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volume 11 Number 2 1983 Nucleic Acids Research NndeotMe sequence of tbe TnlO encoded tetracydine resistance gene Wolfgang Hillen and Klaus Schollmeier Institut far Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRG Received 29 September 1982; Revised 26 November 1982; Accepted 2 December 1982 ABSTRACT The nucleotide sequence of 1530 base pairs of TnlO DNA coding for tetracycline resistance has been determined. The gene start consists of overlapping bidirectional promotors and two operator sequences. One terminator of transcription as defined by the typical terminator sequence is about 1300 base pairs downstream from the promotor. It is preceeded by translation termination codons in all three possible reading frames. The transcript contains an open reading frame coding for a 4 3.3 kDa protein. Two other possible reading frames are discussed. The amino acid sequence of the Tn10 encoded tetracycline resistance gene shows good homology with two proteins encoded in the tetracycline resistance part of the plasmid pBR322. The hydrophobic nature of the 43.3 kDa protein is discussed with regard to it's proposed function. INTRODUCTION The tetracycline resistance determinant of the transposon Tn10 is located on a 2700 bp Hpa I fragment [1]. A reoressor protein controling expression of the resistance gene is encoded on the leftward 700 bp [2]. The promotor and operator reaion for the repressor and resistance proteins consists of two bidirectional overlapping promotors and two operators [2,3]. The TET repressor has been isolated and the mechanism of regulation of the tetracycline resistance evaluated at a molecular level [4]. In addition to the 25 kDa TET repressor, a tetracyclineinducible 36 kDa resistance protein was detected in minicell assays, encoded by a gene which runs in the opposite direction to that encoding the repressor [2]. In addition, a tetracyclineinducible, low molecular weight protein has been detected on R222, a resistance factor bearing Tn10 [5]. Recently, genetic evidence was provided for the existence of two gene loci in the tet resistance part of the tet gene which were called tet A and © IRL Press Limited, Oxford, England. 03O5-1O48/83/1102-O626»2.OO/0 525 Nucleic Acids Research tet B [6). It was assumed that tet B is the 36 kDa protein because it's carboxyterminal coding region extends further than the molecular weight would imply [1,6]. The tetracycline resistance determinants from different R factors can be subdivided into at least four genetically different classes [7]. The tetracycline resistance gene on pBR322, the nucleotide sequence of which is known [0], belongs to class C whereas Tn10 is from class B [7]. It is of interest to compare the nucleotide sequences and the amino acid sequences of these tetracycline resistance proteins. In this article we report the nucleotide sequence of the Tn10 encoded tetracvcline resistance gene. Possible tetracycline resistance proteins are discussed and compared to the tetracycline resistance of pBR322. MATERIALS AND METHODS Materials Restriction endonucleases and T4 DNA ligase were purchased from 3RL, Bethesda, Md. or isolated by a published procedure [9]. Restriction digests were done as described [9]. | P|phosDhate was obtained from MEN chemicals, Dreieich. | P|ATP was synthesized from ADP and | P| by a published procedure [10]. T4 polynucleotide kinase was a gift of V. Eckert and R. Heinzel. Preparation of the DNA Tn10 DNA was prepared from the plasraids pRT61 and pRT29 [1,2]. The 675 bp Hpa I fragment from pRT61 was subcloned into pUR222 [11] to facilitate sequencing. PreDaration of plasmids was as described previously [12]. DNA fragments were either eluted from polyacrylamide gels as described [13] or purified by RPC-5 