Proc. Nati. Acad. Sci. USAVol. 84, pp. 6825-6829, October 1987Genetics

Heterologous enzyme function in Escherichia coli and the selectionof genes encoding the catalytic RNA subunit of RNase P

(hybrid ribonucleoproteins/Ml RNA structure/rnpB gene/Enterobacteriaceae)

NATHAN P. LAWRENCE, ADAM RICHMAN, REZA AMINI*, AND SIDNEY ALTMANDepartment of Biology, Yale University, New Haven, CT 06520

Communicated by Leonard S. Lerman, June 15, 1987 (receivedfor review March 6, 1987)

ABSTRACT The gene for the catalytic RNA subunit ofRNase P has been isolated from several Enterobacteriaceae bycomplementation of an Escherichia coli strain that is temper-ature-sensitive for RNase P activity. The selection procedurerelies on the ability of the heterologous gene products tofunction enzymatically in E. coli. This procedure obviates theneed for positive results in DNA blot hybridization experimentsor for the purification of holoenzyme to identify the RNAcomponent of RNase P and its corresponding gene fromorganisms other than E. coli. Comparisons of the variations insequences provide the basis for a refined two-dimensionalmodel of the secondary structure of Ml RNA.

RNase P is an enzyme essential for the formation of the 5'termini oftRNA molecules in Escherichia coli. It consists ofa catalytic RNA subunit and a protein cofactor (1). RNaseP-like activities have also been found in extracts of manyorganisms (2). Hybrid RNase P holoenzymes consisting ofthe catalytic RNA subunit from one organism and the proteincofactor from a different, distantly related organism can beformed in vitro (2, 3). This capability to form hybrid ribonu-cleoprotein enzyme can be the basis of a selection techniquein a suitable host for genes from different organisms. Here wereport a rapid method for the selection ofrnpB genes from thebacterial family Enterobacteriaceae, the family to which E.coli belongs. The method of selection depends on the abilityof the product of a subcloned rnpB gene to complement(presumably by forming functional enzyme in vivo) E. coliFS101, a strain temperature-sensitive in RNase P activity.This method is generally applicable to the selection of anyrnpB gene that can function in E. coli, irrespective ofwhetherthe appropriate DNA is capable of yielding a positive signalin a blot hybridization assay.A model has been proposed for the secondary structure of

Ml RNA from E. coli that is based on a base-pairing schemeupon which additional constraints have been imposed by datafrom limited digestion with ribonucleases (4). This model hasbeen refined to incorporate results from the sequence anal-ysis of the rnpB gene from Salmonella typhimurium (5). Toverify the structural features inferred from these data, it isadvantageous to compare the sequences of genes (rnpBgenes) encoding the analog to Ml RNA. For example, amodel of the secondary structure for P RNA from Bacillussubtilis (the analog of Ml RNA) can be drawn with somefeatures that are similar to the structural features proposedfor Ml RNA (6). Comparisons of nucleotide sequences fromhomologous genes from diverse species have indeed beenused to test the validity ofmodels for the secondary structureof stable RNAs (7). Data from this study have revealedaspects of the transcriptional control regions of the genescoding for the analog to Ml RNA from various Enterobac-

teriaceae and have also been used to suggest alternativeconformations for parts of the model of secondary structureof Ml RNA that was proposed by Guerrier-Takada andAltman (4).

MATERIALS AND METHODSBacterial Strains and Vectors. E. coli strains used in this

study were JM101 [supE, thi, A(lac-proAB), (F', traD36,proAB, lac~q ZAM15)] (8) and FS101 (rnpA49, recA) (9).Other bacterial strains were a gift from Y. S. Park (BradyMemorial Laboratory, New Haven, CT). A clinical isolate ofErwinia agglomerans was identified and classified in thelaboratory of Yale New Haven Hospital. Serratia marces-cens and Klebsiella pneumoniae were obtained from theAmerican Type Culture Collection. Plasmid pUC19 andbacteriophage M13mpl9 have been described (10).Enzymes and Reagents. Restriction enzymes (New England

Biolabs) and bacteriophage T4 DNA ligase (BoehringerMannheim) were used under the conditions recommended bythe vendors. Ampicillin, isopropyl ,B-D-thiogalactopyrano-side, and 5-bromo-4-chloro-3-indolyl P-D-galactopyranosidewere purchased from Sigma.

