Vol. 259, No.

3, Issue of February

THEJOURNAL
OF BIOLOGICAL CHEMISTRY
0 1984 by The American Society of Biological Chemists, Inc

pp. 1695-1702,1984

Printed in U.S.A.

Complete Sequence of the Staphylococcal Gene Encoding
Protein A
A GENE EVOLVED THROUGHMULTIPLE DUPLICATIONS*
(Received for publication, August 4, 1983)

Mathias Uhlen$QlI, Bengt GussQ, Bjorn NilssonSTi,
Sten Gatenbeck$, Lennart
PhilipsonQII, and
Martin Lindberg$**
From the $Department of Biochemistry, Royal Institute of Technology, S-100 44 Stockholm, Swedenand the §Department of
Microbiology, University of Uppsala, The Biomedical Center, Box 581, S-75123 Uppsala, Sweden

The gene coding for proteinA from Staphylococcus gene for staphylococcal protein A inE. coli (10). This protein
aureus has been isolated by molecular cloning, and a interacts with the F, (constantpart of immunoglobulins)
subclone containing an 1.8-kilobase insert was found domain of several immunoglobulins from many species into give a functional protein A in Escherichia coli. The cluding man and hastherefore been used extensively for
complete nucleotide sequence of theinsert, including quantitative and qualitative immunological techniques (11).
thestructuralgeneandthe
5’ and 3‘ flanking se- Amino acid sequence analysis of proteinA revealed two
quences, has been determined. Starting from
a TTG functionally distinct regions of the molecule (7, 8). Both
initiatorcodon,anopenreadingframecomprising
regions have remarkably repetitive structures.
1527 nucleotidesgives a preprotein of509 amino acids
The NH2-terminal part contains four or five homologous
and a predicted M, = 58,703. The structural gene is IgG-binding units consisting of approximately 58 amino acids
flanked on both sides by palindromic structures fol- each. The COOH-terminal part which is thought to bind to
lowed bya stretch ofT residues, suggesting transcriptional termination signals. Thus, it appears that pro- the cellwall of Staphylococcus aureus consists of several
repeats of an octapeptide (Glu-Asp-Gly-Asn-Lys-Pro-Glytein A is translated froma monocistronic mRNA.
The sequence reveals extensive internal homologies LYS)(8).
In a previous report (lo), we determined the nucleotide
involving a 58-amino acid unit, responsible for IgG
sequence
of the promoter region, as well as theregion coding
binding, repeated 5 times and an 8-amino acid unit,
possibly responsible for bindingto the cell wall of S. for the NH2-terminal part of the protein. Here we report the
aureus, repeated 12 times. Comparisons between the complete nucleotide sequence of the protein A gene including
repeated regions showa marked preference forsilent the 5‘ and 3’ flanking regions from the S. aureus strain 83254. Thestructural gene is 1,527 nucleotides long giving a
mutations, indicating an evolutionary pressure to keep
the amino acid sequence preserved. The structure of preprotein consisting of 509 amino acids and a M, = 58,703.
the gene alsosuggests how the gene hasevolved.
The repetitive structure of the gene has been clarified which
suggests how the gene has evolved.
Evolution by gene duplication is a well known phenomenon
among eukaryotic genes. The globin clusters, the immunoglobulins, and theinterferon genes probably all have ancestral
genes which have been duplicated and then diverged into
functionally distinct genes (1). Examples of internally, repetitive sequences have also been reported; rabbit skeletal tropomysin contains a 7-residue amino acid periodicity throughout the molecule (2), andsimilar repeats have been reported
for chicken fibronectin (3) and mammalian serum albumin
(4). Among prokaryotes, most reports of duplicated genes
have involved in vitro constructions (5), which seem to be
stable inEscherichia coli, but dramatically unstablein Bacillus
subtilis (6). However, the amino acid sequences of a few cell
wall-bound proteins from Gram-positive bacteria have revealed remarkable periodicity, i.e. staphylococcal protein A
(7,8) andstreptococcal M protein (9).
We have earlier reported on the molecular cloning of the

* 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
solely to indicate this fact.
IT Supported by grants from the Swedish National Board for Tech.
nical Development.
11 Present address, European Molecular Biology Laboratory, Hei.
delberg, Federal Republic of Germany.
** Supported by grants from the Swedish Medical Research Council and Pharmacia Fine Chemicals, Uppsala.

