The Nucleotide and Partial Amino Acid Sequence of Toxic Shock ...

Vol. 261, No. 33, Issue of November 25, pp. 15783-15786,1986 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY rc) 1986 by The American Society of Biological Chemists, Inc.

The Nucleotide and Partial Amino Acid Sequence of Toxic Shock Syndrome Toxin-l* (Received for publication, May 23, 1986)

Debra A. Blomster-Hautamaazg, Barry N. Kreiswirthlf, John S. Kornblumlf, Richard P.Novicklf, and Patrick M. Schlievertz From the $Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 and 7The Public Health Research Institute of the City of New York, Znc., New York, New York 10016

likely responsible for the symptomsof TSS (5-8). The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, The biological activities of TSST-1 have been examined one-third of the predicted amino acid sequence was extensively to build a relationship between the toxin’s effects confirmed by amino acid sequence analysis of cyanogen and the symptoms of TSS. The biological activities include: bromide-generatedTSST-1proteinfragments.The the capacity to induce fever, enhancement of host susceptipair bility to lethal shock by endotoxin, nonspecific T lymphocyte DNA sequencingresultsidentifieda708-base open reading frame starting with ATG, an 7 base pairs mitogenicity, suppression of immunoglobulin M synthesis downstream froma Shine-Dalgarno sequence, and ter-against sheep erythrocytes, enhancementof delayed type hyminating ata UAA stop codon. Amino acid analysisof persensitivity reactions, and induction of immunological tolthe intact protein defined the NHZ terminus of the erance in certain rabbits (9). The TSS model proposed by mature protein and located the cleavage point for the Schlievert (10) correlatesthese biological functionstothe signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic struc-pathology seen in TSS patients. To date, TSST-1 has been purified and biochemically chartural similarities with other bacterial signal peptides. acterized (11). In addition, the TSST-1 structural gene has The coding sequence of the mature protein was 585 base pairs (194 aminoacids) in length, and themolec- been cloned from the bacterial chromosome (12). The toxin ular weight of the predicted protein was 22,049. This was purified to homogeneity by differential precipitationwith is ingood agreement with the previously reported mo- ethanol and resolubilization in water followed by successive lecular weight of TSST-l (22,000), as determined by electrofocusing in pH gradientsof 3-10 and 6-8 (11).TSSTsodium dodecyl sulfate-polyacrylamide gel electropho- 1, thus isolated, migrated as a single band in SDS-polyacrylper- amide gel electrophoresis gels to a molecular weight of 22,000. resis. NH2-terminal amino acid sequence analysis formed on isolated TSST-1 CNBr fragments deterThe toxin gene was localized within a 10.6-kb chromosomal mined the position of the peptides in the TSST-1 seinsertcontained in plasmid pRN6100 and was previously quence and verified the predicted amino acid sequence shown t o produce TSST-1. Analysis of subclones of the 10.6in those positions. Computer analysesof the aminoacid kb insert in pBR322 assigned the structural gene to the leftsequence showedthat TSST-1 has little or no sequence most 3.2-kb ClaI fragment (12). homology with biologically related toxins, streptococHere we report the nucleotide and partial amino acid secal pyrogenic exotoxin A, and staphylococcal entero- quenceanalysis of TSST-1. These experiments were contoxins B and C. ducted to determine the complete amino acidsequence of TSST-1 as an approach to understanding the multiple structure-function relationshipsof this toxin molecule. Toxic shock syndrome(TSS’)is a multisystem illness charMATERIALSANDMETHODS acterized by the acute onset of highfever, hypotension or dizziness, rash,desquamation of skinupon recovery, and M13 and DNA Sequencing-DNA sequencing was performed by variable multisystem involvement (1-4). Staphylococcus au- the dideoxynucleotide chain termination method of Sanger et al. (13), reus producing TSST-1 has been isolated from nearly 100% following the subcloning of isolated DNA fragments from pRN6101 of menstrual-associated TSS patients (5,6). Since bacteremia into M13 vectors mplO and m p l l (12). A universal M13 sequencing primer (17 nucleotides, Boehringer Mannheim) was used in the rarely occurs in TSS, it was proposed that a staphylococcal annealing reaction. exotoxin was causing thewidespread systemic effects. TSSTPreparation of TSST-1 CNBr Fragments-TSST-l was purified 1 has been cited by several investigators asa major toxin most as previously described (11).The CNBr reactions were performed as * This work was supported by National Institutes of Health Research Grant A122159. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide seqwnce(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank withaccessionnumber(s) 502615. § Supported by National Institutes of Health Training Grant HLI 07114. The abbreviations used are: TSS, toxic shock syndrome; TSST1, toxic shock syndrome toxin-1; SDS, sodium dodecyl sulfate; bp, base pairs; kb, kilobase pairs.

