Gene. 94 (1990) 137-138 Elsevier
137
GENE 03725
Brief Notes Sequence comparison of mecA genes isolated from methici!!in-resistant Staphylococcus ureus and Staphylococcus epidermidis* (Penicillin-binding protein P B P 2 ' ; recombinant D N A ; gene conservation; point mutations; staphylococci)
Cristina Ryffel', Wolfgang T e s c h ' , lan Bireh-Machin b, Peter E. Reynolds b, Luisella Barberis-Maino', Fritz H. Kayser" and Brigitte Berger-Bgehi" ° Institute of Medical Microbiology, University of Ziirich. CH-8028 Z~rich (Switzerland) and b Department of Biochemistry. Uniwrsily of Cambridge, Cambridge, CB2 I Q.W (U.K.) Received by T.A. Bickle: 3 February 1990 Revised: 18 June 1990 Accepted: 19 June 1990
The mecA gene on the Mc R determinant mec codes for the P B P 2 ' , which is essential for the phenotypic expression of Mc R in staphylococci. Here, we present the complete nt sequences of mecA isolated from the Mc R S. aureus strain, BB270 (mecA BB270), and from Mc R S. epidermidis strain, WT55 (mecA WT$5) (Tesch et al., 1988), and we compare them to the E M B L GenBank sequence of the mecA gene from S. aureus strain TK784 (mecA TK784) (Song et al., 1987). In strain BB270, mecA was expressed constitutively, whereas in WT5$ and TK784, mecA was inducible by /~-lactam antibiotics (Song et al., 1987; C.R. and B.B.-B., unpublished results). Sequence analysis revealed ~hat the mecA genes and their promoter were well conserved among coagulase-positive, as well as coagulase-negative staphylococci. The 3' end of the genes showed an accumulation of point mutations. At the deduced aa sequence level, there was a deletion of two aa at positions 303-304, and single aa substitutions at positions 168, 169, 170, 246, 574, and 612. In m e c A W T 5 5 an insertion of an A and subsequent deletion of a T at nt 833 and 882, respectively, lead to a frameshift encompassing 17 aa from 198-214. These changes did not occur
Correspondence to: Dr. C. Ryffel, Institute of Medical Microbiology, University of Zttrich, Gloriastrasse 32, CH8028 Z0rich (Switzerland) Tel. 01/2572650; Fax 0041/12528107. * On request, the author(s) will supply detailed experimental evidence for the conclusions reached in this brief note. Abbreviations: aa, amino acid(s); bp, base pair(s); UWGCG, University of Wisconsin Genetics Computer Group, Madison, WI; kb, kilobase(s) or 1000bp; Mc, methicillin; mecA, gene coding for PBP2'; nt, nucleotide(s); PBP2', low-affinitypenicillin-bindingprotein; R, resistant/resistance; 8., Staphylococci. 0378-1119/90/$03.50© 1990ElsevierScience PublishersB.V.(BiomedicalDivision)
in the penicillin-binding domain of the PBP2' protein and did not affect its biological activity. These findings support the hypothesis that the differences observed in regulation of PBP2' in the different strains might be due to additional genetic factors, and not to mutations within the promoter region or structural mecA gene.
REFERENCES
Song, M.D., Wachi, M., Doi, M., lshino, F. and Matsuhashi, M.: Evolution of an inducible penicillin.target protein in methicillin-resistant Staphylococcusaureusby gene fusion. FEBS Lett. 221 (1987) 167-17I. Tesch, W., Strgssle, A., Berger-Blehi, B., O'Hara, D., Reynolds, P. and Kayser, F.H.: Cloning and expression of methi¢illin resistance from Staphylococcus epldermldls in Staphylococcus carnosus, Antimicrob. Agents Chemother. 32 (1988) 1494-1499.
