Proc. Natl. Acad. Sci. USAVol. 84, pp. 6516-6520, September 1987Genetics

Autogenous control of the S10 ribosomal protein operon ofEscherichia coli: Genetic dissection of transcriptional andposttranscriptional regulation

(translation regulation/attenuation)

LEONARD P. FREEDMAN*, JANICE M. ZENGEL, RICHARD H. ARCHER, AND LASSE LINDAHLDepartment of Biology, University of Rochester, Rochester, NY 14627

Communicated by Robert K. Selander, June 5, 1987 (received for review April 29, 1987)

ABSTRACT The S10 ribosomal protein operon is regulat-ed autogenously by the product of one of the genes of theoperon, the gene encoding ribosomal protein L4. We have usedsite-directed mutagenesis to isolate leader mutations affectingIA control. The phenotypes of these mutants demonstrate thatIA regulates both transcription and translation of the S10operon. Several mutations abolish both levels of L4 control;others eliminate either transcriptional or translational controlwith little or no effect on the other mode of regulation. Weconclude that L4-mediated transcriptional and translationalcontrol share some sequence requirements, but the two regu-latory processes recognize somewhat different features of theS10 leader. Primary as well as secondary structures within theS10 leader appear to be involved.

The S10 ribosomal protein operon in Escherichia coli isautogenously regulated by ribosomal protein L4, the productof the third gene in the operon (1-3). Protein L4 regulates theexpression of the S10 operon by modulating the level oftranscription (2, 4) and translation (3). The molecular mech-anisms by which L4 inhibits both transcription and transla-tion of the S10 operon are not known. Our in vivo experi-ments have shown that transcriptional control results fromL4-mediated premature termination (attenuation) within theS10 leader (Fig. 1 and ref. 4). The nucleotide sequenceimmediately upstream of the premature termination site canbe drawn as a stem-loop structure followed by severaluridine residues (Fig. 2a and ref. 4), typical of factorp-independent terminators (5). Results from digestions of theS10 leader with nucleases specific for single- and double-stranded RNA are consistent with the proposed terminator-like structure (P. Shen, J.M.Z. and L.L., unpublished ex-periments). Therefore, it is tempting to speculate that thisstructure is an attenuator.The effects of protein L4 on transcription and translation

presumably result from a direct interaction with the S10message. Since L4 is a rRNA-binding protein, its interactionwith the S10 transcript may involve recognition of a primaryor secondary structure in the message that is homologous tothe region of 23S rRNA to which L4 binds. Indeed, theproposed stem-loop structure contains a 9-base sequencealso found at the L4 binding site in 23S rRNA (Fig. 2a and ref.4).To learn more about the autogenous control of the S10

operon, we have made a series of mutations in the leaderregion and examined their effects on L4 regulation. We findthat the stem-loop region of the S10 leader is essential notonly for L4-mediated control of transcription but also for L4control of translation. However, while both types of control

involve the same region of the S10 leader, transcriptional andtranslational control can be separated genetically and there-fore require somewhat different sequence features. At leastseveral of the bases within the 9-base homology sequence aredispensable for L4 control.

MATERIALS AND METHODSStrains, Plasmids, and Growth Media. The host for M13

phage work was E. coli K-12 JM103 (6). Luria-Bertani (LB)medium was used for phage experiments. Expression of thefusion-protein genes was analyzed in strain LL308 (1) car-rying plasmid pLF17. This plasmid carries a gene for ribo-somal protein L4 under the control of the lac UV5 promoter(7). For labeling experiments cultures were grown at 37°C inAB minimal medium (8) supplemented with 0.2% glucose.Plasmid pLF1 (Fig. 1) has been described (7). M13 phage J11(probe I) carries a 180-base-pair Sst I-Bam HI fragment fromthe proximal end of the S10'/lacZ' fusion gene. M13 phagesSUM6 (probe II) and SUM18 (probe III) were described byAksoy et al. (9).