chromatography [14]. Sequencing The nucleotide sequence of DNA fragments was determined by the procedure of Maxam and Gilbert [13]. Reagents were obtained from NEN chemicals, Dreieich. The products of the strand cleaving reactions were separated on denaturing polyacrylamide gels [13] which were 0.5 mm thick and 80 cm long. The qels were exDosed to Cronex low dose plus film from DuPont. The combination of 12.5%, 8%, and 6% polyacrylamide gels allowed us to read ap- 526 Nucleic Acids Research proximately 400 nucleotides from one reaction in the best cases. Generally about 300 nucleotides were readable [15]. Restriction sites were found in the sequence by screening it using a computer program provided by L. Altschmied. RESULTS Sequencing strategy. Figure 1 shows the sequencing strategy used to determine the nucleotide sequence from the Xba I site to the Hind III site [1]. Except for a small portion around the Eco RI site the sequence of both the plus and minus strands was determined to reduce reading errors. For the same reason each part of the 1560 nucleotides was analyzed in at least three independent experiments. The leftmost Hinc II site in figure 1 characterizes the promotor-operator region for tetracycline resistance as it is protected against cleavage both by RNA polymerase [1] and TET repressor [4]. The numbers on the top panel of figure 1 refer to the published restriction map of Tn10 [1]. Figure 2 displays the complete nucleotide sequence from the Hinc II to the Hind III site. The leftmost 100 bp of the sequence including the overlapping promotor-operator region were communicated to us by K.P. Bertrand and W.S. Reznikoff [3] and also determined during the course of this study. The sequence was analyzed to yield the positions of cleavage sites from restriction endonucleases currently commercially available. The position of the cleavage sites are listed in table I. Transcriptional control signals. Figure 3 shows the promotoroperator region of the tetracycline resistance gene. It had been 12 75 675 Xbol Figure 1 Sequencing strategy for the Tn10 encoded tetracycline resistance gene. The lengths of the DNA on the top panel is taken from the Tn10 restriction map [1]. 527 00 u, C T T T A C G T T T G C T T ' T C A T T G C C G A ' T T A G G G G C A A T T T T G C A G G A T A A A T A T T G T C G G G C G G G G G G ' C A G G G A T C A C ' A G G A G T T T T T G C C C C T T A T T T G C C G T G T G T G G G G C T G T C J T G f G c i A A C G T T A T T ^ G G C G T A T T G C T G C T T G G A A A A A i A T T A A T A G G C 1 C T T T G G A T G C ' i I ' A C C A C C T C A G C T T C T C A A C G C G T G A A G T G L G T T T T G G G C T ' F G G T T T A A T A G C G G G G C C T A G A G A T T T C A C C G C A T A G T C C 6 T T T T T T A T C ^ A C T T T C C T T G T G G T T A T G T T T T G G T T C C G ' I T A C G G Figure 2 400 650 TOO 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1530 450 500 550 600 DNA sequence from the Hinc II site to the Hind III site as shown in figure 1 [1] A C C A A A A A T A C A C G T G A T A A T A C A G A T A C C G A A G T A G G G G T T G A G A C A T C G A A T T C G G T A T A C A T C A C T T T A T T T A A A A C G A T G C C C A T T T T G T T T A T T T A T T T T T C A G C G C A A T T G A T A G G C C A A A T T C ( J : C G C A A C G G T ( J G T G C T A T T T A C C G A A A A T C G T T T T G G A T G G A A T A G C A T G A T G G T T G G T T C A T T A G C G G G T C T T G G T C T T T T A C A C T C A G T A T T C C A A G C C T T T ( J C A G G A A G A A T A G C C A C T A A A T G G G G C G A A A A A A C G G C J A G T A C T G C T C T T T A T T D C A G A T A G T A G T G C A T T T G C ^ T T T T T A G C G T T T A T A T C T G A T T G G T T A G A T T T C C C T F F T T T T A A T T T T A T T G G C T G G T G G T G G G A T C G T A C C T G C A T T A C