Fig. i. Sequences of mecA isolated from: (A)$. aumus BB270 Mcn ll~ (EMBL data library accession number X52594);(B) $. eptdenntdisWT55 McR(X52593); (C) $. auneusTK784 McR(Y00688).The sequences were compared by using the UWGCG program package. Sequences identical over 100 nt are represented once only. Differencesin the nt sequences are boxed. Convergent arrows indicate inverted repeats. The putative ribosome-binding site (S/D), the -10, and -35 sequences are indicated. The start and stop codons are overlined. In strains BB2?0 and WT55 an in-frame deletion of 6 bp within the structural gene lead to the loss of a recognition site for Sail, Accl and Taql. In BB270 an A --. G transition at nt 877 created a new restriction site for Xbal, known to be a hallmark for the mecA gene. At the 3' end of mec.Awe found an accumulation of point mutations. Fig. 2. The aa sequences of PBP2' from: (A)S. aureus BB270 McR; (B) S. epidermidisWT55 McR; and (C) $. aureusTK784 McRdeduced by the UWGCG program package. Differences in the aa are shown by asterisks. The active sites of the protein are indicated by arrowheads.
138 -
35
A A'~AC'~.,AT TATAC~'~A&C A~AAAJ~AG &']~A'~AACAC CTTC'I'ACACC TCCA?AT.AC A.qAA&ATT&TJ~ACA'I'~&'ITI''/~qCATAAAT -1o aID ~mm A T R A T A T A ~ A C A R A ~ T A G T CTTATATRAG GACC~TATTG ATGAAR&AGATAAA/~TI%'T TCCACTT&TT T T A A T ~ T/tG~'%"TC~ G'I~fGGT&T& AATAATRCTA TTG&TGCAAT 'I~AA~ATAAAAATT'/CAAACAAGT'/~AT/U~A~,ATA~CAGT TATATTTCTA
100 200 30Q
A
TATTTTTATG CT2CRAAAGA T ~ T T
k
RAAGCqAT&A 2~.GTGRJ4~fAGA~ATGACTG AAC~TCCGAT AAAAATATAT&ATAGTTTAGGCG'Z'TAAI~.Jk~&TA.q&CATT~ q ~ G ' f A
AAATAAAA/~
400
k
AGT&TCTRAA AATARARAR~ GR~fAGATG¢ TCAAT&TARA &TTAAAACAAA~&C~'rA& CA'Z'I'GA'I'C~¢ ~CGTTCA~T TTAATTT'I'G~ TAAA~kAGAT
500
&
GGTATGTGGA RGTTAGATTG GGATCATRC~2 L'TCATTATT¢ ~ G C A
GAA&~AC~A AGGqTACATATTG/~AAT~ AAAATCA~A CG2~TAAAA
800
AAAGG4ATC~ GTTCCA/~.,A ATGT&TCTAA/qA&~TT&T ~ T C G ~&GGCATC G T ~ ¢ ¢ ~ &TGTATCTAAAAAAGATTAT AAAGCAATC~
700 700
AAT~ATCAA AATT~.,L'T~ AAGATGATACCT~[¢~ICCA C~AAIU~C G'I~AAAA/t~ AATGGATCAAAATT~GTAC &J~GATGATACCTT¢GTTCCA ~ C C GTTAAAAA~ AAT~GATCAA AATTGC~TAC/U4GKTG&TACCT~C~'~TCC& GT?AAAAA~
800 800 800
CACATCTATT AJI~CGACTT CACATCTATT AA~GA¢~TT CACATCTATT
900 900
A AGGTTATGTT ~'Z¢CCA*I~A ACTCTGAA~AATTAAAR~AA ARAGAATATA RAGGCTAT~& A~k~L~ATGC& GTT&TTGGT& ARARC,GGACT ¢~AAARJ4CTT
1000
A
TTTTAGACCG AARC.AAT~"fGG~ATTGGC¢& &TACAC.GAR~ B TTT~AG~CCGAAACJ~kTG'I'GGN~TTGGCCA&TACAGGAR~ C 'I'TTT~,~CCG ,~C~TGTG G~ATTGGC~A ATACRGGAR~