Preparation of Genomic DNA. Genomic DNA from Ser.marcescens, Erw. agglomerans, and K. pneumoniae wasprepared according to the procedures of Marmur (11).

Construction of Libraries. Plasmid libraries were construct-ed using genomic DNA from the bacterial strains listedabove. One microgram of each of the genomic DNAs wasdigested with the restriction endonuclease BamHI. Therestriction fragments were then ligated into the BamHIrestriction site of plasmid pUC19 with T4 DNA ligase (12).pUC19 (0.1 ,g) cleaved with BamHI had been dephosphoryl-ated prior to use in the ligation procedure (13). One-tenth ofthe ligation mixture was used to transform JM101 accordingto the procedure of Mandel and Higa (14). One-tenth of thetransformed cells were plated on Luria-Bertani (LB) agarplates supplemented with ampicillin (50 ,ug/ml), 20 ,ul ofisopropyl (3-D-thiogalactoside (100 mM), and 100 ,l4 of 5-bromo-4-chloro-3-indolyl f3-D-galactoside (2%) to verify theinsertion ofDNA at the BamHI restriction site. The remain-ing transformed cells were added to 1.0 liter of LB brothsupplemented with ampicillin (50 ,ug/ml) for use in a large-scale plasmid preparation according to the procedure ofThompson et al. (15). Plasmids prepared by this procedureconstitute the libraries that were used to transform FS101.

Selection of rnpB Genes. FS101 was transformed with eachof the libraries according to the procedure of Mandel andHiga (14) with the following modifications. Incubation priorto plating was at 30°C instead of 37°C, for 24 hr instead of 1hr. Transformed cells were plated, in duplicate, on LB platessupplemented with ampicillin (50 ,ug/ml). One set of plateswas incubated at 30°C for 48 hr, and the other set ofplates was

*Present address: Department of Biochemistry, School of Medicine,Medical Sciences Center of Iran, Tehran, Iran.

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incubated at 43°C for 48 hr. Plasmid DNA isolated fromtransformants that grew at 43°C was examined further.

Southern Hybridization. Plasmid DNAs isolated fromtransformants that grew at the restrictive temperature weredigested with restriction enzymes and transferred to nitro-cellulose filters (16). The DNAs were hybridized to isotopi-cally labeled RNA transcripts derived in vitro from either acloned rnpA gene (coding for C5 protein) from E. coli or acloned rnpB gene (for Ml RNA) from E. coli in an SP6transcription vector (12). Probes were a gift from M. Baer(Department of Biology, Yale University).

Nucleotide Sequence Analysis. Nucleotide sequence analy-sis of the rnpB genes was facilitated by the subcloning ofrestriction fragments into bacteriophage M13mpl9. Nucleo-tide sequences were determined by the dideoxy chain-termination method (17). Two different primers were used togenerate sequence data: the universal sequencing primer,provided with the Bethesda Research Laboratories kit, and a19-base oligonucleotide, specific for the mature domain of theE. coli rnpB gene. This primer anneals between positions 190and 209. The rnpB-specific primer was a gift from N.Lumelsky (Department of Biology, Yale University).

Labeling of Ml RNA in Vivo. Uniformly labeled Ml RNAand Mi-like RNAs were prepared by the procedures ofLawrence and Altman (18).

RESULTSSelection of rnpB Genes. E. coli FS101 was transformed

with plasmid libraries representing total genomic DNA fromSer. marcescens, K. pneumoniae, and Erw. agglomerans.FS101 is temperature-sensitive for RNase P activity becauseofa mutation in the gene coding for C5 protein (19). However,after transformation with the plasmid libraries, discretecolonies of FS101 formed overnight on LB/ampicillin platesat 43°C. In comparison, at the permissive temperature abacterial lawn was formed after transformation with each ofthe libraries.

