Sequence specificity in aflatoxin B1-DNA interactions

Proc. Natl Acad. Sci. USA Vol. 80, pp. 6-1), January 1983 Biochemistry

Sequence specificity in aflatoxin B1-DNA interactions (nucleotide context effect/chemical modification of nucleic acids/alkali-labile lesion/nmutagenesis and carcinogenesis)

KEVIN F. MUENCH, RAvINDRA P. MISRA, AND M. ZAFRI HUMAYUN* Department-of Microbiology, New-Jersey Medical School, Newark, New Jersey 07103 Communicated by Gerald N. Wogan, September 13, 1982

sequence. We have used this technique to investigate the relative extent ofmodification of guanine residues by AFB1 in several DNA fragments ofknown sequence obtained from prokaryotic and eukaryotie sources. Our results suggest that the sequence environment of target sites has a strong and predictable influence on the extent of modification by AFB1.

ABSTRACT The activated-form of aflatoxin B1 (AFB1) causes covalent modification primarily of guanine residues, leading to alkali-labile sites in DNA. A simple extension of the Maxam-Gilbert procedure for sequence analysis permits the identification of alkali-labile sites induced by AFB1 and determination of the frequency of alkali-labile AFBI modifications at-particular sites on a DNA fragment of known sequence. Using this strategy, we have investigated the influence of flanking nucleotide sequences on AFB1 modification in a number. of DNA fragments of known sequence. Our results show-that certain guanine residues in doublestranded DNA are preferentially attacked by AFB1 over others in a manner predictable from a knowledge of vicinal nucleotide sequences. The observed in vitro sequence specificity is independent of a number oftested parameters and is likely to occur-in vivo.

MATERIALS-AND METHODS DNA Preparations. Methods for preparing 4X174 replicative form-(RF) DNA (7), plasmid DNA (8), and restriction fragments and for 5'-end labeling of DNA (9) have been described. Single-stranded (ss) DNA fragments were isolated from the corresponding double-stranded (ds) DNA by electrophoresis on

Most chemical and physical carcinogens are also mutagenic and appear to act by inducing chemical modifications in DNA. The interactions ofmany carcinogens with DNA are complex, giving rise to a number of different types of lesions, some ofwhich may lead to mutagenesis. One of the variables that influence any given type of DNA modification is the relative accessibility of the potential site(s) of modification in a given region of DNA. It has long been suspected that the actual nucleotide sequence surrounding a potential site ofmodification (within an accessible domain of DNA) may influence the extent of action by DNAmodifying-agents. Here, we report that the extent of a particular type of modification of target residues in double-stranded (ds) DNA by a mutagenic carcinogen is strongly and predictably influenced by flanking nucleotide sequences. Aflatoxin B1 (AFB1) is a highly toxic, mutagenic, and carcinogenic metabolite produced by certain strains of the fungus Aspergillus flavus, found as a naturally occurring food contaminant (1), that has been implicated in the etiology of liver cancer in certain geographical regions. Chemically, AFB1 is a highly substituted coumarin, capable of causing covalent modification ofnucleic acids and proteins on activation. Activation is believed to involve the conversion of A7FBL into the highly reactive AFB1-2,3-oxide (2, 3). AFB1 can be activated in vitro either by crude liver enzyme preparations or by oxidation with a mild organic oxidant (4); this activated compound then can react with DNA -to give adducts identical to those obtained in vivo. The principal DNA adduct both in vivo and in vitro has been identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-AFBL (2, 3). The primary N7-guanine AFB1 adduct, like N7-alkyl guanines, is expected to weaken the N-glycosidic bond of the nucleotide (2) such that elevated pH and temperature-would result in depurination followed by strand- scission. Using an extension of the Maxam-Gilbert technique for sequence analysis (5), D'Andrea and Haseltine (6) have shown that AFBi modification of DNA does indeed result in alkali-labile sites that correlate with the occurrence of guaninse residues on a DNA fragment of known