B CTAAA~q~CTAA~'TA'FJ~¢T C ¢TA~q~qACT ~ & ~ T T C T GA~,ACTATA ~ J ~ f d ~ . A A 8
TGGATGRATA TTTAR~T~AT TTCGCARARA AATTTCATCT
TG~&TC~ATA TTTAR~TGAT T T C ~ A R A A A RATTTCATCT TACAAC~AAT GAAACAGAAAG'JlCe~'~AACTA C TGGAT~&ATA TTTARGTOAT TTCGCARARA RATTTCATCT TACAACTAAT GAAACAGAAAGTCG'TAACTA
A TACGATAAAA~qGc'~¢CAACATGAJ~&T~.,C TAqC~T~TCJ~ 8 TAC~ATAJ~A ~ C T C C R A C A T~%AGATGGC T&TCGTGTCA ¢ TACGATAAAA A G C T ~ A A C A TGARG&Tr~-C TATCGTGTCA
UTJ~.~..A&TA C/UP*TCGCACATGq~J~UTA G,qs~A.q,qJq~.~k 1100 UTAGCAATA CAATCGCACATACATTUTA ~ llOO
A AAAAAGA']~.~CAARF~TATT CAAC~AAC~A'~GAT~CTAA AGTTCAAAA~ AGTATTTATAACAAC&TGAAAAAT~qTTAT ~TCAGGTA CT~TAT¢CA 1200 A CC¢'ICAAACA~GTORATTAT TA~CACTTGT AAGCACACCT TCATATGACG TCTATCCATT TAT~TAT~ ATGAGTAAO~AJ~AATATAATAAATTAA~C 1300
GNqGAT;~J~ /~GAN:C~CT GC~C,~qENqG TTCCRGATTA C&~C'~'I"CAC¢ J~GTTCAACT CAAAAJ~T&TTA/¢AGCAAT GATTG~TTA AATAJ~AAAA 1400
A
A ¢ATTAGACGATAJ~J~AAGT TATAAAATCG ATGGTAA&~ TTGGCA&A/~ GATAAATCTTG~GG~GG'1~ACA~J~TACA &GAq'&'I~AAGT~"TAAATGG IS00 A TAkTATCGAC TTAARACA&G CAATAGA~TC &TCAGATAAC Aq~T]L'C~TTG CTAGAGTA~ AC'~/qA41~A GGCAL'TAAGAAAT~ChqJU~ A~.~CATGAAA 1800 & JU~CT~GGTGTTGGTGNkGAT&TACCAA~T GATT&TCCAT TTTAT~ATGC TCAAATTTCA AACAR~4AATT TA~ATAATGA AATATTATT& GCTG&~TCA~
1700
A GTTACGGJ~CAAGGTGAAATA C?G&TTAACC CAG?ACAG&T ¢CTTTCAATC TATAG¢GCATTAGAA&ATAATGGCAATATTAACGCACCT¢ ACTTATTAAA 1800
A AGACACGAAk~ACAJ~I'~J~ G~q,GAk,qAA TATT&TTTCC A~GAJqJ~TA ?CAA'~TA'rT A ~ A T ~ T &TGCAACA~GT¢GTAAATAAAA~.ACATAAA 1900 . . 8 A~CACGA/~ AACAJ~O?'i~ GGAJ~A.J~q.~TA'~&T'~CC AAAGAAJ~TA '~AA?CT&?T A ~ & T ~ T ATGCAACAAGTCGTAAATAAAACACATAAA 1900 C &~ACACGAAAkACA&AO?~ GGAk=AAJQ~A?A'~&?'~CC A,V~GANkA?A ?¢AA?¢TA?? AJ~ATOOT ATOCAACA~ TCGTAM?AA AACACA?AAA 1900 A GAAOATAT?TATAOATC?TATOCAAAC2~AAT?OOCAAAT CC~O?AC?OC AO/~¢~CAAA A?OAAACAAOOAOAJ~GO I=AG~CAAATTOGOTOGT~?A |000 B OAA~TAT~T &TAGATC?T& TOCAJ~C~A &T?GOCAJ~T CCGQ?AC?GC k=AACTCAAJ4A?GA.qACAAQ~ O ~ CAOIqCA*~ATT0GG'~T~TA 11000 C OAAGATA~tT&TA~A?CTTA TOCA,t~CT~A A?TOOCA~AT CCGO?ACTGO AOAAC~CAAA&~fdqJ~N~O C~]q~N~TT GGGT(IGT~T& 2000 A TATCATA?O&TAAAQ&?AATCCAJ~OA~A ~&TOGCT&T T A A ~ F J ~
GATO?ACAJ~ &TANq~N~T ~ C T N :
k &G'~'t&TGAT QAQCT&T&'~ &0AACOOTk~,?kkMt,t,kTAC ~&T&?AOA?G kA~HUqA~CkG?OAJ~¢: B AO?~TATGATQA~TATA?G AGAACGOT~.AT / ~ A T k C C A~I'O?&TQATGAOCTATA?OAOAACOGTAATAAAJI~IqTAC A A??ATGAATTA?TAATkAOT OC?O??AC?T C?¢~?A/~4 0 A~I~&TO~J4?T&TTAA?~4~t OCTO?TA~? CT~,~T~d4AJl '~q,F-'C'~lF~4~'~ C A~&TOAATT A?TAAT~O? OC~O~T&C?T C?.¢~TAAA k B ¢