Mutagenesis. Oligonucleotides with the desired mutationsin the S10 leader were synthesized and purified by gelelectrophoresis (10). Each purified oligonucleotide was thenused to mutate the S10 leader sequence carried by an M13recombinant phage (11). The pLF1-A49 deletion mutant wasmade with an oligonucleotide containing 18 bases on eachside of the deletion. The base substitution mutants were madewith oligonucleotides 14-16 bases long. Each mutant wassequenced (12); a fragment carrying the mutated S10 leaderwas then excised by restriction endonuclease digestion of thereplicative form of the mutant M13 DNA and transferred toplasmid pLF1. Successful transfer was verified by recloningthe leader region in a new M13 vector and determining thenucleotide sequence. The pLF1-A72 mutant was constructedby deleting the leader region between HincII and SnaBI sites.RNA and Protein Synthesis Determinations. To measure

mRNA synthesis rates, cultures were labeled with [3H]uri-dine for 45 sec. The cells were lysed in boiling buffercontaining 2% sodium dodecyl sulfate. Total nucleic acid waspurified by extractions with phenol and chloroform/isoamylalcohol. Incorporation into mRNA from the S10'/lacZ'fusion gene was then measured by hybridization to filter-bound single-stranded M13 DNA containing inserts from theproximal, middle, or distal regions of the fusion gene (Fig. 1).The rate offusion protein synthesis was measured by labelingwith [35S]methionine for 1 min followed by sodium dodecylsulfate/polyacrylamide gel electrophoresis of extracts of thelabeled cells. See ref. 7 for details.

*Present address: Department of Biochemistry and Biophysics,University of California, San Francisco, CA 94143.

6516

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Proc. Natl. Acad. Sci. USA 84 (1987) 6517

RESULTS

L4 Regulation of Transcription and Translation. We havebeen analyzing L4-mediated regulation of the S10 operon,using plasmid pLF1 containing the S10 promoter and leaderfollowed by a fusion between the S10 gene and lacZ (Fig. 1and ref. 7). This plasmid coexists with another plasmid,pLF17, carrying the L4 gene under control of the lacpromoter. Addition of isopropyl P-D-thiogalactopyranoside(IPTG) to cells harboring both pLF17 and pLF1 results in a

4-fold increase in the synthesis ofL4 (2) and a strong decreasein both mRNA and protein synthesis from the S10'/IacZ'fusion gene (7).By comparing the extent of L4 inhibition of protein

synthesis and mRNA synthesis, we could detect two levels ofL4 action, an effect on transcription and a further effect ontranslation. As shown in Table 1, when protein L4 was

oversynthesized in cells carrying pLF1, mRNA synthesisfrom the fusion gene decreased to 23% of the preinductionrate (part a), while fusion protein synthesis decreased to 3%(part b). That is, transcription was reduced by a factor of 4-5,and translation of the residual mRNA transcripts was re-duced by a further factor of 6 (Table 1, part c), so thatfusion-protein synthesis was reduced by a factor of 30. Thephenotypes of mutants described below support our conclu-sion that L4 regulates both transcription and translation.

Deletion of a Region Essential for L4-Mediated Regulation.A deletion removing all but 30 bases of the 173-base leaderfrom pLF1 abolishes L4 control of the fusion gene (7). Sincethe region just upstream of the site for L4-stimulated tran-scription termination contains a structure resembling a tran-scription terminator (Fig. 2a), we studied the role of thisspecific region by constructing a derivative of pLF1, calledpLF1-A49, from which a 49-base-pair region containing thissequence has been deleted (Figs. 1 and 2a). We thenmeasured the effect ofL4 oversynthesis on both transcriptionand translation of S10'/lacZ' on the deletion plasmid. Incontrast to the wild-type leader, the A49 mutation eliminatedautogenous control by L4: synthesis of both mRNA andfusion protein was unaffected by L4 oversynthesis (Table 1,parts a-c). Thus, the stem-loop region of the leader isessential for both transcriptional and translational control ofthe S10 operon.