EXPERIMENTALPROCEDURES

Bacterial Strains and Plasmids-E. coli strains HBlOl (12) and
GM161 (13) were used as bacterial hosts. The plasmid vectors were
pBR322 (14),pTR262 (15), and pEMBL9 (16).
DNA Preparations-Plasmid DNA was prepared by the alkaline
as
extraction method (17). Transformation of E. coliwasmade
described by Morrison (18). Restriction endonucleases, T4 DNA
ligase (New England Biolabs), alkaline phosphatase, and T4polynucleotide kinase (Boehringer-Mannheim) were used according to the
suppliers’ recommendations.
Isolation of the 2.15-kilobase DNAfragment containing the entire
protein A gene was made by digesting the plasmid pSPA3 (10) with
EcoRV. The digested material was electrophoresed on a 5% polyacrylamide gel, and the 2.15-kilobase fragment was eluted electrophoretically. The isolated fragment was passed over an anion exchange
column, eluted, and precipitated with ethanol. The precipitated material was washed in 80% ethanol, dried, resuspended in water, and
used for DNA sequence analyses.
DNA Sequencing Determinutions-DNA fragments were sequenced by the method of Maxam and Gilbert (19) or Sanger et al.
(20). The samples were analyzed on 6, 8, and 20% denaturing polyacrylamide gels using the thermostatic LKB Macrophor system.
Computer Anulysis-All the sequencing analyses were performed
on a Hewlett-Packard desktop computer (HP-85) equipped with a
HP7225A plotter. The software was constructed by M. Uhlen.
RESULTSANDDiSCUSSION

DNA Sequence-We have earlier reported that theprotein
A gene from S. aureus strain 8325-4 is located ona 1.8kilobase insert of staphylococcal DNA cloned in the plasmid