strandseparation gels (5). Chemical Activation Procedure. A slight modification of the procedure of Martin and Garner (4) was used. In a typical reaction, 25 /.Ci.(14 Ci/mmol; 1 Ci = 37 GBq; Moravek Biochemicals, Brea, CA) of [3H]AFB1 was dried in a vacuum dessicator in a glass tube (10 x 75 mm) and then dissolved in 0.2 ml of ice-cold dichloromethane. Next, 10-100 ng of the appropriate end-labeled DNA preparation in 0.2 ml of 20 mM sodium phosphate buffer (pH 7.4) was added to the reaction tube, followed by 0.01 ml of a freshly prepared solution of chloroperoxybenzoic acid (Aldrich) at a concentration of 8 mg/ml of dichloromethane. The reaction tube was tightly stoppered and shaken in an air-bath shaker at an angle of,45' at 370C for 30 min. The reaction tube was cooled on ice and the phases were separated by brief centrifugation in a- table-top centrifuge. The aqueous layer was aspirated into a 1.5-ml Eppendorf tube and the organic phase was extracted twice with 0. 1-ml portions of the sodium phosphate buffer. The combined aqueous layers were extracted three times with equal volumes of chloroform and the DNA was recovered by three successive ethanol precipitations (9). The final pellet was air dried and suspended in an appropriate volume of buffer. Mock experiments were carried out in the same way except for the omission of chloroperoxybeizoic acid. The average number of guanine residues modified per DNA molecule (one or-twb per strand for most of the experiments) was calculated as the difference in 3H radioactivity between test reactions and mock reactions, which usually were 2-2.5% ofthat found in the test. Unlabeled AFB1 (Calbiochem) was used -in some experiments. Microsome Activation Procedure. DNA fragments were treated with AFB1 essentially as described by D'Andrea and Haseltine (6) except that the AFB1 concentration was held at 0.1 mM and no carrier tRNA was used. Analysis of AFB1-Modified DNA Fragments. DNA sequence analysis was carriedout-according to Maxam and Gilbert (5). AFBL-modified DNA was degraded by heating at 900C for 30 min in freshly- prepared 1 M piperidine and was prepared

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: AFBI, aflatoxin B1; RF, replicative form; ds, double stranded; ss, single stranded. * To whom reprint requests should be addressed. 6

Biochemistry: Muench et aL for sequence analysis as described (5). Autoradiography was carried out at --70'C with the aid of intensifying screens.

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RESULTS primary DNA lesion induced the principal N7-Guanine-AFBL, by AFB1, creates alkali-labile sites. When appropriately endlabeled DNA fragments of known sequence are modified with AFB1 and then treated with alkali (or an organic base) at high temperatures, the resulting strand-scission products will run at the same positions as those seen for guanine residues in a stan. dard Maxam-Gilbert sequence analysis gel. This has been shown recently by D'Andsea and Haseltine (6) who used a DNA fragment modified by microsome-activated AFB1. Recently, Martin and Garner (4) reported that AFB1 activated in vitro by peracid oxidation reacts with DNA to give an N7-guanine adduct identical to that given by metabolically activated AFB1. An autoradiograph of a sequence gel on which degradation products obtained by piperdine/heat treatment.of a DNA fragment modified by chemically activated AFB1 have been resolved is shown in Fig. 1. Comparison of the AFB1 DNA lane with the standard guanine-specific Maxam-Gilbert dimethyl sulfate-treated DNA lane shows that AFBI treatment results in creation of alkali-labile sites at guanine residues, confirming that chemically activated AFB1 reacts with DNA in a manner analogous to metabolically activated AFB1. In the type of analysis of AFB1modified _DNA represented by Fig. 1, band position on the gel defines the site of an alkali-labile lesion, white band intensity defines. the frequency with which such a lesion occurs at a particular residue. The examples shown in Fig. 1 a and c (in conjunction with the nucleotide sequences shown in Fig. 2; correlations between reactivity and sequence context are considered below) show that the extent of modification ofguanine residues by chemically activated AFB1 is nonrandom in OX ds DNA. Fig. lb shows that the sequence-related variation in extent of guanine modification by AFB1 is apparently abolished when the same DNA is in. the ss form. In addition, we have observed that reactivity with guanine residues in ss DNA is depressed, in comparison with ds DNA, so that; at a given concentration of AFB1, less ss DNA is degraded (unpublished data). Note that the guanine residues occurring in the minus strand between OX sequence positions 1,126 and 1,111 have been analyzed in ss DNA (Fig. lb) and ds DNA-(Fig. la). Specifically, note that the bands corresponding to positions 1,126, 1,123, 1,120, 1,117, and 1,113 are weak. in Fig. la (lane A) with respect to other bands in the same channel, whereas the bands corresponding to these residues in Fig. lb (lane A) appear to have approximately the same intensity as other bands in the same channel. Also, comparison of lane A in Fig. lb with lane A in Fig. Ic makes- it clear that, in ss DNA, reactivity of guanine residues is more or less uniform; Using this method, we tested the effects of a number of parameters on AFBl-DNA interactions (data not shown). It was found that, for a given duplex restriction fragment, the relative reactivity of the guanine- residues with AFB1 was independent of(i) the method of activation of AFB1 i..e., the chemical procedure vs. microsomal enzyme procedure; (ii) the dosage of AFB1, from 3&2 to- 160 nmol-at higher concentrations, there. was more degradation, but the basic pattern of weak and strong sites was not affected; (iii) the reaction time, from 5 to 30 minat longer times, there was more degradation but no effect on the basic pattern; (iv) NaCl concentration, from 0 to 1.5 M; (v) whether the DNA was from coding or noncoding regions (+X174); and (vi) whether the DNA was prokaryotic (+X174 and pBR322, Figs. 2 and 3) or eukaryotic (human repeat, Alu; data not shown).