Base Substitutions Affecting L4-Mediated Regulation. Wenext examined the effects of specific base changes within thestructure removed by the A49 mutation. Since we wereparticularly interested in the 9-base sequence that is alsofound at the L4 binding site in 23S rRNA, we constructed fourmutants (shown in Fig. 2b) containing two base substitutionsaffecting this region of the stem-loop structure. In two ofthese mutants the 9-base homology sequence was changed,

S10 operonPS10 L23

XN

either in the loop (pLF1-117,118) or in the stem (pLF1-122,123). The latter mutation presumably abolishes the pu-tative terminator structure. The other two mutants werechanged outside the 9-base homology sequence, one in theloop (pLF1-113,114), the other (pLF1-106,107) in the regionof the upper stem to which the homology sequence isbase-paired. Finally, we combined the two stem-disruptingmutations to make a "compensatory" mutant (pLF1-106,107,122,123) with four base substitutions that allow formationof a stem similar to the wild-type structure.The complex phenotypes of the base-substitution mutants

indicate that the region of the S10 leader containing theproposed stem-loop structure is involved in determining threeparameters of gene expression: L4 regulation of transcription,L4 regulation of translation, and translation efficiency duringexponential growth. As summarized in Table 1 (parts a-c), L4regulation of the S10 operon was altered in all five base-substitution mutants. By comparing the effects of excess L4 onmRNA synthesis and on protein synthesis, we could identifymutations affecting only transcription or only translation, aswell as mutations affecting both levels of L4 regulation (seebelow for details). In addition, we found that several of themutations resulted in a significant decrease in the rate offusion-protein synthesis in the absence of excess L4, without a

corresponding decrease in the rate of transcription of the fusiongene (Table 1, part d; discussed in more detail below).The base substitutions in the stem part of the 9-base

sequence (pLF1-122, 123) eliminated L4-mediated regulationof both transcription and translation. However, the mutationdisrupting the other side of the stem (pLF1-106, 107) alsoabolished both levels of L4 control (Table 1, parts a and c).Thus, the 9-base homology sequence per se is not sufficientfor L4 control of transcription or translation of the S10operon. When the two stem mutations were combined in a

"compensatory" mutant that should restore the secondarystructure but not the primary sequence, L4 regulation oftranslation, but not transcription, was restored. With thismutant, oversynthesis of L4 did not inhibit transcription (ifanything, the rate offusion-message synthesis was increased;Table 1, part a), yet translation of the message was decreasedby a factor of 5 (Table 1, part c). The phenotype of this mutantindicates that L4 can regulate translation without inhibitingtranscription. The compensatory mutant also shows that atleast 2 of the 9 bases within the homology sequence are

dispensable for translational control by L4.Finally, both mutant leaders containing base substitutions

in the loop (pLF1-113,114 and pLF1-117,118) exhibitednormal transcriptional control by L4, but translational con-trol was abolished. Fusion-mRNA synthesis was decreasedby a factor of 3-5 as a result ofL4 oversynthesis (Table 1, part

1 kb

I lacZ' \

FIG. 1. Maps of the S10 operon (Top)and the fusion plasmid pLF1 (Middle).The S10 leader and proximal end of theS10'/lacZ' fusion gene are expanded 5-fold below the main map of pLF1.Hatched bars indicate fragments clonedin M13 phage to create hybridizationprobes I (J11; see Materials and Meth-ods), II (SUM6; ref. 9), and III (SUM18;ref. 9) used for transcription measure-ments. Open bars indicate regions re-moved in the pLF1-A49 and pLF1-A72deletion mutants. The vertical arrowshows the site of L4-mediated attenua-tion. kb, Kilobase.

S19 L29 S17

12 N 1L221 S3 6116 `

IpLF1

lacZ'

II III

Ps10S10'

A72 A49

I 0. I0.1 kb

Genetics: Freedman et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

Table 1. Expression of the S10'/lacZ' fusion gene from mutant plasmids

Effect of L4 oversynthesis Translation

Part a: Part b: Part * efficiency§Plasmid mRNA synthesis* Protein synthesist Prt(L4 not induced)__________________ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _Prot in ( 4 norin d c e d

Name Mutation Probe n Mean Average (SE) n Average (SE) per mRNAt n Average (SE)

pLF1 None (wild I 4 0.22type)

II 4 0.23 0.23 (0.014) 7 0.034 (0.006) 0.15 4 1.0III 4 0.23

A49 Stem-loop I 2 1.07deletion

II 2 0.75 1.06 (0.149) 5 1.05 (0.049) 0.99 2 0.79 (0.24)III 2 1.36

106,107 Stem, left I 2 1.17II 2 1.47 1.26 (0.140) 5 0.94 (0.060) 0.74 2 0.08 (0.02)III 2 1.15

122,123 Stem, right I 2 0.95II 2 1.04 1.09 (0.102) 5 1.01 (0.059) 0.93 2 0.25 (0.08)III 2 1.30