1695

\ (a few basic residues followed by a stretch of 23 hydrophobic residues). 3. Restriction map and sequencing strategy of the inalso indicated inFig. A.. First. B. aureus are also presented in Table I.Theseareindicatedin Fig. J. the entire insertwas Amino Acid Composition-Attempts to determine the prosequenced according to the method of Maxam and Gilbert (19). The gene is both preceded and followed by palindromic sequences indicating transcriptionterminations. According to Sjodahl (27) and Lind183-185. B. coli. FIG. The protein A gene is contained in a 1. similar to other Gram-positive genes (25).Second. there is an open reading frame of 1. M. andthe B= 0 1 kb possible mRNA hairpin structures that can beformed are schematically drawn in Fig. also similar to other Gram-positive genes (24. Thepartialamino acid pBR322 (21). = 58. U h l h . schematic drawing of the gene coding for protein A with its different regions. Boxes show the positions of the replication origin (OR0 and thegenes coding forprotein A (PROT the start codon is sevennucleotides. unlike E. B. 3. only a few amino acids NHZ-terminal mark et al. Biochem.8 kilobase TuqI. and C. . aureus. and X is the COOH-terminal part the 235 amino acids comprising all four regions vary. Third. Sjoquist. 3. 25). 1. The sequence of the promoter region ent. this startcodon gives a putative signal Pet I peptide with a reasonable size (36 amino acids) and structure \ .. there are several reasons to postulate this. It was not possible to obtain sequence on both strands tein sequence of protein A have involved digestion of staph(28) or analyzing protein in all parts of the gene. thespace between the lastG in thissequence and EcoRV insert in the plasmid pBR322.. (10) lacks one of the three thymidines position isolation of the protein. coli in which it is pSPA8 fl very rare (22). consists of 509 amino acids giving a M . = 52. 8 out of 11 nucleotides are complementary to the 3' end of B..' M . including the putative signal peptide. (1984) Eur. C. do not have any experimental data to show where the tranEcoRV scription of the protein A mRNA starts or terminates. Hellman.1696 DNA Sequence of Staphylococcal Protein A Starting from a TTG codon at nucleotide 184.Theplasmid was designatedpSPA8andis to thededuced sequence of region X also shows high similarity shown schematically in Fig. 1. B. the sequence a few hundred nucleotides compositions of different parts of the protein..527 nucleotides terminating ina TAG stop codon at nucleotide 1. unpublished results.' The amino acid numbering starts with the alanine at and the 5' end of the structural gene has been reported (10) nucleotide 292 which has been shown to be the first amino as well as the sequence of therepetitive region X which acid of the mature protein A. In to confirm the sequence in these parts. 4. Structure of plasmid pSPA8 with relevant restric.In addition. and therefore additional sequencing ylococcal cell walls with lysostaphin using the enzymatic method (20. A direct comparison of of structures from deduced and purified proteins is difficult. J.2. Lindberg. Although we have not shown that the codon at nucleotide 184 is the translational start. The acid compositions of purified protein A from different strains complete nucleotide sequence of the protein A gene is shown of S.. and Sjodahl. M.711 thus gives a mature protein A of 473 amino acids and a in S. as deduced from the DNA sequence. The preprotein. sert. (8). U.20). sequencing strategy of DNA sequence was obtained from strain 8325-4 andthe the 1.703. Expression of the gene was sequence although about 10% of the amino acids are differdemonstrated in E. this codon is preceded by a possible ShineDalgarno sequence (23) that has many features in common with other Gram-positive ribosomal binding sequences (24). 2C. A. in press. although the first -35 sequence shows relatively poor complementarity (only three out of six) with TTGACA. Nilsson. J. S is a signal sequence. resulting Using the strategy outlinedin Fig.752. TTGa common is start codon in Gram-positive bacteria (21). A ) and p-lactamase (AMP). Sou3 Amino Acid Sequence-The amino acid sequence deduced Rea1 from the DNA sequence as well as amino acids that differ in EcoRI I the partial protein sequence established in Sjodahl (27) are FIG. a high degree of homology exists and only 4 out of All these E is a region homologous to A-D. it thus Bcl I PstI appears likely that protein A is translated from a monocisHlndI I I tronic mRNA. The amino downstream from the EcoRV site on the originalplasmid pSPAl (10)was determined using both methods (19. 3. there are - 9 Guss.711. protein sequencefrom strain Cowan I. Note that the previouslypublishedsequence due tostrain differences and proteolyticdigestion during at Lofdahl et al. of protein A which lacks IgG-binding activity. Both palindromes arefollowed by a T-rich stretch of residues (TTTATTTT). 16) was performed in order Afrom mutant bacteria which secrete the product (8). Among the IgG-binding regions D. As no palindromic order to compare the sequences deduced from the DNA sesequence indicating transcription termination was found in quence with those obtained experimentally. the divergence is probablydue tostrain variation.8-kilobase insert.' The stop codon at nucleotide probably is responsible for thecell wall binding of the protein 1. partial restriction changes canbe explained by single point mutations.subtilis 16 S rRNA. the amino acid the 3' endof the gene. Two upstream overlapping promoter sequences similar to the consensus sequences (TTGACA and TATAAT) of prokaryotes (26) havebeen indicated in Fig.A-D are IgG-binding regions. tion sites. in Fig. Although we ToqI C. are tabulated in TableI. Since the map of the corresponding DNA sequence.

0 U L UL am a z a J -u u ^ e d "a- m o v c u as u c am ua am a > ad e n am oa e m u- na a3 e u r i am a > a 1 uc am au e no a mrr oac .DNA Sequence of Staphylococcal Protein A * 1' 1 . l 3 4 3 B 25 a > si2 uc am aa a > e u e J u c 8e Xo P U L U L W Y ern as am aa O > c u e J u r uc E %S c L a z I-+ u u e L t L 1697 .a 1o U U " m m a L e m um a r u eaam a r a J u n am Eu -% oa ? L U T a t C L U L ac u > 0 0 0 u c am aa am uma am a > a J 4 y7 u n am ma am a > a J I-> cu U J cL wam u ac e- a- :: +L u > oO W u c am aa e o m i ua 3.