A G G G A G T G A T G T C T A T C C A A A C A A A G A G T C A T G A G G G T G C T T T A C A G G G A T T A T T G G T G A G ( L : C T T A C C A A T ( F C A A C C G G T G T T G G C C C A T T A C T G T T T A C T G T T A T T T A T A A T C A T T C A C T A C C A A T T T A T G G C T G G A T T T G G A T F A T T G G T T T A G C G T T T T A C T G T A T T A T T A T C C T A T C G A T G A C C T T C A T G T T A A C C C C T C A A G C T C A G G ' G G A G T A A A C A G A C A A G F G C T T A G T T A T T T C G T C A C C A A A T G A T G T T A T T C C G C G A A I A A T G A C C C T C T T G A T A A C C C A A G A G G ^ C A T T T T T T A C G A T A A A G A A I J T A G C T T C A A A T A A A A C C T A T C T A T T T T A T T T A T C T T T C A A G C T C A A T A A G C C G C G G T A A A T A G C A A T A A A T T G G C C T T T T T T A T C G G C A A G C T C T A G G T T T T T C G C A T G T A T T G C G A T A T G C A T A A A C C A G C C A T T G A G T A T T T T A A G C A C A T C A T C A T C A T A A G C T T T T G T T G C T T A A A A G T G A G T A T G G A T T A T T A G G G T A C C T A G A G C T G T G C T T G G A A G C T C A T A G G C'T G G T A A T A A T T A G C G T C T C C T T A T C A T G c i c A G A T A T C G C T A A A G G T T A T C T T T G C G C C C A G T G C T G G C T G G C T T T T T C G C T G A ' j ' G G G G A T T G G A T T G C T T C G G A i - G C G T T A A T G d G A T T T G G T C G G G A T T A C T T A T + T A T A C G G T A T C T T C T C T T C C T T A C G G A T G G 1 5 ° 200 250 300 350 T A C T C C G G G C T C T T G A G A C T G A A A A G T G A A A T G A A T A G T T C G A C A A A G A T C G C A T T G G T A A T T A C G T T A C G T C T A 50 100 G A C A C T C T A i C A T T G A T A G A G T T A T T T T A i c A C T C C C T A T C A G T G A T A G A > W 3) m - -• a O_ c z Nucleic Acids Research Table I Position of restriction endonuclease cleavage sites Restriction endonuclease Position Alu I Cla I Cfo I Dde I Eco RX Eco RI Eco RV Fok I Hae II Hae III Hga I Hha I Hind III Hinf I Hpa I Hpa II Hph I Mbo I Mbo II Mlu I Msp I Pal I Sau 3a Sau 96 I Sst II Taq I Tha I 1357, 1395, 1448, 1528 384, 429, 1233, 1206 270, 299, 337, 718 1262 426, 831 , 1235, 90, 155, 658, 7 2 2 , 735, 902, 975, 1146, 1427 657 172 344, 778, 1152 269 1 18, 358, 494, 7 3 0 , 1105, 1431 648 270, 299, 337, 718 1527 1041 1221 1094 1074 , 1275 526, 78, 375, 996 169, 857 438 1094 118, 358, 494, 730, 1105, 1431 78, 375, 996 1105 493, 1409 70, 101 , 655, 899, 1207 1294, 1410 399, 439, demonstrated previously that transcription starts in both d i rections from a common starting area [2-4]. The functions of this control element are indicated in figure 3. It consists of three promotors, one is directed towards the tetracycline r e sistance gene and two are directed towards the tetracycline repressor gene. The -10 homologies of two promotors overlap [16]. The third promotor is directed towards the tetracycline repressor gene and is 20 bp to the left in figure 3 of the overlapping promotors. This arrangement of transcriptional start signals is supported by in vitro transcription studies. The -10 region of the two overlapping promotors is flanked by two palindromic sequences which serve as binding sites for the TET repressor. This interpretation is concluded from protection and DNAse I footprint experiments (W. Hillen, C. Gatz, and K. Schollmeier, 529 Nucleic Acids Research -35 region Hind II -10 region raRNA ATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAA TAAAAACAACTGTGAGATAGTAACTATCTCAATAAAATGGTGAGGGATAGTCACTATCTCTTTT raRNA -10 ""mRNA -10,-35 -35 region region region Figure 3 The tet gene regulatory region as defined by in vitro transcription and protection experiments as well as DNAse I footprinting with TET repressor (W. Hillen, C. Gatz, and K. Schollmeier, manuscript in preparation). The -10 and -35 regions of three