7

Proc. NatL Acad. Sci. USA 80 (1983)

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c and 8% for b) on which strand.-scission products obtained by AFB1 treatment of various OX (RF, ds) or ss DNA fragments were resolved.

(+) and (-), specification of labeled strand. (a) The 38-base-pair-long ,AX (RF) DNA fragment obtained by Hpa II (sequence position 1,103 of XX; ref. 10)/Hha I (position 1,143) digestion. The AFB1-treated'

fragment was subjected to piperidine/heat degradation and electrophoresed adjacent to, standard Maxam-Gilbert sequence gel lanes of DNA nottreated with AFBI. Lanes: A, aflatoxin-treated DNA; D, dimethyl sulfate-treated DNA (this lane represents the usual guanine-specific lane on sequence gels). Guanine residues are identified by numbers indicating their position on the OX genome (10). (b) The 121-nucleotide-longminus strandfragment isolated byHhaI (position 1,020-1,143) digestion and subsequent strand separation (the first 38 nucleotides from the 5' end are the same as those analyzed in the ds fragment shown in a). The AFB1 degradation products-(lane A) are compared with those (lane D) obtainedby dimethyl sulfate treatment of the ds 121-base-pair-long fragment labeled in the minus strand. (c) The 149-base-pair-long ds DNA fragment obtained by Hha I (position 1,143)/Hpa I (position 1,292) digestion of OX DNA. Variable nonspecific background seen in gels is not due to AFB1^ degradation.

The data obtained by analyzing the relative extent of guanine modification by AFB1 in a number of restriction fragments from various sources are summarized in Fig. 2, and Fig. 3 presents a compilation of the G-C-containing oligonucleotides and the A-T-containing oligonucleotides having a single guanine residue in the sequences analyzed. In both figures, guanine residues have been rated as weak, intermediate, or strong sites. These data reveal the following pattern. (i) The relative frequency of creation of an alkali-labile guanine site by AFB1 is strongly influenced by the flanking nucleotide sequences in ds DNA; all guanine residues in ss DNA (which does not form stable secondary structures under our experimental conditions; see Discussion) are equally accessible to AFB1. (ii) In most cases examined (18 out of 21), single guanine residues flanked by (A.T)rich sequences are poor-targets. In the three exceptions (T-GT-A, T-A-T-G-T-T-T, and T-T-T-T-T-A-T-G-T; Fig. 3) the guanine residues are moderately accessible. It is curious that in all three cases the guanine occurs as a member of a tetranucleotide with alternating purine/pyrimidine residues and that dimethyl sulfate reactivity with each residue is also enhanced (e.g., Fig.