. Chromosomal genes are represented by four Bacillus quence were scanned by a computer program. isolated by lysostaphin treatment of bacteria. aureus from different strains of A-T 6-C T-A C-G C C T-A CT-A A 5' f5 I ~ T -A T T T-A. The per cent G/C of the if region E is omitted (A-E). Their hypothesis predicts that efficient in-phase translation is faof region D in protein A isolated from cell walls of Cowan I. similar to theoverall GC content difference in size and amino acid composition is due to proof the Bacillus species involved. In contrast. the plasmid-coded genes have a marked preference of the protein or if it reflects genomic differences. At present. amino acids 57-473. 3. the DNA sequence and its deduced amino acid segenes. . reported for A676 (8). . amino acids 1-473 in Fig. The protein A gene of Cowan I has recently been cloned in our laboratory. . Although the repetitive nature of the protein Agene makes statistical analysis risky. it is unclear if this degenerate third base is 42%. and AGC (Ser). by highly expressed genes. The NH2. Total a genes and plasmid-coded genes by the four putative proteins encoded by the staphylococcal plasmid vector pC194 (26). FIG. In teolysis both in the NH2-terminal andCOOH-terminal parts contrast.TTAAGCC ' B. AAC (Asn). The numbers to Grosjean and Fiers (33).. selection for C is DNA sequence starting at nucleotide 292 in Fig.. . it appears likely that the secreted form of protein A from strain A676 does contain region E.. However.are the codon pairs which. it seems which will help to clarify this point. terminal sequence of this protein (8) fits well with the NH2. -3 ' C -T. 8 From Lindmark et al.a few exceptions. extracellular protein A produced by a methicillin-resistant strain. are most likely to be preferred or refer to nucleotides in Fig. T T. Furthermore. Hypothetical secondary structures at the 5' and 3' Also indicated by or . Table I shows that the imal codon-anticodon interaction energy.. /T A-T C-G A-T A-T C -G G-C A-T G-CA C-G A-T C -G G-C T-A A-T A-T A-r 69 Lysine Histidine 7 Arginine 6 Aspartic acid 105 10 Threonine Serine 22 25 78 Glutamic acid 31 Proline 33 Glycine 42 Alanine 15 Valine Methionine 6 10 13 14 18 Isoleucine Leucine 31 36 41 9 Tyrosine 14 1412 Phenylalanine 65 7 5 103 85 7 78 30 28 18 38 12 6 62 6 4 91 7 17 18 67 2727 26 3131 10 5 27 8 7 14 13 45 3 5 2 20 6870 2426 3136 4 3 28 29 5 12 52 4 5 82 5 65 27 30 22 34 5 2 911 27 5 12 53 4 4 83 6 16 48 3 4 82 4 16 64 30 8 3 12 7 3 4 4 473 417 366 381 366 395 509 Protein A including the signal peptide. 4. . aureus which is 30-33% (34). this nucleotide is indeed chosen 64% of the time of protein A from A676 would then indicate that the protein (67/105). . only 22% G/C. . Amino acid composition of deduced protein A gene or purified protein S.codon pairs marked in Table I1 are most dependent on maxtained due to a blocked terminus (27). The amino acid composition. the DNA sequence does not adapting to theoverall GC content of the host cell with some contain the COOH-terminal -Val-Ala-Lys which has been exceptions. of a mature protein A lacking 107 amino is 32%. . the exact NHAerminal sequence could not be ob. 3. .. Therefore. to exhibit aclear preference for third position A/U bases with In contrast. dMature protein A except COOH-terminal part. Mature protein A except region E.DNA Sequence of Staphylococcal Protein A 1698 TABLE I A. similar to theGC content of chromosomal DNA from acids in the COOH-terminal part shows good agreement with S. The GC content at the thirdbase of the codons the DNA sequence. . 'From Lindmark et al. as deduced from can be found.ATCATCT/" " TTTATTTTAC. Homology Plot Analysis-In order to search for homologous Codon Usage-The codon usage for the preprotein of protein A is compared in Table I1 with other Gram-positive regions. The size preferred. and the However. not preferred. according regions flanking theprotein A coding sequence. according to the theory. isolated by lysostaphin treatment of bacteria. (8). * Mature protein A. size of the deduced protein from 8325-4 is larger than two Table I1 shows that among the chromosomal genes the independent determinationsof the protein from Cowan I even codon usage is randomly distributed. cilitated by proper choice of degenerate codewords. amino acids 1366. e From Movitz (2). . for A/U bases. . among the four codon both by Edman degradation of the purified protein' and by pairs in which. respectively. which is 42-47% (34). - 851 Amino acids Deduced protein A from 8325-4 Purified protein A Prot-A" Mat-Ab A-E' A-Xd Cowan I' Cowan I' A67W - ~ TTTATTTTAT . 3. the codon usage the composition of purified protein A from strain A676 as of the proteinA gene shows a preference for A/U bases shown in Table I. . the four codon pairs with predicted is truncated at theCOOH-terminal lacking approximately 80 selection for U show a reversed ratio. Every point in 5 I . UUC (Phe)..3 8 + . mainly following the Grosjean-Fiers (33) rules for highly expressed genes.Two of these exceptions can be explained by the Grosjean terminus of protein A from strain 8325-4 when determined and Fiers (32) hypothesis. (8). and only 21% C (18/85) amino acids.