Fig. I.
|100
/~COTAAO QP,T~?TOC, ?CkCq~Tq'q~J~ |~100
~cac,~
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TTTTATOOA? ?TCTT&TTTO TAAT~TC~O ~ATAAC~TAC TTTT&TG0~T TTCTTATTTG T~ATTTC~C GATAACQTAC TTTT&TGOA? T?CTT&TTTO TAkTTTCAGC 0ATkACOTAC
k CTACAACTTC T T C T C ~ T A T TTACCTTCTT CTACCCATAA TTTAAATGAT &TTGAAR~TQ T & ~ C A T O C N i l 0 CT&CAJ~'JLVl'C'J~CTCC~TAT TTACCTTCTT CTACCCATAA TTTAAATGAT ATTGAAJ~T~ T&~.pca~= C ¢TACAA¢~kC 'Z~CTC¢~J~AT TTA~CTTCTT C?ACCCAT~ TTTARATGAT ATTGARA~TG TAq~CATGC
2470 ~4~0
~470
loo 100 100
a 8 C
~4xKzx zvp Lx~xvvvvcrozy~yAY~o~x ~ x D a X E o ~ Q v ~ o u v x S~80HG~VZM~X~Xy~S~CV~OXMZ00~X~8~V0aQm Ik0qql KI VPL1Ll VVVVGFGI YF¥&8KDnXlqN~IOAZEOI~'NFKQ~D80Yl8 K B D N O ~ Z K I ~ 8 ~ D ZNIQO~I~0~¥OAQ~ F0qKI R[VPLI LIVVVVGFGIy'Fy&SR01~IHHTIO&IEOiTI~KQVYKO88yI ~ R 8 0 H G E ~ I K I ~ S ~ 0 1 H I OO~ I ~ | ~ V O A 0 ~
& n C
I I¢~[q4~/GNI 0RKVQFNFVIq]~0GIqI~LDHOHSVI IIPGIqlQlq0 *** q 8XHIEHLlqSEP'GK 1LOKHHY~LAH"~MRLG |VPlTHVSKIqOY1KA/r/Ul~I,| 18|0YZ*e* ~ |00 I KTH¥GH1DRHV~:'I~VI~OGHHXLO~OH8VnPGH~OQBI HIEH~SERGRIL O R ~ & ~ I G I ~ S n O ~ I ~ L S I 8EOyIK~ |00 : : t t1~t~:tD:,l~l ~:h'FI~IqEOGI4V#I~LI)HOHSV11PCA4QIqOQ81H1EHLlqSEP'GIqI LOI~Nh'~~ ~ IW ~ 8 ~ D ~ ' ~LB1880¥1 ~ |00
&
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IV IV ~ H S H ~ A H T L ~ E K K ~ q ~ K ~ L T ~ J ¢ V ~ q 8 ~ Y H ~ Y G ~ G ? & X H ~ E ~ ` U ~ L V ~ 8 Y ~ F ~ E ~ L ~ L ~ T 0OHSNTl AHTLI EI¢lq~qOGKOIQL?I OAXVQK81yI~I~OqNI)¥GSOTAII ~ E L L A L V S ~ 8 ¥ O ~ S ~ L ~ D ~ L ~ I 1VRVDD~SHTI &HTL|EIqXI~OGIqD2QLTXDAXVQIqSI~I~[~qHO¥GSG?&2~ B L ~ L V S ~ S Y O ~ G H S ~ L ~ D ~ L L ~ Q |
8A C
TSPGY~]~ILTNqlGLh'I~TLDOK~SyIqI0GIqGNQK01qSWGGyIWTRYEVVIqGH i DLlq~ I ESSOH1 F F ~ V A ~ 8 ~ ~ 0 1 TSPCSTQKl LTAHIGLHHI~LOl)l~Sylqi 0GKCIIQIqOK8tlGGYHV~Ay~FliGHi D~ 18880HtFF~V&~ ~ 8 ~ ~ G E O Z P TSPG8T~RI LTAHIGLIq4KTLDOKTSYiql DGRG~QIqOK8NGGYIW'FRYRVWtGHZ DLK~XESSOH1 F F ~ V & ~ ~ V ~ D | P S D ~
A
LSO~qXXFHLTIq48TESI~gypl~ydqTSflLLGYVGP1HSEEl,X~lq~qqG¥1q0O&VlGl~GL~L¥DIqXLQI.~OOyRV~ 300
•
B c
YHAQI SHIqlqLOIqElLLAOS~YG~GEI 1,II~VQILSIYB&LEHHGHI tti~pHLLKO~qHIWMlqKNi Z8 ~ H I H L ~ D ~ D YNAQ/,'g~qKNLONE/LLAOSCyG~JEI LINPVQIL8IYSALENNGHINAPHL L K O ~ 11S~HINLL~~o YHAQI SHKHLDNEXLLAOSGyGQ~EILINIPVQILSIYSALENNGHZ~APHLLKDTKNKV.~i I ~ H I H L L ~ D ~ D
A