8

Proc. Nad Acad. Sci. USA 80 (1983)

Biochemistry: Muench et al. 1050

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FIG. 2. Nucleotide sequences of relevant portions of OX (RF) (a-d; ref. 10) and pBR322 (e; ref. 11) DNAs showing relative frequencies of alkalilabile sites induced by AFB1. *, Guanine residues at which alkali-labile sites are induced most frequently; o, least.favored guanine residues; o, guanine residues that fall between the most frequent and least frequent categories in ds'DNA; , equivalence in reactivity of guanine residues in ss DNA fragments. Symbols were assigned by visual inspection of autoradiographs on the basis of band intensity compared with neighboring bands in the AFB1 lane and on the basis of densitometry (see below). For this assignment, bands were not compared with bands from dimethyl sulfate-treated DNA, which were only used as markers. Examination of the band patterns shows that, in some segments, all bands are weaker or stronger in comparison with other segments. In general, there is a "fade-up" effect imposed on the whole pattern such that the smaller degradation products give a more intense autoradiographic signal when compared with the larger products. Nevertheless, it is possible to make assignments by comparing each band with its neighbors. For example, guanine residues in positions 1,135, 1,122, and 1,167 (Fig. 1 a and c) are rated as mostfavored sites while positions 1,120, 1,113, 1,165, and. 1,178 are least-favored sites and positions 1,123, 1,193, and 1,214 are intermediate. Each of the DNA fragments was subjected to-AFBl degradation analysis two to six times under different conditions (extent of modification by AFB1, ranging from one to six; strength of analysis gels; film exposure time) and the assignments for each residue were reproducible.'Although visual inspection of the autoradiographs alone-shows that the relative modification of the guanine residues are sufficiently different (see Fig. 1c) to make the qualitative assignments valid, we have confirmed these. assignments by densitometric analyses of most of the autoradiographs that form the basis for this figure. When band intensities are expressed as percentages of total undegraded material, semiquantitative figures that are a measure of the frequency of scission at each guanine residue can be obtained. For example, applying such methods to the' data in Fig. la (lane A), we can obtain percentage results by using an empirical correction factor (12) to take multiple hits on the same strand (fade-up effect) into account. The correction .factor used assumes random multiple hits and, because the AFB1 reaction is nonrandom, these figures are semiquantitative. Nevertheless, the symbolic assignments for the guanine residues are in reasonable agreement with these results. Thus, the following assignments were made (numbers in parentheses are percentages): 1,138 (strong, 2.08); 1,137 (strong, 1.92); 1,135 (strong, 2.38); 1,126 (intermediate, 0.4); 1,123 (intermediate, 0.54); 1,122 (strong, 1.28); 1,120 (weak, 0.23); 1,117 (weak,0.28); 1,113L(weak, 0.2); 1,111 (intermediate, 0.72). In relative terms, when compared with poor sites, intermediate sites have reacted 2-3 times faster and strong sites have reacted 6-10 times faster. At lower AFB1 concentrations, the reactivity of AFB1 is at least 10 times faster at strong sites than at poor sites. (Other supportive densitometric data are available on request.)

lc, lane D, guanine 1,193). (iii) In most cases examined, the second guanine (from the 5' end) in contiguous guanine di- and trinucleotides is most favored while the first and third are moderately favored. (iv) A guanine residue in a C-G sequence is

moderately favored while. that in a G-C one is a poor target. (v) A guanine residue in C-C-G is a strong target site while one in the sequence G-C-C is a poor target. The patterns observed for G-C-containing di- and trinucleotides are essentially. re-

Biochemistry: Muench et aL

Proc. NatL Acad. Sci. USA 80 (1983)