reported by Gross (14) using a 2OOO:l molar ratio of CNBr:TSST-1. The formic acid and excess CNBr reagent were removed by lyophilization. As a control, toxin was incubated with formic acid alone and then analyzed by gel electrophoresis. No significant protein degradation occurred due to the acidic reaction conditions. Separation of CNBr Fragments byGel Filtration-Lyophilized TSST-1 fragments (5-10 mg) were solubilized in 30% acetic acid and then loaded onto aSepbadex G-75 column (1.7 X 120cm) equilibrated with 10% acetic acid. Fractions (2.0 ml each) were eluted with 10% acetic acid at a flow rate of 12 ml/h. The acetic acid was removed from each fraction by lyophilization. After lyophilization, column fractions containing a visible amount of material were suspended in 0.2 ml of water. The protein content of each fraction was then analyzed on a 10-18% linear gradient SDS gel(1-5 pgllane) to

15783

Sequence of Toxic Shock Syndrome Toxin-1

15784

ascertain which toxin fragments were present ineach sample. Protein concentrations were estimated by use of the Bio-Rad assay. ap~ r e a - S ~ ~ - G r a d ~ e n ~ PGet o ~Electrophoresis-The y~~~mide proximate molecular weight of the CNBr-generated TSST-1 fragments were determined using a 10-18% lineargradient SDS gel containing 7 M urea and a cross-linking ratio of 20:l (15, 16). The gel bands were visualized by silver staining (17). Estimates of fragment sizes were obtained by comparison with standards: ovalbumin (43,000), a-chymotrypsinogen (25,700), 8-lactoglobulin (18,400), lysozyme (14,300), bovine trypsin inhibitor (6,200), and insulin chain A (3,000). Amino Acid Sequencing-Amino acid sequences were determined by automated Edmandegradation in agas-phase sequenator (Applied Biosystems Model 470A, under program 03RPTH) as described by Hewick et al. (18). The purity of each CNBr fragment and intact TSST-1 analyzed was assessed by SDS-gel electrophoresis. Purified pept.ide and intact toxin samples were immobilized in the reaction cartridge by means of Polyhrene-impregnated glass fiber filters. The amount of peptide loaded in each run was in the range of 80-400 pmol. An aliquot (YIz) of the phenylthiohydantoins a t each cleavage was injected directly into a high performance liquid chromatograph (Model 120A, Applied Biosystems, Foster City, CA). Resolution of the phenylthiobydantoins was accomplished on a reverse-phase column (Brownlee “Spheri-5” C18, 0.21 X 22 cm) with a complex gradient composed of 114 mM acetate, pH 4.0, in 5% tetrahydrofuran (solvent A) and acetonitrile (solvent B), as specified in the technical bulletins of Applied Biosystems. Computer Analysis-The amino acid sequences of TSST-1, staphylococcal enterotoxins B and C (19, 20), and streptococcal pyrogenic exotoxin type A (21, 22) were compared using the computer program Fast Protein Data Base (FASTP) written by Lipman and Pearson (23; sequence analysis programs distributed by the National Institutes of Health). This program is based on modificatjons of the algorithm of Wilbur and Lipman (23). T o determine the statistical significance of sequence similarities, we employed Monte Carlo analysis using another algorithm written by Lipman and Pearson. RESULTS AND DISCUSSION

Nue~otideSequence Analysis of the TSST-1 Gene-Previously, subclones of pRN6101 were isolated which failed to express the TSST-1protein (12).These results indicated that a 300-bp BamHI-HincII fragment was required for expression and established the sequencing strategy (Fig. 1) since it could be assumed that this region was within or very close to the TSST-1struct.ura1gene. Using the dideoxy chain termination method of Sanger (131, the overlapping 1.1-kb HincII and the 1.0-kb BamHIfragments, which bracket the gene-specific region, were separately cloned into coliphage M13 and sequenced. The completion of the entire nucleotide sequence of the tst gene and its controlling regions required additional cloning and sequencing of subfragments depicted in Fig. 1.