CTAELlql~Q~ESGRQI G~F1 ~Y01~NP~t~ I NV~OVQDKC44ASY~iA~I8GIqVYDELyEI~NXI~01DE GT&£I.KM~QGETGRQIGq~l :'/0KONPI'~4M~qI NVKOVQ01q(R4~q~ A K I $GI~DELYENCNIq~OIDE GTA~I,XI~G£TGRQIGHFIS~r0KD~I~)~qHI~IIWXDVQI)KGI~SyNAKI S~DEL~C~OIDE
B
Fig, 2.
i,~o~
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C
400 T 400 T 400 ~80~ 80~
S00 SOO SOO
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~70 1~70
137
GENE 03725
Brief Notes Sequence comparison of mecA genes isolated from methici!!in-resistant Staphylococcus ureus and Staphylococcus epidermidis* (Penicillin-binding protein P B P 2 ' ; recombinant D N A ; gene conservation; point mutations; staphylococci)
Cristina Ryffel', Wolfgang T e s c h ' , lan Bireh-Machin b, Peter E. Reynolds b, Luisella Barberis-Maino', Fritz H. Kayser" and Brigitte Berger-Bgehi" ° Institute of Medical Microbiology, University of Ziirich. CH-8028 Z~rich (Switzerland) and b Department of Biochemistry. Uniwrsily of Cambridge, Cambridge, CB2 I Q.W (U.K.) Received by T.A. Bickle: 3 February 1990 Revised: 18 June 1990 Accepted: 19 June 1990
The mecA gene on the Mc R determinant mec codes for the P B P 2 ' , which is essential for the phenotypic expression of Mc R in staphylococci. Here, we present the complete nt sequences of mecA isolated from the Mc R S. aureus strain, BB270 (mecA BB270), and from Mc R S. epidermidis strain, WT55 (mecA WT$5) (Tesch et al., 1988), and we compare them to the E M B L GenBank sequence of the mecA gene from S. aureus strain TK784 (mecA TK784) (Song et al., 1987). In strain BB270, mecA was expressed constitutively, whereas in WT5$ and TK784, mecA was inducible by /~-lactam antibiotics (Song et al., 1987; C.R. and B.B.-B., unpublished results). Sequence analysis revealed ~hat the mecA genes and their promoter were well conserved among coagulase-positive, as well as coagulase-negative staphylococci. The 3' end of the genes showed an accumulation of point mutations. At the deduced aa sequence level, there was a deletion of two aa at positions 303-304, and single aa substitutions at positions 168, 169, 170, 246, 574, and 612. In m e c A W T 5 5 an insertion of an A and subsequent deletion of a T at nt 833 and 882, respectively, lead to a frameshift encompassing 17 aa from 198-214. These changes did not occur