A. G-C CLIGOLICLEOTIDES 00 0 000 0 * @000 GG/CG/GC/GGG/CGC/CCG/GCG/GCC/GGC/CCGG/GGCG 0 0 0 0 0 0 0 00 0 1 00

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FIG. 3. Compilation of G-C-containing oligonucleotides (flanked by one or more A-T pairs) and A-T containing oligonucleotides having one internal guanine residue (flanked by one or more G-C pairs) occurring in the nucleotide sequences analyzed (Fig. 2) for induction of alkali-labile sites by AFB1 in the ds configuration. Symbols are as in Fig. 2. Oligonucleotides are written in 5' to 3' order. Some of the listed oligonucleotides occur several times in the sequences shown in Fig. 2. Where a particular oligonucleotide is represented more than once, the AFB1 reactivity of individual guanine residues is identical or similar.

tained (except for certain additive effects) in longer runs of GC sequences, as exemplified by the pBR322 nonanucleotide CC-G-C-C-G-C-C-G, in which all three residues are strong sites (cf. C-C-G). Also, in contiguous guanine tetranucleotides (data not shown), the 5' and 3' guanine residues are intermediate in reactivity while the second and third react strongly. Similarly, the 5' guanine residues in G-C, G-C-C, G-C-C-C, and G-C-GG-C are poor targets. (vi) Within the limits of the experimental variations used, the sequence-specific effects are independent of the method of AFB1 activation, source of DNA, AFB1 concentration, reaction time, and ionic strength.

D'Andrea and Haseltine (6) have observed that the rate of reaction of individual guanines with AFB1 in a single restriction fragment is variable, although they did not attempt to correlate the variation with the sequence context, which, in the case ex-

amined, would have been difficult because the single short sedid not have sufficient sequence variation. It is worth noting that some ofthe context effects we have deduced are also identifiable in their data (figure 4 left in ref. 6). Although the reactivity of dimethyl sulfate with ds DNA is also not quite random, the sequence-specific effects seem to be less pronounced (Fig. lb; unpublished data) when compared with those of AFB1 and appear to follow somewhat different rules. quence

DISCUSSION

We have sought to determine context effects on the relative particular type of guanine modification caused by AFB1. The guanine modifications we have analyzed are those that result in the creation of alkali-labile sites. Although we have used such terms as accessibility of particular guanine residues, we emphasize that our analysis concerns the creation of a particular type of lesion at the guanine residues. We have not yet addressed the possibility that all guanine residues are modified to an equal extent in ds DNA and that the observed sequencespecific effects are due to secondary reactions (see refs. 13 and 14) of AFB1 differentially affected by flanking nucleotide sequences. Despite this consideration, our data show that alkalilabile lesions at guanine residues caused by AFB1 in ds DNA are by no means random and that flanking nucleotide sequences have a marked influence on the extent of such modifications. In the data presented here, a pattern is discernible that permits extent of a

9

prediction ofthe guanine residues most frequently modified by AFB1 with induction of alkali-labile lesions in a DNA of known sequence. In general, all guanine residues in ss DNA are equally liable to this type of AFB1 lesion, whereas in the ds form, modification of the same guanine residues is strongly influenced by flanking sequences. Scattered guanine residues, in particular, guanine residues flanked by (A-T)-rich sequences are poor targets for the induction of alkali-labile lesions by AFB1, whereas certain guanine residues occurring as members in G-C clusters are highly favorable to this type of modification. Some specific "rules" that determine a favorable site have been presented above. Moreover, in ss DNA capable of forming stable secondary structures (hairpins), the AFB1 reaction is uniform but suppressed in the ss portion and is stronger and follows the "rules" in the hairpin stem region (unpublished data). Our data suggest that the observed sequence-specific effects are independent of the method of activation of AFB1, source of the DNA, reaction time, and concentration of AFB1 but rather represent an intrinsic feature of AFBI-DNA interactions likely to occur in vivo. If one assumes that the observed sequence