The final percentage of the sequence determined from both strands was 70%. The toxin sequence, presented in the 5‘ to 3‘ orientation, starts in theAluI fragment and extends to the 3’ end in the HincII fragment (Fig. 2). The sequencing data identified a 705-bp open reading frame starting with an ATG at nucleotide position 478 and terminating at a TAA nucleotide number 1180. A good consensus Shine-Dalgarno site lies 7 bp upstream of the ATG start position (dotted line in Fig. 2). Amino acid sequence analysis of intact TSST-1 identified the NE, terminus of the matureTSST-1protein as: Ser-Thr-Asn-AspAsn-Ile-Lys-Asp-Leu, thus confirming previous studies of Igarashi etal. (24). In addition, thesedata defined the cleavage point for the signalpeptide at an Ala/Ser sequence and determined the length of the signal peptide to be 40 amino acids. The signal peptide is indicated by a solid line in Fig. 2. The TSST-1signal peptide containedthe characteristic structural homologies found in other bacterial signal peptides: (a) 1-3 basic amino acids at theamino terminus, (b) a hydrophobic region of approximately 15 residues, ( e ) a Pro or Gly in the hydrophobic core, (d) a Ser or Thr near the carboxyl terminus of the core, and (e) an Ala or Gly at the cleavage site (25). The coding sequence of the mature protein was 585 bp in length (194 amino acid residues), and thecalculated molecular weight was 22,049, which is in complete agreement with the molecular weight of TSST-1 determined by SDS-polyacrylamide gel electrophoresis, 22,000 (11). Generation and Isolation of CNBr Peptides-The TSST-1 protein fragments generated by CNBr cleavage are shown in Fig. 3. CNBr cleaved the toxin at its 2 methionine residues, producing five peptide fragments. The estimated masses of the fragments were: CN1,18 kDa; CN2,17 kDa; CN3,14 kDa; CN4,6-8 kDa; and CN5,4kDa. The mixture of CNBr-generated toxin fragments was then applied to a Sephadex G-75 gel filtration column. The elution profile of the column fractions showed one broad protein peak. Therefore, each column fraction containing a visible

“ ACA ACT GCTACA GAT 111 ACC CCT GTT CCC TTA TCA TCT M T U A ATA ATC AM ACTGCA AAA GCA Thr I l e A l a The Asp PheThr Pro Val Pro Leu Ser Ser A l n Gln l l e I l e LYS Thr A l a LYS A l aI

TCT ACA MC GAT AATATA MG GAT TTG CTA GAC TGG TAT RGT AGTGGG TCT W C ACT T T l ACA M T Ser Thr A m Asp Arn I l e Lys Asp Leu Leu Asp Trp Tyr Ser Ser G l y Ser Asp Thr Phe Thr Asn .22

0

AGT GAA GTT TTA GATAAT TCC TTA GGA TCT ATGCGTATA AM M C ACAGAT GGC K C ATC AGC TTG Ser G l u Val Leu Asp A m Ser LeuGlySet *ET Arg I l e Lys A m Thr Asp Gly Ser Ile Set Leu

+u

*.e

m

8

H

ACA MT ACT M A R I A TTA CCT ACT CCA I T A GAACTA CCT TTA AM GTT M G GTT CAT 667 A M GAT lhr A m Thr Glu Lys Leu Pro Thr Pro Ile Glu Leu Pro Leu Lys Val Lys V a l His G l y Lyr +$*a Asp

NX CCC TTA WG . TAT ujc CCA R I G TTC GAT A M AAA Ser Pro Lee Lys Tyr T r p Pro Lys Phe Asp Lyr Lys

A

A

A A

CAA TTA GCT ATA T U ACT TTA GAC TTT 6U Gln Leu ala I l e Ser Thr Leu Asp F-he r130 Glu

C I A ACT CAR RTA CAT GGA TTA TAT CGT TCA AGC GAT A M ACG GGT GGT TAT TGG ATT CGT CAT CAG I l e Arg H i s G l n Leu Thr Gln I l e H i s G l y Leu Tyr Arg Ser Ser Asp Lys Thr Gly G l y T y r *m. Trp

”

D

D I

”

FIG. 1. Restriction map and sequencing strategy for the tst gene. The top line depicts the nucleotide positions of the restriction sites on a pRN6100 subclone. The cross-hatched line indicates the signal sequence and theblackened line designates the coding sequence of mature TSST-1. Thesolid lines below showthe subclones generated for sequencing and therestriction enzymes used. A, AluI; B, BamHI; L), DdeI; H , tiincif. The arrows indicate the direction and distance of the sequence analysis.