promotors are indicated. The bars indicate the TET repressor binding sequences. Details will be discussed elsewhere. manuscript in preparation) . A typical transcription termination sequence [16] is found around position 1320 of the sequence displayed in figure 2. The possible secondary structure of the terminator region is displayed in figure 4. This signal is preceeded by termination codons in all three possible reading frames within the last 80 nucleotides. Inspection of the sequence reveals the possibility T AT A A C C A C GA T r G T T A T T C GC G-C C-G T-A T-A T -A A-T T-A 5'TAGT TAATGA T CATTTTTTA 3' C-G C-G C-G T-A C-G T-A T-A G-C A T C C A A Figure 4 Possible secondary structure of the transcription terminator. Details are discussed in the text. 530 Nucleic Acids Research Reading frame A B C Sequence Start Stop codon codon T G A T A GAG A A A A G T G A A A T G . . . TAG T T G G A T G G A A T A G C A T G A T G , . . TAG G C T T C T C A A C G C G T G A A G T G . „ . TAG Figure 5 Comparison of the Shine-Dalgarno sequences preceeding three possible reading frames. Homologies of three or more nucleotides are underlined [18]. In addition, the start and stop codons are shown, of a second hairpin structure in the transcript ahead of the terminator. The stem and loop sizes are quite similar to those found in attenuators [17], however, the possibility to form an alternative structure necessary for attenuation is not found [17]. It is not clear whether this feature of the sequence, which is also shown in figure 4, is of biological importance. Possible reading frames for tetracycline resistance proteins. A single open reading frame extends over the entire sequence and codes for a 43.3 kDa protein. It starts 45 bases downstream from the overlapping promotors i.e. at position 60 of the DNA sequence. A second translation start may occur at position 793 in the same reading frame giving rise to a 17.6 kDa protein containing the 157 carboxyterminal amino acids of the 4 3.3 kDa protein. Both of these translation starts exhibit a Shine-Dalgarno sequence preceeding the initiation codon (Fig. 5) [18]. A third possible reading frame on the transcript starts at position 446 and terminates at 533. It lacks good Shine-Dalgarno homology and has, thus, only a low probability to be expressed. This latter protein would consist of 29 amino acids and have a molecular c | A_ P.O i i HincO ECBRI Figure 6 Location of three possible reading frames in the tetracycline resistance part of Tn10. P,0 denotes the regulatory sequence and T shows the transcription termination signal. 531 Nucleic Acids Research weight of 3.7 kDa. It would, however, be in a different reading frame from the previously discussed proteins. Figure 5 compares the DNA sequences at these possible gene starts and figure 6 gives the location of the three reading frames on the Tn10 DNA. In the analysis to follow only the 43.3 kDa protein is considered because it completely embodies the 17.6 kDa protein, while expression of the 3.7 kDa product is considered unlikely anyway. Codon usage and amino acid composition„ Table II shows the result of a codon usage analysis of the tetracycline resistance protein Table II Codon usage and amino acid composition of the 43.3 kDa protein Codon usage Phe uuu uuc Leu UUA UUG CUU cue lie Met Val Ser CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG UCU UCC UCA UCG AGU AGC Pro ecu Thr CCC CCA CCG ACU ACC ACA ACG Trp 532 UGG total content 22 6 23 13 11 2 4 7 22 10 5 13 7 4 6 8 4 9 7 9 2 5 3 5 1 5 9 6 6 12 28 60 codon usage Ala Tyr His 37 13 Gin Asn Lys 25 Asp Glu 31 Cys Arg 14 26 12 Gly total content GCU GCC GCA GCG UAU UAC 13 5 13 9 4 3 CAU CAC CAA CAG 3 2 8 5 AAU AAC AAA AAG 9 1 6 3 GAU GAC GAA GAG UGU UGC CGU CGC CGA CGG AGA AGG 9 1 5 2 1 1 1 0 10 GGU GGC