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Physical Analysis of Spontaneous and Mutagen-Induced Mutants Of Copyright 0 1990 by the Genetics Society of America Physical Analysis of Spontaneous and Mutagen-Induced Mutantsof Escherichia coli K-12 Expressing DNA Exonuclease VI11 Activity Suresh K. Mahajan,’ Charles C. Chu? David K. Willis,” Ann Templin and AlvinJ. Clark Department of Molecular and CellBiology, University of Calqornia, Berkeley, Calgornia 94720 Manuscript received January25, 1989 Accepted for publication February28, 1990 ABSTRACT We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VI11 (ExoVIII). One mutation,sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and thetrg gene. Western blot analysis showsthat the protein produced is larger than wild type ExoVIII. The nucleotide sequenceshows that a translational gene fusion has occurred. The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call SfcA. Analysis of the protein sequence encoded by SfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme. We discuss the possibility that SfcA is identical tomaeA which encodes NAD-linked malic enzyme from Escherichia coli. Restriction nuclease analysis ofa second deletion,sbcA81, by Southern blot technique indicates that about105 kb of DNA have been deleted anda transcriptional gene fusion has occurred between recE and the regulatory region of an E. coli chromosomal gene. We also examined eight other sbc mutations that result in ExoVIII production. Five have no effect on restriction nucleotide fragment sizes detected by complementarity to X rev as probe. These are presumed point mutations. Three seem to produce additional restriction nucleotide fragments complementary to X rev. The possible nature of these sbc mutations is discussed. UTATIONS suppressing recB21 and/or recC22, on the E. coli genetic map (FOUTSet al. 1983; WILLIS M called sbc mutations, have beendetected in four et al. 1983), and is the defective prophage Rac (LOW different genetic backgrounds Escherichiaof coli K-12. 1973). Rac carries the recE gene (GILLENet al. 1977; Such backgroundscan be nicknamed to avoid writing KAISERand MURRAY1979) and orij (DIAZand PRIT- the complete genotype each time theyare referred to. CHARD 1978; DIAZ,BARNSLEY and PRITCHARD1979). In this case the backgrounds were designated EndoI-, Thus the 1157 background carries a deletion muta- Hfr, Su- and 1157 (TEMPLIN,KUSHNER and CLARK tion which removes recE and hence cannot host recE- 1972). In the first three genetic backgrounds the sbc expressing suppressor mutations. mutationslead to detectable levels ofexonuclease Because the sbc suppressors fell into the recE-ex- VI11 (ExoVIII), an ATP-independent DNA exonucle- pressing and nonexpressing groups, TEMPLIN,KUSH- ase not found in the original strains (BARBOURet al. NER and CLARK(1972) called the first sbcA and the 1970; TEMPLIN,KUSHNER and CLARK1972). The second sbcB. sbcB was found to be the structural gene authors interpreted this to indicate that the structural for DNA exonucleaseI (KUSHNER, NACAISHIand gene for ExoVIII(called re&) had become expressed CLARK,1972, 1974). sbcA, however, has not been so as a result of the suppressor mutation. In the fourth easy to characterize because different types of muta- background (1 157) sbc mutations do not result in recE tions lead to expression of recE, possibly by different expression. KAISER and MURRAY(1979) found that mechanisms. Of five sbcA mutations examined,KAISER several strains with the 1157 background lacked a 27- and MURRAY(1 980) found thattwo, sbcAI and sbcA23, kb elementof DNA present in the otherbackgrounds. showed no change in Rac detectable by the Southern Where it is present, this element is partly complemen- blot method and presumed them to be point muta- tary toand can recombine with phagelambda tions. sbcAl was later found to be cis-acting and dom- (GOTTESMANet al. 1974; KAISER and MURRAY1979). inant to sbcA+ (FOUTSet al. 1983) indicating the pos- The element comprises the region from 29.6 to 30.2 sibility that it alters an operator or promoter forrecE. One mutation, sbcA8, consisted of a large deletion of ’ Present address: Molecular Biology and Agriculture Division, Bhabha most of Rac, includingattR and theimmunity repres- Atomic Research Center, Bombay 400 085, India. sor region (KAISERand MURRAY1980). Because the ‘ Present address: Laboratory of Immunology, National Institute of Al- lergy and Infectious Diseases, Bethesda, Maryland 20892. leftward promoter and operator, presumed to control ’ Present address: ARS/USDA Plant Disease Resistance Unit and Depart- ment of Plant Pathology,University of Wisconsin,Madison, Wisconsin recE transcription by analogy with phage X, were also 53706. absent, KAISERand MURRAY(1 980) hypothesized that Genetics 125: 261-273 (June, 1990) 262 S. K. Mahajan et al. sbcA8 fused recE to a promoter from a gene outside were transferred to nitrocellulose filter paper by the proce- Rac. To explain recessivenessof sbcA8 to sbcA+ (LLOYD dure of SMITHand SUMMERS (1 980).Hybridization of radio- active Xrev probe DNA to the