and C (7. and B. 6 the sequence of the regions are aligned to enable comparisons. There exists a nine-nucleotide insertion in region E’ giving three amino acid residues (59-61) not homologous to the otherregions. more than half of the nucleotides (8/15) are homologous. 27). UGG (Trp). the homology plots represents an identical residue (1). As the start codons are yet to be identified. licheniforrnis penicillinase (31). The eight codon pairs which aremost likely to be preferred (+) or not preferred (-) by highly expressedgenes (331. In order to achieve maximal homology.1699 DNA Sequence of Staphylococcal Protein A TABLE I1 Phe UUU uuc Leu UUA UUG cuu CUC CUA CUG Ile AUU AUC AUA Met AUG Val GUU GUC GUA GUG Ser UCU ucc Pro UCA UCG CCU ccc CCA CCG Thr ACU ACC ACA ACG Ala GCU GCC GCA GCG Prot-A” Chromb Plasmid‘ 2 12 20 5 7 1 6 2 8 9 1 6 5 2 6 2 5 0 3 2 21 45 20 34 22 31 7 3 31 38 30 12 29 21 21 21 30 20 21 31 22 16 11 11 25 13 16 48 45 29 36 40 38 39 11 35 13 10 4 5 4 27 5 18 12 12 1 14 4 16 1 7 4 10 5 3 1 14 4 15 5 0 8 2 5 1 4 0 25 1 11 5 . The same holds for the other end of the repetitive region located in the beginning of region E’. Thus. and homologous repeats show up as parallel lines. B . The codons AUG (Met). Although the first three amino acids are different from region D’. 5. A and 8. e Four putative proteins of pC194 (32). The cleavage points for trypsin are marked with arrows. which disappear when no homology exists. although the lastfive amino acids of region C’ (292296) are changed compared to region B’. _ Prep 9 1 6 1 Tyr UAU UAC Term UAA UAG His CAU CAC Gin 33CAA CAG Asn AAU 31AAC Lys AAA AAG Asp GAU 35GAC Glu 59GAA GAG UGU Cys UGC Term UGA Trp UGG Arg CGU CGC CGA CGG Ser AGU 17 AGC Arg AGA AGG GGU Gly 36GGC GGA GGG Sum Per cent G/c‘ 29 9 8 1 0 0 6 1 38 2 20 45 51 18 21 19 37 1 0 0 0 0 3 3 0 0 3 12 0 0 18 14 1 0 49 33 27 8 46 20 17 1 16 6 43 12 56 12 22 5 19 10 7 4 9 4 1 3 0 13 3 11 4 11 2 3 3 509 32 1654 42 655 22 35 68 79 26 81 35 2 2 - 35 18 5 10 9 19 11 14 22 - Protein A including the signal peptide (preprotein). the part of the gene coding for the signal peptide (S) as well as the promoter region (5’) seems to be totally unrelated to theIgGbinding regions (E. A changed nucleotide compared to region B’ in Fig. Recently. 5. A . flanked by unique sequences without homology in the 5’ and the 3’ ends of the structural gene. amyloliquefaciens a-amylase (25). 5B. the boundary of these regions has been moved 15 nucleotides towards the 3’ end of the gene. These results strongly support the previously suggested hypothesis (27) of an evolutionary pressure in these regions keeping the amino acid sequence preserved. e Per cent G/C in the third degenerate base. the total open reading frames are taken into account. 5A are more broken than those in Fig. As already pointed out in the homology analysis (Fig. D. A. The sum of four Bacillus chromosomal genes. indicating a relationship. The nucleotide triplets and thededuced amino acids are compared in Fig. subtilis SpoOF (30). This choice is of course arbitrary as the5’ end and the 3‘ end of the repetitive region have diverged slightly. we showed (10)that strain 8325-4 also contains a fifth region E homologous to the four repetitive regions earlier identified by protein sequencing. five out of nine nucleotides are identical. However. B and C ) located in the middle of the gene. B. As the sequence is compared with itself. In Fig. B.respectively. 6 is . B. The plots reveal two structurally distinct regions with internal homology. which means that many of the nucleotide changes between the codons in the homologous regions have occurred in bases giving no amino acid change. The partof the gene coding for the COOH-terminal part of region X as well as the 3’ flanking sequence seems to be unrelated to both the repetitious region X and the IgGbinding regions. Structure of IgG-bindingRegions-The IgG-binding regions of protein A have been defined by trypsin cleavage of the mature proteininto functional IgG-binding units D. a line of identity occurs from the left upper corner to theright lower corner. A and B ) these regions seem to be nonhomologous to the IgG-binding regions. Comparisons between the plots show that the homology lines in Fig. and AUA (Ile) are omitted. Also shown in Fig. 6 are the sequences flanking the repetitive regions. subtilis a-amylase (29).