106,107, Compensatory I 4 1.38122,123

II 4 1.84 1.53 (0.105) 7 0.34 (0.034) 0.22 3 0.14 (0.04)III 4 1.38

113,114 Loop I 3 0.37II 4 0.32 0.35 (0.034) 6 0.46 (0.028) 1.31 3 1.2 (0.30)III 4 0.36

117,118 LQop I 2 0.24II 2 0.15 0.18 (0.030) 5 0.44 (0.057) 2.44 2 1.2 (0.17)III 2 0.15

A72 Upstream I 2 1.08deletion

II 2 0.90 1.01 (0.104) 2 0.33 (0.070) 0.33 1 0.91III 2 1.06

Cells carrying the indicated plasmid plus pLF17 were pulse-labeled with [3H]uridine or [35S]methionine before and 10 Min after induction ofL4 oversynthesis. RNA was extracted and analyzed by filter hybridization. Protein extracts were analyzed by gel electrophoresis.*Part a shows the effect of L4 on S10'/lacZ' mRNA synthesis, calculated as the fraction of total radioactivity in RNA hybridizing to the indicatedprobe (I, II, or III; see Fig. 1) in samples labeled after induction of L4 oversynthesis divided by the fraction of total radioactivity in RNAhybridizing to the same probe in samples labeled before L4 induction. The means from the indicated number of experiments with each probe(n) are shown. For each plasmid, the data from all three probes were then pooled to determine the average and standard error (SE). Valuesindicating loss of L4 control are underlined.

tPart b shows the effect of L4 on fusion protein synthesis, calculated from the ratio of 35S radioactivity in the fusion-protein band (normalizedto the radioactivity in the bands of the /8 and 13' RNA polymerase subunits) after and before LA induction. The values shown are the averageof the indicated number of experiments (n). Standard errors are indicated in parentheses. In previous experiments L4 overproduction reducedthe synthesis of the S10'/lacZ' fusion protein by a factor of only 6 (ref. 7), not 30 as observed here. The difference is due to a protein comigratingwith the fusion protein in the previous gel system. We can now resolve the fusion protein from the contaminating protein by reducing theconcentration of methylenebisacrylamide by a factor of 4.tPart c shows the effect of L4 on the amount of fusion protein synthesized per fusion message, calculated as the ratio of values in parts b anda. Since results in part b indicate the effect of L4 on fusion-protein synthesis-i.e., the result of LA regulation at both the transcriptional andtranslational level-and results in part a measure the effect of L4 on transcription only, the values in this column give an estimate of theL4-mediated regulation at the level of translation. Values indicating loss of L4 inhibition are underlined.§Part d shows the translation efficiency-that is, the amount of fusion protein synthesized per mRNA molecule-in exponentially growing cellscarrying the indicated plasmids. These values were calculated by dividing the rate of fusion-protein synthesis by the rate of fusion-messagesynthesis in cells growing in the absence of L4 oversynthesis. The mRNA data are from hybridizations to probe I (Fig. 1). The resulting ratiosare normalized to the value for pLF1. Results are averages of the indicated number of experiments (n), except for A72, which was analyzedonly once. Standard errors are shown in parentheses. Values showing a decrease in translation efficiency by a factor 24 are underlined.

a), but protein synthesis in the loop mutants showed only amodest inhibition by excess L4 (Table 1, part b). In otherwords, translation of the fusion mRNA still being synthesizedafter L4 induction showed no inhibition by L4 (Table 1, partc). In fact, for the 117,118 mutant it appears that L4stimulates, rather than inhibits, translation of fusion mRNA.These results show that L4 can regulate transcription in theabsence of translational control. The two loop mutations,together with the compensatory mutation, demonstrate thatthe two modes of L4 regulation can be separated genetically.And the 117,118 mutant again shows that several bases withinthe 9-base sequence are dispensable, in this case for tran-scriptional control.

Deletion of the Upstream Portion of the S10 Leader. Todetermine if the portion of the leader upstream of theproposed stem-loop structure is also involved in L4 control,we deleted 72 bases of the leader upstream of the 49-basedeletion discussed above (Figs. 1 and 2a). Analysis ofa fusionplasmid carrying the 72-base deletion revealed that this partof the leader is in fact required for L4 control. The deletioneliminated transcriptional control after L4 oversynthesis(Table 1, part a), although translational control was stillalmost normal (Table 1, part c). This result suggests that theregion of the leader containing the proposed secondarystructure is necessary, but not sufficient, for L4-nmediatedattenuation control of the S10 operon.