immobilized restriction frag- and BARBOUR 1974), KAISER and MURRAY (1980) ments wasby the method of WAHL,STERN and STARK further hypothesized that an operator anda repressor (1979). gene controlled the promoter to whichrecE was fused. Sequencing the endpoints of sbcA8 Samples of 1.8 pg Furthermore, they hypothesized that sbcA8 had de- chromosomal DNA fromJC5412 (sbcA8) and1.2 pg leted the repressor gene. Two final mutations,sbcA5O pBR322 DNA were digested separately with HindIII, mixed, treated with T4 DNA ligase and used to transform and sbcA51 were associated with EcoRI nuclease diges- JC5519 (recB21recC22) to resistance to ampicillin (50 pg/ tion fragments of Rac which could have been derived ml) and mitomycin (1 pg/ml). Six transformants were ob- from tandemly arranged multiple copiesof Rac, either tained of which one, JC15901, was stably MitCR UVK and onthe chromosome or inthe cytoplasm, or from Ret+. This transformant contained a12.4-kb plasmid having monomeric circular copies of excised prophage (KAI- an 8.1-kb HindIII fragment inserted into the HindIII site of pBR322. The plasmid was named pSKM1. pSKM2 was SER and MURRAY 1980).Additional unusual EcoRI constructed by digesting pSKM1DNA with EcoRI and fragments of Rac made it seem possible that small BamHI, treating the mixture withligase and using it to deletions occurred in someof the copiesof Rac in the transform DH 1 to ampicillin resistance. Subsequent restric- sbcA51 strain (KAISERand MURRAY1980). The phys- tion enzyme analysis of plasmidDNA from fiveof the ical nature of sbcA5O and sbcA51 could not be conclu- transformants revealed one 5.2 kb plasmid which contained a 1.2 kb EcoRI-BamHI fragment inserted between the EcoRI sively determinedpartly because of thenumerous and BamHI sites of pBR322. The strain containing pSKM2 copies of Rac apparently present and partly because was named JC15903. neither sbcA+ strain from which the two mutants were pSKM1 and pSKM2 were used as sources ofDNA to derived was examined. make M 13 bacteriophage strains for sequencing (Figure la). In this paper we extend the studies of KAISERand Most phages were made by digesting plasmid DNA withone or two restriction nucleases, adding the mixture to appro- MURRAY(1 980)by determining thatsbcA8 is a trans- priately digested phage DNA, treating the mixture with lational fusion of part of recE with part of another ligase, transfecting JM103 with the mixture and screening gene approximately 140 kb distant. We also deter- surviving cells in colorless plaques for the desired phage by mine the characteristics of another deletion, sbcA81, restriction digests of DNA minipreps. Techniques used were and hypothesize that this produces a transcriptional those of MESSING (1983). Enzymes used were those whose sites mark the termini of cloned fragments(Figure la). fusion of recA to a promoter about 105 kb distant. Phages JCM 1591 1to JCM 15916 were derived frompSKM 1 Additionally we examine the Rac prophage in mutants and MI3 mp8. Phages JCM15919 to JCM15923 were de- carrying sbcA1, sbcA23, and six other sbcA mutations rived from pSKM2 and M13 mp8.JCM15927 was con- not examinedby KAISERand MURRAY(1 980). Finally structedfrom pSKM2 and M13 mp9. Finally, phages we discuss the appropriateness of labeling as sbcA all JCM 15658 andJCM 15659 were derived respectively from gel-isolated 505 bp and 684 bp BamHI-EcoRI fragments of mutations which suppressrecB and recC mutations and JCM15914 RF DNA and M13 mp8. lead to ExoVIII activity. Template ssDNA generated from the above phages was employed in the chain termination method (SANGERet al. MATERIALS AND METHODS 1977) to obtain the sequence. The extents of each insert actually sequenced are illustrated in Figure 1b. The univer- Bacterialstrains, abbreviations and general genetic sal primer was used except where noted. Three oligonucle- methods: All bacterial strains analyzed in this work are otide primers were also used. prJC22 has the sequence 5' derivatives of Escherichia coli K-12. The derivations and ACGATCGGCAATGCCCAT 3'. It has two mismatches to relevant genotypic markers of most are listed in Table 1. 5' ACCGATCGGAATGCCCAT 3' from coordinates1000 Genotype symbols are those used by BACHMANN(1983). to 983 of the final sequence because it was designed with Question marks indicate that allele and plasmid numbers reference to an earlier inaccurate version of the sequence. have not been stated or that there is a question about the prJC23 has the sequence 5' TTGAGGAGTTAAAATGAA presence of an allele. We also used DH 1, recAI, endAl 3' corresponding to coordinates 172 to 189 andprJC27 has gyrA96 thi-1 hsdR17 supE44 relAI(?) F-X- (MANIATIS, the sequence 5' TCTGCTTTATACTTG 3' correspond- FRITSCHand SAMBROOK1982) and JM103, de (lac-proAB) ing to coordinates 389 to 403 of the final sequence. thi-? supE? rpsL? endA? sbcBI5 hsdR4 [F? traD36 proAB+ lac' de(lacZ)M15], [PI] (MESSING 1983). Tet abbreviates tetra- RESULTS cycline and MitC abbreviates mitomycin C. Superscripts R and S indicate resistance and sensitivity respectively.
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