a

Plasmid DNA purified from transformants that grew at therestrictive temperature was used to retransform FS101.Following this second round of transformation, FS101formed a lawn at both 30'C and 43TC. This indicates that theplasmids selected from the three libraries were responsiblefor complementing the RNase P mutation in FS101 and thatgrowth at the restrictive temperature was not due to areversion of the RNase P mutation. The mutation in FS101can be complemented by plasmids carrying either an rnpAgene (the gene coding for C5 protein) or an rnpB gene (thegene for Ml RNA) (9, 19). To determine which gene or geneswere responsible for supporting the growth of FS101 at 430C,plasmid DNA obtained from each of the temperature-resist-ant isolates was transferred to nitrocellulose filters andhybridized to an isotopically labeled probe in a Southernhybridization assay. The probes were derived by transcrip-tion in vitro from the DNA of either the rnpA gene or the rnpBgene from E. coli. Only the probe derived from the E. colirnpB gene hybridized to the isolated plasmids (data notshown). These data, analogous to the results of Motamedi etal. (9), indicate that all of the temperature-resistant trans-formants that were tested harbor a plasmid that carries anrnpB gene.

Sequence Analysis for the rnpB Genes. For further refer-ence, the plasmid isolated from the Ser. marcescens, K.pneumoniae and Erw. agglomerans libraries were namedpSrnpB, pKrnpB, and pErnpB, respectively.The nucleotide sequence of rnpB gene isolated from the K.

pneumoniae library was identical to the sequence determinedfor the corresponding gene from Erw. agglomerans. Further-more, transcripts derived in vivo from FS101 cells that harborpKrnpB or pErnpB have the same mobilities in denaturingpolyacrylamide gels (data not shown). However, restrictionmaps of the DNA upstream from the rnpB genes are different(data not shown). This difference may be due to variations inthe nucleotide sequences of the genomes flanking the rnpBgenes or to the spontaneous deletion ofDNA during cloningin the case of pKrnpB. The rnpB genes from these twospecies appear to be identical, but one must recall that there

b

IV

I

FIG. 1. A model of the secondary structure of Ml RNA from E. coli with phylogenetic variations in the nucleotide sequence indicated onthe model. (a) Specific structural features discussed in the text are designated by Roman numerals. Arrows pointing away from the model indicatedifferences in the nucleotide sequence of the transcript derived from the rnpB gene from K. pneumoniae and Erw. agglomerans. An arrowpointing toward the model indicates the addition of a nucleotide at that position relative to E. coli. The double arrow at structure III indicatesa substitution of eight nucleotides for four nucleotides that are found in Ml RNA from E. coli. (b) Designations are the same as in a. Thedifferences in the transcript derived from the rnpB gene from Ser. marcescens are shown. The letter D indicates the deletion of a nucleotideat that position relative to Ml RNA from E. coli.

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have been problems in distinguishing between Erw. agglom-erans and K. pneumoniae (20).

Analysis of the rnpB genes isolated from K. pneumoniaeand Erw. agglomerans revealed 12 differences (Fig. la).There are two additions to the E. coli sequence of rnpB, acytosine after position 38 and a guanine after position 42,constituting a new base pair near to the loop end of stem I.A tetranucleotide at positions 111-114 in E. coli is replacedby an apparently unrelated octanucleotide in an unpairedregion. There is a G-IA transition at position 87 of Ml RNAin stem II and a T-IC transition in a short helix at position223. The net result of all these changes is the addition of sixnucleotides to the primary transcript, consistent with therelative mobility in a denaturing polyacrylamide gel of atranscript derived from pErnpB and KrnpB in vivo.The differences in nucleotide sequences in the rnpB gene

from Ser. marcescens compared to that from E. coli includea total of 21 transitions, 2 transversions, 2 additions, and 1deletion, relative to the rnpB gene from E. coli. The net resultis a transcript one nucleotide longer than Ml RNA.The changes in the rnpB gene from Ser. marcescens do not

appear to be randomly distributed throughout the gene.Rather, there are regions within the gene where the changesare clustered (Fig. lb). For example, within the final 10nucleotides of the region of the gene corresponding to themature RNA, there are five transitions and a deletion relativeto the rnpB gene from E. coli. Another region of complexvariability occurs at the position of tetra- to octanucleotidesubstitution in the rnpB gene from K. pneumoniae and Erw.agglomerans (see Fig. la), where Ser. marcescens replacestwo adjacent guanines at positions 110 and 111 by twocytosines, one after position 110 and the second after position111. There are also four transitions, occurring at positions108, 114, 117, and 123.