6518 Genetics: Freedman et al.

Proc. Natl. Acad. Sci. USA 84 (1987) 6519

a b

u cA G

A UC A

110 *U--AA--U 120C--B6C--BC--6

U CU u

C--BU--AC--B

A B* 130100 'C--B A

A--UU--A

80 90 U--A' 6--U

BBBBAGCUACGUAABAACB- -CAUUUUCBUUUAUAA

A72 - 49

U CA B A

A U AC A 4U--AA--UA- - UC--B

106. 107

U C

C AU- -AA--U

c cIc ^

U C6 A 6U A U

c AU--AA--UC--BA - -

122,123 106,107,122,123

Av C 6C BC AU--AA--UC--BC--BC--B

113, 114

U CA B

A

U--AA--UC--BC--BC--B

117.118

FIG. 2. (a) Proposed structure of the S10 leader transcript in the region upstream of the site of L4-induced premature termination oftranscription. The line extending from base 117 to base 125 indicates the 9-base sequence also found in 23S rRNA at the L4 binding site. Theregion deleted in pLF1-A49 and the distal endpoint of the A72 deletion are also shown. (b) Various substitution mutants derived from pLF1.Base changes are enclosed by boxes.

Translation Efficiencies of the Mutated S10 Leaders. Be-sides their effect on L4 control, base changes in thestem-loop region of the S10 leader affected the translationefficiency of the message during exponential growth. Thetranslation efficiency was calculated as the ratio of the rate offusion protein synthesis (before induction of L4) and the rateof fusion message synthesis (also in the absence of excessL4), for the wild type and each of the mutant plasmids (Table1, part d). Base changes in the upper stem (mutants 106,107and 122,123) resulted in a significant decrease in the rate offusion protein synthesis, even though mRNA synthesis wasnot affected. In particular, the left stem mutation reduced thetranslatability of the mutant message by a factor >10. Thesevariations in the expression of the fusion gene in the variousmutants can be readily seen in the preinduction samples in theautoradiogram shown in Fig. 3. Note that translation wasonly slightly improved in the compensatory mutant, and thatneither of the deletion mutations had a significant effect ontranslation efficiency.

DISCUSSIONWhen L4-mediated autogenous control was first observed(1), it seemed to be a simple repression of transcription ortranslation. However, further analysis has revealed that themechanism is complicated, involving regulation of bothtranscription and translation. The work reported here iden-

tifies a region in the leader of the S10 operon that is essentialfor L4-mediated autogenous control. The phenotypes ofdeletion and base-substitution mutations in this region arecomplex, affecting differentially three aspects of the expres-sion of the S10 operon: L4-mediated regulation of transcrip-tion termination (attenuation), L4-mediated regulation oftranslation of the S10 gene, and the efficiency of translationof the S10 gene.We have previously proposed (4) that a specific secondary

structure just upstream of the L4-mediated termination sitemay be important for transcriptional control by L4 (Fig. 2a).Our analysis of mutations in the S10 leader suggests thattranscriptional control may involve more than simply astem-loop structure: the sequence within the stem (butapparently not within the loop) may also be important.Sequences upstream of the stem-loop also appear to beimportant, since the A72 deletion, which removes basesentirely upstream of the proposed attenuator structure,eliminates transcriptional control. The phenotype of thismutant suggests that L4-mediated attenuation control mayinvolve alternative secondary structures or perhaps a specifictertiary structure within the S10 leader. Or perhaps a mini-mum transcript length is required for efficient termination(13). Unfortunately, our understanding of the attenuationprocess in the S10 operon is now hampered by limited generalknowledge about protein-RNA interactions, RNA secondaryand tertiary structure, and transcription termination.

pLF1 compens. 113,114 AES A49 106,107 117,118 122,123

L4: - + - + - + - + - + - + - + - +

_4 a_ "_ _ _

_M ....,AM _a_4_m- M__-_ ___Mo . - m -.