A. and direct repeats appear asparallel lines across the grid. The comparison is based on region B'. 6. A comparison of the five regions with respect to mutual relationship reveals a pronounced "homology gradient" along the protein molecule. thus generating slightly dissimilar nucleotide and amino acid sequences. Dot matrix comparisons of the protein A sequence. The cleavage points for trypsin are marked with arrows. the deduced amino acid sequence compared with itself. 5. and a changed amino acid is underacid changes and Table lined. Each dot represents the center of a three-base identity. the entire nucleotide sequence and the immediate 5' and 3' flanking sequences are compared with itself. As a result of these evolutionary events.the higher the degree of homology. A. The fact thatcodons acids (Table (Table IV) have changed much faster than amino 111) indicates that anevolutionary pressure exists tokeep the . the closer the location of two regions. Table I11 summarizes the amino IV the codon changes between the regions. marked with an asterisk. one interpretation of thisphenomenonisthatthe primordial structural gene coding for the IgG-binding part of protein A has been subjected to stepwise gene duplications involving only oneregion followed by a period in which point mutations have occurred.e. i. REGION C' FIG. 5' 5' 3' S E D A B FIG. R.DNA Sequence of Staphylococcal ProteinA 1700 B. and a nucleotide is marked with a n asterisk and an aminoacid is underlined when different from the B' region. As already pointedout by Sjodahl (27). a homology gradient will evolve. Comparisons of the IgG-binding regions and flanking regions. The sequences of the repetitive regions have been aligned to achieve maximal homology.