Correspondence to: Dr. C. Ryffel, Institute of Medical Microbiology, University of Zttrich, Gloriastrasse 32, CH8028 Z0rich (Switzerland) Tel. 01/2572650; Fax 0041/12528107. * On request, the author(s) will supply detailed experimental evidence for the conclusions reached in this brief note. Abbreviations: aa, amino acid(s); bp, base pair(s); UWGCG, University of Wisconsin Genetics Computer Group, Madison, WI; kb, kilobase(s) or 1000bp; Mc, methicillin; mecA, gene coding for PBP2'; nt, nucleotide(s); PBP2', low-affinitypenicillin-bindingprotein; R, resistant/resistance; 8., Staphylococci. 0378-1119/90/$03.50© 1990ElsevierScience PublishersB.V.(BiomedicalDivision)
in the penicillin-binding domain of the PBP2' protein and did not affect its biological activity. These findings support the hypothesis that the differences observed in regulation of PBP2' in the different strains might be due to additional genetic factors, and not to mutations within the promoter region or structural mecA gene.
REFERENCES
Song, M.D., Wachi, M., Doi, M., lshino, F. and Matsuhashi, M.: Evolution of an inducible penicillin.target protein in methicillin-resistant Staphylococcusaureusby gene fusion. FEBS Lett. 221 (1987) 167-17I. Tesch, W., Strgssle, A., Berger-Blehi, B., O'Hara, D., Reynolds, P. and Kayser, F.H.: Cloning and expression of methi¢illin resistance from Staphylococcus epldermldls in Staphylococcus carnosus, Antimicrob. Agents Chemother. 32 (1988) 1494-1499.
Fig. i. Sequences of mecA isolated from: (A)$. aumus BB270 Mcn ll~ (EMBL data library accession number X52594);(B) $. eptdenntdisWT55 McR(X52593); (C) $. auneusTK784 McR(Y00688).The sequences were compared by using the UWGCG program package. Sequences identical over 100 nt are represented once only. Differencesin the nt sequences are boxed. Convergent arrows indicate inverted repeats. The putative ribosome-binding site (S/D), the -10, and -35 sequences are indicated. The start and stop codons are overlined. In strains BB2?0 and WT55 an in-frame deletion of 6 bp within the structural gene lead to the loss of a recognition site for Sail, Accl and Taql. In BB270 an A --. G transition at nt 877 created a new restriction site for Xbal, known to be a hallmark for the mecA gene. At the 3' end of mec.Awe found an accumulation of point mutations. Fig. 2. The aa sequences of PBP2' from: (A)S. aureus BB270 McR; (B) S. epidermidisWT55 McR; and (C) $. aureusTK784 McRdeduced by the UWGCG program package. Differences in the aa are shown by asterisks. The active sites of the protein are indicated by arrowheads.