AAT GAC GGA TCC ACA TAT C A I AGT GAT TTA TCT AAA M G TTT GAA T I C AAT ACT AAAATAACAATG Lys I l e Thr MET Asn Asp Gly Ser Thr Tyr G l n Ser Asp Leu Ser LysLys Phe Glu T y r Asn Thr +IS0 *$I6 &%A A M CCP, CCT AT& AAT AT1 GAT GAIl ATA AAA ACTATA

GAA GCAGAA

ATT ART TAA TTTMCACTTT

Glu Lyr Pro Pro l i e Afn l l e Asp Glu lie LYS l h r l l e G l u A l a G l u l l e Asn Ter t1 0 1

FIG. 2. The nucleotide sequenceof TSST-1 and the deduced amino acid sequence. The boxed area indicates those predicted residues which were verified by amino acid sequence analysis of the intact TSST-1 and the CNBr-generated fragments. The solid line identifies the signal sequence. The dotted line designates the ShineDalgarno sequence.

Sequence of Toxic Shock Syndrome Toxin-1 I

*0

2

43

x

t . I =

.-0 25.7

2 18.3

-I3

14.3

$

6

=

3

JCN I \CN2 - CN3

u

-CN4 -CN5

FIG. 3. 10-18% linearSDS-gradientpolyacrylamide gel electrophoresis. Lune 1 purified TSST-1 (3.5 pg), lane 2 purified

TSST-1 cleaved by CNBr (4.0 pg). The protein bands were detected by silver staining. The proteins used as molecular weight standards were ovalbumin (43,000), a-chymotrypsinogen (25,700), 0-lactoglobulin (18,400),lysozyme (14,300), bovine trypsin inhibitor (6,200), and insulin A chain (3,000). I Ser

33

I 58 Met

Met

68

34

I59

34 Arg

57

quenced protein. A sample containing CN1 and CN2generated two unambiguous peaks at each amino acid position (24 cycles, repetitive yield 91%). These two signals were easily resolved by examining the inferred aminoacid sequence; one signal depicteda peptide originatingat the NH2 terminus, the second signaldescribed a fragment starting at methionine residue 33. The positions of CN1 and CN2 were determined by comparing the SDS gel estimated molecular weights and the molecular weights calculated from the predicted amino acid sequence (Table I). Peptide CN5 was not retrieved from the gel filtration column and, therefore, was not sequenced (indicated by the completely open box in Fig. 4). The position of CN5 is proposed based on the SDS gel estimated molecular weight and the calculated molecular weight of the predicted sequence. All of the SDSgel estimated molecular weights are in good agreement with the predicted amino acid sequence values except for CN4 (Table I). CN4 is an acidic fragment, and it isknown that SDS-polyacrylamide gel electrophoresis overestimates the molecular weight of several other acidic proteins (26, 27). Each of the samples described above was analyzed once, but analysisof the CN1, CN2 preparationverified the earlier CN3 results, and intact toxin analysis confirmed and extended the CN2 assignments. The boxed sequences in Fig. 2 identify the inferred amino acid sequence which was confirmed by amino acid sequence analysis of intact TSST-1 and CNBr-generated fragments. Approximately one-third of the nucleotide sequence was con-

CN4

CN5~4

seu

184 Asp

Asn -3

I I

134

Asn

TABLE I 7

Molecular weights derived for the CNBr fragments

CN2

Thr

I I

34

15785

CNBr fragment

SDS gel estimated"

Calculatedb

CN1 CN2 CN3 CN4 CN5

18,000 17,000 14,000 6,000-8,000 4,000

18,246 17,777 14,172 4,123 3,605

CN I

TSST- I

FIG. 4. The location of the TSST-I CNBr fragmentsin the primary structure. The top line represents the TSST-1 inferred amino acid sequence. The solid area of each box represents the sequenced region of each fragment and of intact TSST-1. The open area indicates the remaining unsequenced peptide.

Values were estimated from the SDS gel analysis shown in Fig. 3. bValues were calculated for each fragment from the predicted amino acid sequence listed in Fig. 2.