Structuralstudies of protein A have suggested that 11 amino acids of the IgG-bindingregions are essentialfor binding to theF. Therefore. 6. not clearly defined since the 12 last nucleotides. a u r e u s Cowan I and 8325-4. 20) have suggested that region X starts at amino acid 292 which differs five amino acids from the boundary chosen in Fig. A comparison nucleotide by nucleotide reveals that 14 out of 18 bases are identical between these two regions. region.Again. in regions E’. giving an approximately 300-base pair repetitive region (X. which has been observed in protein A both fromS. The boundarybetween region C’ and region X is. As seen in Fig. 188-189 (Phe-Tyr). This pressure is evenmore pronounced when comparing the residues in these a-helices that interact with IgG. Region E’ D’ A’ B’ E’ D‘ 0 31 A’ B‘ C‘ 25 26 36 31 0 21 25 28 2536 2128 0 14 30 26 25 14 0 20 C’ Total 30 20 0 118 105 101 86 115 amino acidsequencepreserved. 5. Apart from the mutual homology between the five regions. Comparison of the repetitive units of region X and flanking regions. and A’. Hence. D’. to Asn-Met. This subregion (residues 57-62) is possibly related to other regionslike amino acids 4-9 in the beginning of region E’. The six first amino acids (Lys-Pro-Gly-Lys-Glu- . Clearly. there are striking homologies in these two a-helices between the different regions. are identical with the corresponding amino acids of region X1 (Fig. the nucleotide sequence coding for amino acids 179 (Lys) to 188 (Phe) and196 (AAC)to 205 (Phe) all withinregion B contains 24 identical outof 30 nucleotides. The comparison is based on region XI. Region E’ D‘ A’ E‘ D’ A’ B‘ C‘ 0 11 14 11 0 12 14 21 11 17 12 7 0 5 15 7 B‘ C’ Total 57 46 11 5 0 10 21 17 15 10 0 40 41 64 TABLE IV Comparison of codons of the ZgG-binding regions The values listedrepresentthenumber of changednucleotide triplets of identically positioned codons when the regions are campared in pairs. structurally the octapeptideof region X seems tobe repeated 12. 6.1701 DNA Sequence of Staphylococcal ProteinA TABLE 111 Comparison of amino acids of the ZgG-binding regions The values listed represent the number of changed amino acids of identically positioned residueswhen the regions are compared in pairs. the corresponding residues are 183-192 and 198-211. Since the number of total changes of codons is lowest for region B’ (Table IV). 7. a changed nucleotide is marked with an asterisk. 7) which is directly followed by the constant 209 C‘ 2’37 x1 305 x2 313 x3 32 1 x4 329 x5 337 X6 345 x7 353 X8 361 x9 369 x10 377 x11 385 x12 3’) 3 FIG. Since region C’ terminates at amino acid 296. coding forthe last four amino acids of region C‘. The sequences of the repetitive region have been aligned to achieve maximal homology. 192 (Leu).The numbers refer to the amino acids in Fig. The 3’ end of the repetitive region is obviously located at amino acid 392 (see Fig. however. thereis a serine insteadof aspargine at position 70. 7). these amino acids are 184-186 (Gln-Gln-Asn).In Fig. generating a few amino acids identical with region XI. Another subregion of interest is the nine-nucleotide insert. 203 (Asn). and an altered nucleotide is marked with an asterisk and an altered amino acid is underlined.) followed by a constant region coding for 81 amino acids (Xc). 206-207 (Ile-Glu). As discussed above. The changes observed are often outside the two helical areas. for instance. there is a strong pressure tokeep these amino acids preserved. at position 193-194 of region B’. In region B’. giving the amino acids 5961. part of the immunoglobulins (35). and 210 (Lys). suggesting an evolutionary pressure to keep these residues intact. but this region has obviously diverged in the COOH-terminal end. 5. the repetitive part of region X consists of exactly 12 units each with a length of 24 nucleotides. The cleavage point for trypsin which defines region X (7. the end of region C’ is probably related to the otherIgG-binding regions. 6. 7. 20) is immediately before amino acid 292 (Glu). and a changed aminoacid is underlined. S t r u c t u r e of Region X-The repetitive nature of region X is indicated as multiple lines in Fig.5 times. there also seem to exist internalhomologies in each region as revealed by traces of lines in Fig. the changed His-Leu. 3. In region B’. Acomparison of the 12 repeated units reveals striking homologies. As seen in Fig. A and B. this region was chosen for the comparison in Fig. but all the other 49 residues are identical. Mostof these amino acids are assumed tobe located in two a-helical regions (35). A and B . 7. the 24-nucleotide repeats are aligned and a mutual comparison was performed. Structural studies based on the cleavage with trypsin (7.