138 -
35
A A'~AC'~.,AT TATAC~'~A&C A~AAAJ~AG &']~A'~AACAC CTTC'I'ACACC TCCA?AT.AC A.qAA&ATT&TJ~ACA'I'~&'ITI''/~qCATAAAT -1o aID ~mm A T R A T A T A ~ A C A R A ~ T A G T CTTATATRAG GACC~TATTG ATGAAR&AGATAAA/~TI%'T TCCACTT&TT T T A A T ~ T/tG~'%"TC~ G'I~fGGT&T& AATAATRCTA TTG&TGCAAT 'I~AA~ATAAAAATT'/CAAACAAGT'/~AT/U~A~,ATA~CAGT TATATTTCTA
100 200 30Q
A
TATTTTTATG CT2CRAAAGA T ~ T T
k
RAAGCqAT&A 2~.GTGRJ4~fAGA~ATGACTG AAC~TCCGAT AAAAATATAT&ATAGTTTAGGCG'Z'TAAI~.Jk~&TA.q&CATT~ q ~ G ' f A
AAATAAAA/~
400
k
AGT&TCTRAA AATARARAR~ GR~fAGATG¢ TCAAT&TARA &TTAAAACAAA~&C~'rA& CA'Z'I'GA'I'C~¢ ~CGTTCA~T TTAATTT'I'G~ TAAA~kAGAT
500
&
GGTATGTGGA RGTTAGATTG GGATCATRC~2 L'TCATTATT¢ ~ G C A
GAA&~AC~A AGGqTACATATTG/~AAT~ AAAATCA~A CG2~TAAAA
800
AAAGG4ATC~ GTTCCA/~.,A ATGT&TCTAA/qA&~TT&T ~ T C G ~&GGCATC G T ~ ¢ ¢ ~ &TGTATCTAAAAAAGATTAT AAAGCAATC~
700 700
AAT~ATCAA AATT~.,L'T~ AAGATGATACCT~[¢~ICCA C~AAIU~C G'I~AAAA/t~ AATGGATCAAAATT~GTAC &J~GATGATACCTT¢GTTCCA ~ C C GTTAAAAA~ AAT~GATCAA AATTGC~TAC/U4GKTG&TACCT~C~'~TCC& GT?AAAAA~
800 800 800
CACATCTATT AJI~CGACTT CACATCTATT AA~GA¢~TT CACATCTATT
900 900
A AGGTTATGTT ~'Z¢CCA*I~A ACTCTGAA~AATTAAAR~AA ARAGAATATA RAGGCTAT~& A~k~L~ATGC& GTT&TTGGT& ARARC,GGACT ¢~AAARJ4CTT
1000
A
TTTTAGACCG AARC.AAT~"fGG~ATTGGC¢& &TACAC.GAR~ B TTT~AG~CCGAAACJ~kTG'I'GGN~TTGGCCA&TACAGGAR~ C 'I'TTT~,~CCG ,~C~TGTG G~ATTGGC~A ATACRGGAR~
B CTAAA~q~CTAA~'TA'FJ~¢T C ¢TA~q~qACT ~ & ~ T T C T GA~,ACTATA ~ J ~ f d ~ . A A 8
TGGATGRATA TTTAR~T~AT TTCGCARARA AATTTCATCT
TG~&TC~ATA TTTAR~TGAT T T C ~ A R A A A RATTTCATCT TACAAC~AAT GAAACAGAAAG'JlCe~'~AACTA C TGGAT~&ATA TTTARGTOAT TTCGCARARA RATTTCATCT TACAACTAAT GAAACAGAAAGTCG'TAACTA
A TACGATAAAA~qGc'~¢CAACATGAJ~&T~.,C TAqC~T~TCJ~ 8 TAC~ATAJ~A ~ C T C C R A C A T~%AGATGGC T&TCGTGTCA ¢ TACGATAAAA A G C T ~ A A C A TGARG&Tr~-C TATCGTGTCA
UTJ~.~..A&TA C/UP*TCGCACATGq~J~UTA G,qs~A.q,qJq~.~k 1100 UTAGCAATA CAATCGCACATACATTUTA ~ llOO