Phylogenetic Comparisons of DNA Flanking rnpB Genes. Acomparison of the nucleotide sequences of the regionsupstream and downstream from the mature domain of thernpB genes suggests that the basic organization of the gene ismaintained in each of the species studied. Alignment of theflanking sequences upstream of the rnpB genes reveal -35and -10 consensus sequences in excellent agreement withthose of E. coli promoters (Fig. 2a) (24). However, in Ser.marcescens, there is a T->C transition at position -8 relativethe the rnpB gene from E. coli. The spacer region betweenpositions -35 and -10 is more variable than the nucleotidesequences of the mature domains of the genes.Located between the -10 consensus sequence and the

start of the mature domain of the rnpB genes is a G+C-richregion that fits the criterion for the consensus sequence ofgenes under stringent control (25). The sequences in thisregion are identical in E. coli, S. typhimurium, K. pneumo-niae, and Erw. agglomerans. In this region, Ser. marcescenshas a single nucleotide that differs from the sequences in thespecies listed above. This sequence is still in agreement withthe consensus sequence (Fig. 2a). The data further show that,at approximately positions -70 to -80, relative to the maturedomain of the gene, a sequence homologous to the -10consensus sequence of E. coli promoters is present (26, 27).In both Erw. agglomerans and Ser. marcescens, a G+C-richsequence is also present that is similar to the consensussequence associated with stringent control. In the subclone ofthe rnpB gene isolated from the K. pneumoniae library, DNAcorresponding to this region of the gene is absent (27).Comparisons of DNA immediately downstream from the

mature domain of the rnpB genes show that nucleotides inthis region are subject to greater drift (Fig. 2b). Nonetheless,this region is transcribed as part of the precursor to Ml RNA.Although it is within this region that processing ofthe primarytranscript occurs to yield mature Ml RNA, there is nocompletely uniform sequence downstream from the mature

a-35 -10 +1

1 CGCAACGCGGG GTGACA AGGGCGCGCAAACCCTC TATACGCGCGCC G

2 CGCAGCGCAGG GTGACA MGTCGCGCGAACCCAC GCGCGCC G

3 TACCACTAGGG ATGACA ACGGGCGGTAAACCCTC TATAC GCGCGCC G

4 CCGTAAAGCGG TTGACC TGCCGCCGTCGCGACGC ACTA CCGCGCC G

bCAGTTTCACCTGATTTACGTAAAMCCCGCTTCGGCGGGTTTTT

GCTTT~ ~AAGAAGATGAGAATG [ORFS]*

2

3

CAGTTTCACTTCTTCATAAAACCCGCTTCGGCGGGTTTTT

GCTTTTAC2ogCGGCAGGATGAATG [OFs 1

CAGTTTCACCTTTTACGCAAACCCCGCTCCGGCGGGTTTTTGC

TATTCGMTAGCIEGA4GGATGAATG

4

[ORrasI

iTCAACTCCCTCGACAAAAAGMAACCCCGCGCAACGCCAGTTGTGGTTTTTCGTTT

CAGCCG.AC G CC

A AC C

C G UC UC C

UC UCC C UcG C CCCc C C CcCC ltic C C C UCC U C CC AUCC ' Cc AUCC z~~C CC A

AiU CG AU AUAU Cc AU AUAU CC AU AU CAU AU AU AUAU AU AU AUAU AU AC AU

1 CGCUUUU 2 UAUUCCUUUI 3 CC-CtAUU 4 CArmX

FIG. 2. Comparisons of regions flanking the sequences of themature RNAs encoded by the rnpB genes from various Enterobac-teriaceae. (a) DNA sequence of the flanking regions upstream fromthe rnpB genes of E. coli (row 1; ref. 21), S. typhimurium (row 2; ref.5), K. pneumoniae and Erw. agglomerans (row 3), and Ser. mar-cescens (row 4). The sequences are aligned to facilitate comparisonof specific sequence elements. The -10 consensus sequences areshown in boxes. The -35 consensus sequences are underlined. Thesequences associated with stringent control are in italics. Thenumbers above the sequences define the position of the nucleotidesrelative to the mature domain of the gene. (b) DNA sequence of theflanking regions downstream from the rnpB genes of E. coli (row 1;ref. 22), S. typhimurium (row 2, ref. 5), K. pneumoniae and Erw.agglomerans (row 3), and Ser. marcescens (row 4). The sequenceshown starts with the final 10 nucleotides of the mature domains ofthe genes to facilitate alignment of the sequences. The arrowindicates the last nucleotide of the mature domain of the gene.Inverted repeats are underlined. Shine-Dalgarno sequences (23) arein boxes and the first potential ATG initiation codon is designated byan asterisk. Sequence of the region downstream from the invertedrepeat in Ser. marcescens is not available for comparison and isindicated by dots. ORFs, open reading frames. (c) Potential p-independent terminator structures formed by the inverted repeatsshown in b. Row 1: E. coli (21). Row 2: S. typhimurium (5). Row 3:K. pneumoniae and Erw. agglomerans. Row 4: Ser. marcescens.

