that it has chaperone activity that may play a role in virus infection.     

Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase

A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.     

Production of pea lectin in Escherichia coli

In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin. This system is based on the production of pea lectin in Escherichia coli. The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E. coli (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E. coli cells (2-5 mg/liter). Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex. This procedure yields a homogeneous 28,000-dalton protein. Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.     

Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex. Mapping of the E1 beta-binding region on E2.

A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha-keto acid dehydrogenase complex was isolated from a lambda ZAP expression library. The bovine E1 beta cDNA is 1,393 base pairs in length. It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues. The calculated molecular mass of the mature bovine E1 beta subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04. The mature bovine E1 beta subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG). When expression was induced by IPTG at 37 degrees C, the soluble recombinant E1 beta subunit existed as a single high molecular weight form (Mr congruent to 3.5 x 10(5)), which sedimented during sucrose gradient ultracentrifugation at 2 x 10(5) x g. However, lowering the induction temperature to 25 degrees C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1 beta protein. The low molecular weight form (Mr congruent to 9.1 x 10(4)) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins. The results showed that the E1 beta subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain. In contrast, the recombinant E1 alpha subunit did not bind the E2 component. The data suggest an apparent binding order of E2-E1 beta-E1 alpha, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M.L., Xu, W.-X., Oliver, R.M., Wall, J.S., Hainfeld, J.F., Mullinax, T.R., and Reed, L.J. (1989) Biochemistry 28, 6816-6821). The relatively inaccessible topology of E1 beta may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex.     

Transferring redox regulation properties from sorghum NADP-malate dehydrogenase to Thermus NAD-malate dehydrogenase

NADP-dependent chloroplastic malate dehydrogenase (E.C.1.1.1.82) is regulated by thiol disulfide-interchange with thioredoxin. It displays two regulatory disulfides per subunit, located in specific sequence extensions respectively at the N- and C-terminal ends of each subunit. In the present study, attempts were made to transfer the regulatory properties of sorghum NADP-malate dehydrogenase to a constitutively active NAD-dependent malate dehydogenase (E.C.1.1.1.37) from the thermophilic bacteria Thermus flavus, by grafting the regulatory extensions of the former to the latter. The results demonstrate that a successful transfer of redox regulation properties requires the grafting of both full-length extensions, but also the introduction of specific hydrophobic residues in the core part of the protein. These residues are very likely involved in the interaction between monomers, and structural changes at the active site.     

Plants under Climatic Stress: VI. Chilling and Light Effects on Photosynthetic Enzymes of Sorghum and Maize

The activity of several photosynthetic enzymes was unaltered by exposure of sorghum or maize to low temperatures (10 C) and light (170 w m⁻²). Two light-activated C₄-pathway enzymes, NADP-malate dehydrogenase and pyruvate Pi dikinase, were reduced in activity, and this was largely attributable to a loss of enzyme rather than to incomplete enzyme activation. Loss of NADP-malate dehydrogenase was more marked in sorghum than in maize, and in both species no loss occurred at 10 C when light levels were reduced from 170 to 50 w m⁻². A light-dependent, low temperature-induced loss of catalase activity was also observed in maize leaves.     

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.     

Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli. Molecular cloning of rat ornithine transcarbamylase

A cDNA clone corresponding to the mature form of ornithine transcarbamylase (OTCase) was selected from a rat liver cDNA library constructed in bacteriophage lambda gt10. OTCase clones were selected using a synthetic DNA probe of 15 bases corresponding to the 3' end of the OTCase mRNA [Horwich, A. L., Kraus, J.P., Williams, K., Kalousek, F., K?nigsberg, W. & Rosenberg, L.E. (1983) Proc. Natl Acad. Sci. USA, 80, 4258-4262]. Putative OTCase clones were subcloned into the expression vector, pUC9, and the identity of inserts confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose filters followed by probing with monospecific anti-OTCase antibodies and 125I-labelled protein A. A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from Escherichia coli beta-galactosidase) was obtained and the identity of the clone was confirmed by comparison of the 5' sequence with a limited N-terminal amino acid sequence [Lusty, C., Jilka, R. L. & Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036]. A sequence discrepancy between the published sequence (Lusty et al.) and the sequence predicted from the cDNA structure is noted.     

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.     

Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase

We have used a previously characterized mouse cDNA clone for the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1300-1304), which we designate C alpha, to isolate cDNA clones coding for a second isoform of the C subunit, C beta. C alpha cDNA clones hybridize to a 2.4-kilobase mRNA on Northern blots whereas C beta cDNA clones detect a 4.3-kilobase mRNA. Nucleotide sequence comparison between C alpha and C beta cDNA clones shows that the C beta cDNA codes for a protein which shows 91% identity with C alpha. Determination of mRNA levels for C beta in various tissues shows that it is most highly expressed in brain although it is detectable in all tissues examined. The presence of two genes coding for the C subunit of cAMP-dependent protein kinase may explain past reports of heterogeneity in C subunit protein preparations.     

Enzymatic properties of the protein encoded by newly cloned human alcohol dehydrogenase ADH6 gene.

Five non-allelic genes which encode five types of alcohol dehydrogenase subunits have been identified in humans. An additional gene (ADH6) and cDNA, whose coding sequences were not highly analogous to any of the known alcohol dehydrogenase subunits, were recently cloned (Yasunami et al., Proc. Natl. Acad. Sci. USA 88, 7610-7614, 1991). The full-length ADH6 cDNA was expressed in the E.coli expression system and in the in vitro translation system of rabbit reticulocytes. The protein produced had its isoelectric point at pH 8.6, optimum pH at pH 10, and a lower Km for benzylalcohol than for ethanol and propanol. These characteristics are compatible to the properties of mu- or sigma-alcohol dehydrogenase isozyme existing in human stomach, indicating that ADH6 gene encodes the mu- or sigma-alcohol dehydrogenase subunit.     

Survival and induction of recA protein in mitomycin C-treated Escherichia coli rec, lex, or uvr strains

The capability to synthesize recA protein has been tested for Escherichia coli treated with mitomycin C. recA protein was assayed using an immunoradiometric assay (Paoletti, C., Salles, B., and Giacomoni, P. U. (1982) Biochimie 64, 239-246). Mitomycin C-treated wild type E. coli can express recA gene in a similar quantitative fashion, independently of the growth media used in this work; glucose did not inhibit induction of recA protein in cells growing in synthetic media. Wild type E. coli recovering from energy starvation displays a similar qualitative capability to induce the synthesis of recA protein independently of the stage of growth at which the cells are treated with the drug. At midexponential phase, the cells appear to have an enhanced capability to synthesize recA protein. The relationship between survival and capability to synthesize recA protein was explored for E. coli lex, rec, and/or uvr mutants, after treatment with mitomycin C. A good correlation was found, except for a recB mutant and for an ethidium-sensitive strain, both able to produce as much recA protein as the wild type but 100-fold more sensitive to the drug. A similarly satisfactory correlation was found when plotting the survival after UV irradiation versus the capability of synthetizing recA protein with the exception of an uvrA strain and of a lexA strain.