A AAAAAGA']~.~CAARF~TATT CAAC~AAC~A'~GAT~CTAA AGTTCAAAA~ AGTATTTATAACAAC&TGAAAAAT~qTTAT ~TCAGGTA CT~TAT¢CA 1200 A CC¢'ICAAACA~GTORATTAT TA~CACTTGT AAGCACACCT TCATATGACG TCTATCCATT TAT~TAT~ ATGAGTAAO~AJ~AATATAATAAATTAA~C 1300
GNqGAT;~J~ /~GAN:C~CT GC~C,~qENqG TTCCRGATTA C&~C'~'I"CAC¢ J~GTTCAACT CAAAAJ~T&TTA/¢AGCAAT GATTG~TTA AATAJ~AAAA 1400
A
A ¢ATTAGACGATAJ~J~AAGT TATAAAATCG ATGGTAA&~ TTGGCA&A/~ GATAAATCTTG~GG~GG'1~ACA~J~TACA &GAq'&'I~AAGT~"TAAATGG IS00 A TAkTATCGAC TTAARACA&G CAATAGA~TC &TCAGATAAC Aq~T]L'C~TTG CTAGAGTA~ AC'~/qA41~A GGCAL'TAAGAAAT~ChqJU~ A~.~CATGAAA 1800 & JU~CT~GGTGTTGGTGNkGAT&TACCAA~T GATT&TCCAT TTTAT~ATGC TCAAATTTCA AACAR~4AATT TA~ATAATGA AATATTATT& GCTG&~TCA~
1700
A GTTACGGJ~CAAGGTGAAATA C?G&TTAACC CAG?ACAG&T ¢CTTTCAATC TATAG¢GCATTAGAA&ATAATGGCAATATTAACGCACCT¢ ACTTATTAAA 1800
A AGACACGAAk~ACAJ~I'~J~ G~q,GAk,qAA TATT&TTTCC A~GAJqJ~TA ?CAA'~TA'rT A ~ A T ~ T &TGCAACA~GT¢GTAAATAAAA~.ACATAAA 1900 . . 8 A~CACGA/~ AACAJ~O?'i~ GGAJ~A.J~q.~TA'~&T'~CC AAAGAAJ~TA '~AA?CT&?T A ~ & T ~ T ATGCAACAAGTCGTAAATAAAACACATAAA 1900 C &~ACACGAAAkACA&AO?~ GGAk=AAJQ~A?A'~&?'~CC A,V~GANkA?A ?¢AA?¢TA?? AJ~ATOOT ATOCAACA~ TCGTAM?AA AACACA?AAA 1900 A GAAOATAT?TATAOATC?TATOCAAAC2~AAT?OOCAAAT CC~O?AC?OC AO/~¢~CAAA A?OAAACAAOOAOAJ~GO I=AG~CAAATTOGOTOGT~?A |000 B OAA~TAT~T &TAGATC?T& TOCAJ~C~A &T?GOCAJ~T CCGQ?AC?GC k=AACTCAAJ4A?GA.qACAAQ~ O ~ CAOIqCA*~ATT0GG'~T~TA 11000 C OAAGATA~tT&TA~A?CTTA TOCA,t~CT~A A?TOOCA~AT CCGO?ACTGO AOAAC~CAAA&~fdqJ~N~O C~]q~N~TT GGGT(IGT~T& 2000 A TATCATA?O&TAAAQ&?AATCCAJ~OA~A ~&TOGCT&T T A A ~ F J ~
GATO?ACAJ~ &TANq~N~T ~ C T N :
k &G'~'t&TGAT QAQCT&T&'~ &0AACOOTk~,?kkMt,t,kTAC ~&T&?AOA?G kA~HUqA~CkG?OAJ~¢: B AO?~TATGATQA~TATA?G AGAACGOT~.AT / ~ A T k C C A~I'O?&TQATGAOCTATA?OAOAACOGTAATAAAJI~IqTAC A A??ATGAATTA?TAATkAOT OC?O??AC?T C?¢~?A/~4 0 A~I~&TO~J4?T&TTAA?~4~t OCTO?TA~? CT~,~T~d4AJl '~q,F-'C'~lF~4~'~ C A~&TOAATT A?TAAT~O? OC~O~T&C?T C?.¢~TAAA k B ¢
Fig. I.
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A
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Fig, 2.
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