domain. However, all of the sequences contain invertedrepeats that can form a stable hairpin structure, followed bya series ofuridine residues (Fig. 2c). In E. coli, the rnpB genesfrom Ser. marcescens, K. pneumoniae, and Erw. agglomer-

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ans are transcribed as precursors and processed to yieldshorter, mature RNAs. The sizes of the primary transcriptsare in agreement with the sizes of transcripts that areterminated at the expected stop signals.

DISCUSSIONIsolation of rnpB Genes. The rnpB genes from Ser. mar-

cescens, K. pneumoniae, and Erw. agglomerans were se-lected by a technique that utilizes complementation of an E.coli RNase P mutant. This selection method depends on thecapability of the heterologous gene products to form func-tional enzyme in vivo. Such capability has been demonstratedwith plasmids harboring the rnpB gene from S. typhimurium(5) and recently with plasmids harboring the rnpB gene fromB. subtilis (unpublished experiments) even though there isinsufficient sequence homology between Ml RNA from E.coli and the analogous RNA from B. subtilis to yield apositive signal in a Southern hybridization analysis (21).Therefore, complementation provides a means to rapidlyselect rnpB genes that cannot be identified by the Southernhybridization assay. This selection technique is, of course,limited to rnpB genes that can be expressed and whose geneproducts form functional enzyme in E. coli. Both the proteinand RNA subunits of RNase P are essential for E. coliviability, although we cannot rigorously exclude the possi-bility that the RNA component alone, when present inexcess, can complement FS101 (18). One explanation for thepreferential section of rnpB genes rather than rnpA genes by

the methods described above is that, like the rnpA gene fromE. coli (19), the rnpA genes from the strains used for thisstudy may also contain restriction sites of BamHI, therestriction enzyme used for cloning. In this event, onlypartial, nonfunctional rnpA genes would be included in theplasmid libraries.Analyses of the rnpB genes from Ser. marcescens, K.

pneumoniae, and Erw. agglomerans have shown that thenucleotide sequences coding for promotion and terminationof the Ml RNA analogs are closely conserved. The data havealso been used to reexamine a model of the secondarystructure of Ml RNA. The first model of the secondarystructure ofMl RNA was based on a theoretical base-pairingscheme (21). Model 1 was revised first to incorporate datafrom studies of partial nuclease digestions of the RNA (4) andlater to include data from the study of the rnpB gene from S.typhimurium (M. Baer and S.A., unpublished work), givingmodel 2. In model 2, four of the six nucleotide differences inS. typhimurium can be explained as compensatory changes.All other features of model 2 are consistent with the data thatwas used to generate model 1. To test the validity of model2, the sequence differences found in the rnpB genes from Ser.marcescens, K. pneumoniae, and Erw. agglomerans arepresented at the positions where they occur in model 2 in Fig.1. Although more examples are required to fully test thefeatures shown, the criteria used by Woese et al. (28) andNoller et al. (29) to test specific features of a model of 23SrRNA have been applied. Two independent examples ofcompensatory base changes within a putative helical region