specificity is a

reflection ofdifferential primary reactivity of guanine residues, it is clear that sequence environment in ds DNA is recognized by AFB1. Except for the three single guanine residues that occur as members in three oligonucleotides that have an alternating purine/pyrimidine sequence, as noted in Results, we do not see a positive correlation between the Z conformation of ds DNA and accessibility to AFB1. However, our data are insufficient to answer the question of whether guanine residues in Z DNA are more (or less) accessible to AFBI-induced lesions as compared with B DNA. We can imagine two types of mechanism that could result in preferential modification of guanine residues in a particular sequence environment but not in others. One possibility is that DNA regions containing specific G-C clusters may have conformational features favoring modification by AFB1, which can diffuse freely. Thus, one can imagine either a preexisting conformational feature or a cooperative (or noncooperative) change induced in a neighboring guanine residue by AFB1 modification of a given guanine residue. The second possibility is that all guanine residues (irrespective of their sequence environment) are equally reactive with AFB1 but the relative frequency is determined by variance in the localized preexisting concentration of AFB1 along the helix. Thus, more AFB1 might bind (for example, by intercalation or base stacking) to DNA regions containing certain contiguous G-C sequences as compared with regions that do not, which could account, by mass action, for the observed sequence specificity. Our most recent unpublished data support the latter possibility. Although all guanine residues in DNA are potential targets for AFB1 modification in vivo, the work of Bailey et at (15) suggests that all DNA domains in chromatin are not equally accessible to AFB1. Specifically, these authors have made the important observation that the internucleosomal linker sequences are preferred targets for AFB1 modification. It is significant that the domains favored by AFB1 are those that are accessible to nucleases and presumably to proteins controlling gene expression. The data reported here suggest that, within accessible domains, certain guanine residues may be preferred by AFB1 over others in a manner predicted by the actual nucleotide sequence. It will be interesting to determine whether such strong context effects are observable in the reactivities of other carcinogens that interact covalently with DNA. We will discuss the potential biological significance of AFB1 sequence specificity elsewhere. Briefly, we propose that the deleterious consequences of AFB1 are mediated by a clustering of AFB1 lesions

10

Biochemistry: Muench et aL

on both strands at specific sites predicted on basis of the observed sequence specificity. We wish to thank Drs. C. Cantor, E. Goldman, W. Olson, and M. Ptashne for comments and R. Marotti for typing the manuscript. This work was supported by National Cancer Institute Grant CA27735. 1. Busby, W. F. & Wogan, G. N. (1979) in Food Borne Infections and Intoxications, eds. Feinmann, H. P. & Bryan, R. L. (Academic, New York), 2nd Ed., pp. 519-610. 2. Essigmann, J. M., Croy, R. G., Nadzan, A. M., Busby, W. F., Reinhold, V. N., Buchi, G. & Wogan, G. N. (1977) Proc. Natd Acad. Sci. USA 74, 1870-1874. 3. iUn, J., Miller, J. A. & Miller, E. C. (1977) Cancer Res. 37, 44304438. 4. Martin, C. N. & Garner, R. C. (1977) Nature (London) 267, 863865. 5. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol 65, 499560. 6. D'Andrea, A. D. & Haseltine, W. A. (1978) Proc. Nati Acad. Sci. USA 75, 4120-4124.

Proc. Nati Acad. Sci. USA 80 (1983) 7. Humayun, M. Z. & Chambers, R. W. (1979) Nature (London) 278, 524-529. 8. Clewell, D. B. (1972)J. BacterioL 110, 667-676. 9. Humayun, M. Z., Jeffrey, A. & Ptashne, M. (1977)J. MoL BioL 112, 265-277. 10. Sanger, F., Coulson, A. R., Friedman, T., Air, G. N., Barrell, B. G., Brown, N. L., Fiddes, J. C., Hutchinson, C. A., III, Slocombe, P. M. & Smith, M. (1978)J. Mol BioL 125, 225-246. 11. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. BioL 43, 77-90. 12. Haseltine, W. A., Gordon, L. K., Lindan, C. P., Grafstrom, R. H., Shaper, N. L. & Grossman, L. (1980) Nature (London) 285, 634-641. 13. Groopman, J. D., Croy, R. G. & Wogan, G. N. (1981) Proc. NatL Acad. Sci. USA 78, 5445-5449. 14. Wang, T. V. & Cerutti, P. (1980) Biochemistry 19, 1692-1698. 15. Bailey, G. S., Nixon, J. E., Hendricks, J. S., Sinnhuber, R. 0. & Van Holde, K. E. (1980) Biochemistry 19, 5836-5842.