R. FL 35.. J. G. Marinus. Stephen Fahnestock for a correction of the nucleotide sequence. Mol. 2361-2370 . Biol. and Rabinowitz. Seyer. (1981) Proc. 459-472 Apart from the distinct24-nucleotide repeat. Infect. L.) 12.326-331 A. L. V. S. H. Lofdahl. and between Asn and Gly in regions 5 to 10 (seeFig. the codon coding for the first lysine is changed periodically (1977) Gene (Amst. Nilsson.) 302.. L. J. 1-45 resolving the molecular events causingstepwise multiple DNA 23. Acad.) 13..) 23.95-113 in regions X7 to X12.. and Gilbert.. Vol. 9. 34-38 duplications. Gen. M. 2. R. (1969) J. 24. A. (1977) Proc. Hartley. F. Philipson. M. H. (1973) Mol. Sjodahl.. Sci.) 2. compartments. Tanaka. U...(1979) J. Natl. T. Lindberg. and Manjula. 291-299 29.. the evolution of the repetitive partof region 18. (1982) Semin. (1982) Gene (Amst. 258. Hence. 15131523 24-nucleotide sequence. Betlach. J . Sullivan. and Doly.) 18. New York 2.. and Saito. 139. Zmmunol. McLaughlin. Kawamura. (1983) Nature (Lond. 73..UhlBn. and Coulson. Nicklen. H. Hirano. J. D. Kalkkinen.. S.. (1983) Nucleic Acids Res. C.. Kozak. K. 11. but the nucleotide sequence of Acad.. Asn.. Academic Press.157-252 12.. W. Chem. W. K. and Roulland-Dussoix.M. or Asn-Lys. N. Biochm. Xr although the gradient must 11. The two last 7. B. H. G. J. 32. Dente. pp. Pettersson. Sci. Biochem.623-628 of this extremely conserved octapeptide is not known. S. J.. R. Sci. 12 nucleotides have changed when compar. 815825 33. A. I.. C. M. B.. (1979) Nucleic Acids Res.(1982) Adu. B. M. Inc. I. H. Gatenbeck. Yamada. Shine. Beachey. (1981) Biochemistry 20. S. U. I. (1977) Eur. M. Reu. A . there has been a strong pressure to preserve its amino acid Infect.800-804 27. L. H. Andras Gaal for introducing us to the thermostatic LKBMacrophor system and Dr. Bacteriol. Boyer. J.237-249 30. and aminoacid 7 is changed periodically 15. J. 47.. Ohno. Dis.. S. ed) Vol. (1977) Eur. (1983) Proc. Cesareni. Acad. J... A. region. L. and Cortese. (1982) Semin.and Fiers. W.. Johnson.M. R. S. J .46-50 4. Sci. M. all occurring in a wobble position and therefore representing 11. Crosa. U. s. J.10. Soderlund. How this evolved at 20. Pastan. thewobble base A/G in 14.. Bacteriol. (1983) Gene (Amst. M. clearly 9. 7657-7661 5. Fasman. F. L. Acad. H. B.. Kobayashi. Y.. Acad. Gly-Asn. may help in 22. 199-209 34. A. (1981) in Genetic Engineering (Williamson. 69-183. A.. T. R.. A. W. S... (1977) Eur.5463-5467 the protein A gene from other strains.19. Natl. the molecular level is unclear. (1981) Gene (Amst. Dis. Poteete. Sci. F.. Horinouchi. and Losick. and Sjoquist. 1 2 7 . 74. H.. I.. 80. 4. Boca Raton. Biochem. 11. C. D. 4 7 4 5 signs of a 48-nucleotide repeat. Neugebauer. Langone. A. A. Natl.. and Falkow. U. H. Movitz. Galizzi. (1975) Nature (Lord. Birnboim. J. 80. M.. Sanger. 6). K. Y. (1983) Proc. and Dalgarno. L. Biol.369-378 for proteins with similar repeated structures. Greene. Acknowledgments-We are grateful 50 Dr.or 48-nucleotide long sequence. M.401-410 sequence. B. 68. Grosjean.11283-11291 25. and Kang. 2577-2588 32. Deisenhofer. Moran.(1983) Microbiol. U. andYamada. 80. 658-662 31. 411-418 3. J.. Fishetti. J. P.775-782 Asp) are identical throughout the X. S. (1981) J. D. L. REFERENCES 1. 78. Heyneker. Thus. Boyer. Jeffreys. J. and Gregori. H. P. and Kaariiiinen. (1983) J. Guss. 78. K. S. 347353 Biol. (1982) J. C.DNA Sequence of Staphylococcal Protein A 1702 6. H. (1980) Gene (Amst. Palva. Chem... S. 41. (1979) Methods Enzymol. A. We thank Hans-Olof Pettersson and Bjorn Jansson for skillful technical assistance and ChristinaPellettieri and Gerd Benson for patient secretarial help. Swanberg. Shimotsu. Lindmark.. J.H. (1976) Eur. 68. Natl. Natl. T. 471-490 28.. Natl. and Lindberg.. Rodriquez... Morrison. CRC Press. 4. R.. Genet. X probably involved stepwise gene duplications of an ances. Murray. E. 74. Bachman..1645-1655 be based on a 48-nucleotide repeat rather than the primordial 17. Sprengel.... Sjodahl.. L. aswell as genes coding 21. S. Guss. Roberts. Biochem.. pp. Riedel.. N. Takkinen. (ed) (1976) CRC Handbook of Biochemistry and Molecular Biology: Nucleic Acids Section 3rd Ed. B. M. 256. Maxam. 1-48. gradient throughout the region. Movitz. Acad. U... A. W. J. A. (1977) Proc.)254. Y.560-564 tral 24.. We also thank Dr. In conclusion. C. Bolivar.. 7. R. John Sjoquist for critical comments and advice. D. C.H. F. Although the biological function 74. 150. (1983) Nucleic Acids Res. and Henner. R. 697-701 ing thesix conserved amino acids in the12 X. L. 123-127 There also seems to besomeevidencefor a homology 16. U h l h .. and Weisblum. A. there arealso 13. J. M... and Schaller. Yang. and Philipson. (1983) Proc.10071013 26. I. S. (1981) Nucleic Acids Res. G. de Crombrugghe. Sci. 343-351 amino acids are changed in a regular pattern between Asn8. R. silent mutations.

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