FIG. 3. Autoradiograms of the sodium dodecyl sulfate/polyacrylamide gels of proteins pulse-labeled with [35S]methionine before (-) andafter (+) L4 oversynthesis was induced. The gel contained 7.5% acrylamide and 0.05% N,N'-methylenebisacrylamide. Arrows indicate theposition of the fusion protein. In AES, the fusion gene was inactivated by deleting the promoter through the beginning of the gene.

Genetics: Freedman et al.

Oft go OR 'W-1*1 oi-oh. 400 .

Proc. Natl. Acad. Sci. USA 84 (1987)

Although our mutant analysis was initially directed towardstudying L4 inhibition of transcription, the mutants alsoyielded information about translational control by L4. Trans-lational regulation had been observed in vitro by Yates andNomura (3), but we had not been able to demonstrate in vivocontrol independent of L4-mediated attenuation. However,we isolated two mutants (the compensatory mutant and theA72 deletion mutant) in which transcriptional control hasbeen eliminated but translational control is left intact. Thesemutants and the two loop mutants provide strong geneticevidence that there are two independent molecular mecha-nisms for L4 control, one acting at the level of transcriptionand the other, at the posttranscriptional level. The require-ments for transcriptional and translational control by L4 areclearly different. Translational control requires only se-quences downstream of base 86; the critical features includethe stem structure and the primary sequence in the loop. Thefunctional relationship between transcriptional and transla-tional regulation is not clear, but the two mechanisms maywork in parallel. When the concentration of free L4 exceedssome critical level, transcripts that have recently beeninitiated may be terminated; transcripts that have alreadybeen elongated past the point of the attenuator would beregulated at the level of translation.We were surprised that base substitutions up to 67 bases

upstream of the initiation codon for the S10 gene affect boththe efficiency and the L4-mediated regulation of translationof the operon. Possibly, this upstream region forms anextended binding site for ribosomes and/or initiation factorsthat ensures efficient translation of the S10 gene. However,since the message from pLF1-A49 is translated with near-normal efficiency, this does not seem a likely explanation.Another possibility is that the S10 leader can form two ormore alternative structures that differ with respect to theaccessibility of the Shine-Dalgarno region and/or the initia-tion codon. Binding of L4 to the leader could change theequilibrium between such alternative forms and therebyregulate the overall translation rate. Although we do notunderstand the molecular basis for translational control of theS10 operon, it is important to note that translational efficien-cy and regulation of translation by L4 are not alwayscorrelated. The compensatory mutation restores L4-mediat-

ed translational control without restoring efficient translationof the fusion gene.One important topic for further investigation is the deter-

mination of the L4 binding site in the S10 leader. The resultspresented here do not address this question, since the loss ofregulation could be due to the loss ofL4 binding or to the lossof the regulatory response to the binding. Our original modelpredicted that the binding site was in the region of the 9-basesequence (bases 117-125) that is also found near the L4binding site on 23S rRNA. However, our data suggest that L4recognition may not require the entire 9-base primary se-quence. A further complication is that the proposed second-ary structure containing the 9-base sequence in the leader isentirely different from the secondary structure of the corre-sponding sequence in 23S rRNA (14).

We thank Patrick Fritz and Joseph McCormick for constructingthe J11 probe and the A72 deletion mutant and Richard Gumport forsynthesizing the oligonucleotide used to construct the 113,114mutation. This work was supported by a Public Health Service grantand a Research Career Development Award (to L.L.) from theNational Institute for Allergy and Infectious Diseases.

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241-248.5. Platt, T. (1981) Cell 24, 10-23.6. Messing, J. (1983) Methods Enzymol. 101, 20-78.7. Freedman, L. P., Zengel, J. M. & Lindahl, L. (1985) J. Mol.

Biol. 185, 701-712.8. Clark, D. J. & Maal0e, 0. (1967) J. Mol. Biol. 23, 99-112.9. Aksoy, S., Squires, C. L. & Squires, C. (1984) J. Bacteriol.

157, 363-367.10. Lo, K. M., Jones, S. S., Hackett, N. R. & Khorana, H. G.

(1984) Proc. Natl. Acad. Sci. USA 81, 2285-2289.11. Zoller, M. J. & Smith, M. (1984) DNA 3, 479-488.12. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl.

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6520 Genetics: Freedman et al.