amount of lyophilized material was analyzed on a 10-18% linear gradient SDSgel (gel not shown). The SDS gel analysis TABLEI1 showed that fraction numbers39 and 61 contained separated, Amino acid composition predicted from the nucleotide sequence homogeneous bands of CN3 (14 kDa) and CN4 (6-8 kDa), Amino acid Corrected" Previousb respectively. Fraction number 35 contained both CN1 (18 Aspartic acid 14 13 kDa)andCN2(17kDa)fragments;none of thecolumn 10 Asparagine fractions contained anisolated preparation of CN1 11 andCN2. 18 Threonine 19 CN5 was not recovered from the column. Fraction numbers 19 Serine 21 35, 39, and 61, as well as intact TSST-1, were used in the Glutamic acid 11 11 amino acid sequence analysis. 6 Glutamine 6 Amino Acid Sequence Analysis of the CNBrPeptides10 Proline 10 Glycine 11 10 Purified protein fragments were analyzed by automated Ed7 Alanine 3 man degradation to determine the amino acid sequence of 0 0 Half-cystine each fragment. These data were usedto determine the position Valine 5 5 of each CNBr fragment in the TSST-1 molecule, as well as 1Methionine 2 verifying the inferred aminoacid sequence in thosepositions. 15 Isoleucine 17 Furthermore, defining the position of each fragmentis critical 15 16 Leucine Tyrosine 9 11 for structure-function studies; localizing a specific biological 8 Phenylalanine 7 activity to a mapped fragment will ultimately determine the Histidine 5 6 functional domains of the intact protein. Lysine 21 22 Amino acid sequence analysis of purified CN3 and CN4 3 Tryptophan 3 mapped their positions on the TSST-1 protein as shown in Arginine 4 4 Fig. 4 (CN3, 35 cycles; CN4, 26 cycles). The repetitive yields 195 194 in the CN3 and CN4 analyses were 90% and 93%,respectively. a The amino acid composition predicted from the corrected nucleoThe solid area of each box indicates the sequenced region of tide sequence of TSST-1. bThe amino acid composition previously reported (11). each peptide, and the open area shows the remaining unse-

15786

Sequence Syndrome Toxin-1 Shock of Toxic

firmed. Note, amino acid residues 25-33 were determined by sequence analysisof intact TSST-1 (38 cycles, repetitive yield 97%). Analysis of TSST-I Amino Acid Sequence-Previously, we reported the amino acid composition of TSST-1 predicted from the nucleotidesequence (11). ThesubsequentCNBr fragment amino acid sequence analysis showed a few changes in the predicted aminoacid sequence. The changes occurred in those areaswhere there was some ambiguity in the reading of the gel sequences. The amino acidsequence analysis of the CN1, CN2 preparation andof intact TSST-1 identified three excess nucleotides between amino acid numbers 31-32, 3940, and 43-44. The corrected predicted amino acid composi11. As previously noted, thiscomposition tion is listed in Table correlates closely with other TSST-1 protein compositions ( 5 , 24), excluding the differences in cysteine residues reported t o be present by Reiser et al. (28). The most interesting features of the toxin’s amino acid sequencearetheabundance of hydrophobicresidues, the clusters of proline residues, and the two predicted @-turns. Approximately 25% of the total amino acids in TSST-1 are hydrophobic residues. Also, the TSST-1amino acid sequence had four different areas containing clusters of proline residues; amino acids 48-56,95-101,112-117, and 179-180. Evaluation of the secondary structure of TSST-1 by the ChouFasman method suggested the presence of two @-turns at residues 35-39 and 47-50 (29). TSST-I Amino Acid Sequence Homology with Related Toxins-Previously, studies have shown that staphylococcal enterotoxins B and C1 and streptococcal pyrogenic exotoxin type A have highly significant protein sequence homology (19-22). Since TSST-1 belongs to the same general family, based upon shared biological activities, analyses were performed t o compare the toxin protein sequences. No homology was observed between TSST-1 and enterotoxins B and C1. Minimal homology was seen between TSST-1 and streptococcal pyrogenicexotoxin type A. However, Monte Carlo analysis indicated that this minimal homology was not significant. Furthermore, streptococcal pyrogenic exotoxin A and staphylococcal enterotoxins B and C1 showed some serological cross-reactivity, as detected by Western blot analysis using polyclonal antisera against each toxin (data not shown). In support of the sequence homology results, none of the related toxins showed any cross-reactivity with TSST-1 antiserum, nor did the TSST-1 band cross-react with antisera against the other toxins. In summary, knowing the complete amino acid sequence for TSST-1 and its CNBr fragments now allows for careful examination of the structure-function relationships of this immunoregulatory toxin. We plan to localize the biological activities retained in the CNBr fragments, as well as using DNA technology to further specify those particular amino acids involved.

Acknowledgments-We gratefully thank Yvonne Guptill for typing the manuscript, Dr. Robert Wohlhueter for the amino acid sequence analysis, and Timothy Leonard for artwork and photography. 1. 2. 3.

4. 5.

6. 7. 8. 9. 10. 11. 12.

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