40u cU

U GC GGUOGC G

U A

U eG

U * A

C * GU.AGCo*GG GC o G.. A

soC * G C U oG A C AC 6G U GU A ,, C GG AAGCCGGG CA¢ "*UUCGGCCC CA A AG U A S.A C GC6CoG A A C

C 0CCnGOC C

VI 0

pp ACCU

I cI.0

A C to

U GG CG CA *U cGAGAGWC * GG A

; G A^AACCAC G AC

UU 00A U GGCAUGGG 6G C C G C* A GU CC G 0° C UAv C CarGI CU AAUGGA C C U UGA6 U G A Co cA 8 uC

AACC

A GG*CAUG C

AGU GO C~~~~60

s ~~~AAU CC5CUA CA CAAC CC AA CGAA C

FIG. 3. A new secondary-structure model ofMl RNA from E. coli. This model is based on the results of site-directed mutagenesis experiments(18) and phylogenetic comparisons of the rnpB genes from a variety of bacteria, including structural elements proposed in a study of the rnpBgene of B. subtilis (6). Some of the structural elements shown in this model are retained from the previous model (see Fig. 1); these elementsare indicated by Roman numerals. Sites of compensatory base changes are circled. The'line in structural element I indicates the addition of apair of nucleotides in K. pneumoniae and Erw. agglomerans. The circled pair of nucleotides in structural element IV was determined from thesequence of S. typhimurium (5). The arrow points to the structural element that was suggested by studies of mutations in Ml RNA (18) andpsoralen-cross linking experiments (31)

C,

y COGGoCC *GC *G

&OOA o UC AG AC 6G A

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were considered proof of the existence of the helix.Uncompensated base changes, which result in mismatches(except in the case of G-C to GNU), were considered to beevidence against the helix (7). Thus in model 2 shown in Fig.1, our data strongly support the existence of features I andVI.

Features III and V are not supported by the comparativesequence data. Feature III occurs within a highly variableregion of the molecule. Examples of uncompensated basechanges are found in the rnpB genes of Ser. marcescens, K.pneumoniae, and Erw. agglomerans. Although the basechanges at nucleotide position 114 are transitions that couldform GNU pairs, such base pairs at the beginning of stems addlittle to the stability of the structure (30). These data are inagreement both with site-specific mutagenesis studies (18)and with crosslinking data (31), which suggest an alternativestructure for feature III, as shown in the scheme for model 3illustrated in Fig. 3. Feature III of model 2 is replaced by thestem and loop indicated by the arrow. Feature V of model 2,a stem consisting of four base pairs and a loop of only threebases, shows two examples of uncompensated base changesand is removed from model 3. Evidence for or against theexistence of other specific features in model 2 is limited atpresent to single examples of nucleotide changes and cannotbe considered convincing.A Refined Model of Ml RNA. The data derived from this

study support some aspects ofthe model ofMl RNA that wasbased on theoretical base pairings (32), patterns of nucleasedigestion of the RNA, and comparisons of the nucleotidesequences ofthe rnpB genes from E. coli and S. typhimurium.A refined model of the structure ofMl RNA is shown in Fig.3. This model incorporates the data used to generate previousmodels of Ml RNA and the results of phylogenetic compar-isons presented in this study. The model also incorporatessome structural features proposed by Reich et al. (6), basedon the sequence of the RNA component of RNase P from B.subtilis and a structural element suggested by a site-directedmutagenesis study (18).Model 3, as shown in Fig. 3, can only be considered a way

station on the road to a final structure to be confirmed at somepoint by crystallographic studies. Nevertheless, it providesan immediate basis for incorporating results of analyses bothof mutants in the rnpB gene ofE. coli (M. Baer, L. Kirsebom,N. Lumelsky, and S.A., unpublished work) and of possibletertiary interactions (e.g., nucleotides 11-18 with 303-296; G.McCorkle and S.A., unpublished work). Until a much largercollection of analogous sequences is available, it also pro-vides a new starting point for designing further probes of thestructure of Ml RNA.

We are grateful to Mrs. Donna Wesolowski for excellent technicalassistance. This work was supported by grants from the NationalInstitutes of Health and the National Science Foundation (to S.A.).

N.P.L. and A.R. were recipients of predoctoral training grants fromthe National Institutes of Health.

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6327-6334.5. Baer, M. & Altman, S. (1985) Science 228, 999-1002.6. Reich, C., Gardiner, K. J., Olsen, G. J., Pace, B., Marsh,

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Inst. Health, Bethesda, MD), NIH Publ. No. 79-99, Vol. 2,No. 2, p. 43.

9. Motamedi, H., Lee, K., Nichols, L. & Schmidt, F. J. (1982) J.Mol. Biol. 162, 535-550.

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Cloning: A Laboratory Manual (Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY).

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