GGA GGG 13 9 7 9 38 8 4 1 40 13 10 10 12 Nucleic Acids Research from Tn10 and gives the araino acid composition for the 43.3 kDa protein. The codon usage is clearly non-random and rather different from other E. coll proteins [19]. Very striking is the use of UUU 22 times out of 28 codons for Phe. A similar observation has been made for the transposon Tn3 genes [20,21] as well as others [21]. The general tendency seems to be that codons with AU pairs instead of GC pairs in the wobble positions are preferred in genes with a high rate of expression [21]. This observation is especially clear in the cases of Phe, Leu, lie, Ser, Ala, Gin, Asn, Asp, Glu, and Arg. Distribution of amino acids in the tet resistance protein. Figure 7 displays the distribution of amino acids grouped according to their polarity in hydrophobic, uncharged polar, acidic, and basic residues. The dominance of hydrophobic amino acids, which make up for 58% of the total, is clearly demonstrated in this figure. With two exceptions they are evenly distributed over the entire protein. This result agrees well with the proposed function of the resistance protein as an inner membrane protein [22-24]. Two interruptions of the hydrophobic structure occur at position 180-200 and 320-340. The former does not fall in a region of horaology with pBR322 and may, therefore, not be of functional importance. The latter, however, shows some homology with pbR322 suggesting a possible functional significance (compare also fig. 8 ) . Comparison of the tetracycline resistance genes in TniO and pBR322. Figure 8 compares the amino acid sequences of the pBR322 encoded tetracycline resistance proteins [8] with that encoded by Tn10. Clearly extensive homology exists between the two proteins encoded by pBR32 2 and the one encoded by Tn10. The homology is very good over the first 80 ainino acids of the small 21 kDa pBR322 protein and remains good throughout the next 80 amino acids. The last 30 ainino acids of the small protein are not homologous to Tn10. Around position 160 of the small pBR322 tetracycline resistance protein the homology shifts to the large 40 kDa tetracycline resistance protein (compare fig. 8 ) . This is coded in a different reading frame but is initiated within the reading frame of the 21 kDa protein. It shows no horaology to the Tn10 protein in that part which overlaps the 21 kDa protein to 533 w Figure 7 H.t Trt Ala Ser TSr Trr *i" 6ln Glu Air vai H€t I" ft? Hit Asp 20 I 30 40 I 50 60 I 70 JllllL 80 90 L m JLL J_ J L J 100 110 120 130 140 150 16O 170 180 190 ii i ii i L 200 210 220 230 240 250 260 270 280 290 3OO 310 320 33O 340 350 360 370 380 390 400 410 L 10 L i ill Hi I D i s t r i b u t i o n of h y d r o p h o b i c amino a c i d s i n t h e 4 3 . 3 kDa p r o t e i n Amino Acids Amino Acids Position of Ammo Acid Acidic Basic Ala f/? vS? Uncharged polar fH' Ammo Acids Hydrophobic Ammo Acids Nucleic Acids Research position 160. Good homology with the Tn10 tetracycline resistance protein is observed with the last 260 amino acids. It is interesting to note that two proteins, each of which shows extensive homology to the respective Tn10 protein, are apparently necessary for tetracycline resistance in pBR322. This observation will be discussed below in the light of TniO complementation data [6]. DISCUSSION The finding of a complex, overlapping promotor system for the tetracycline resistance gene carried on Tn10 and described in figure 3 is in agreement with previously published data [2]. A similar arrangement of overlapping promotors has been described for the tet proraotor of pBR322 [25]. The particular advantage of such an arrangement for the regulation of the tet gene is not clear. The transcription terminator structure is quite similar to other terminators [16]. It is preceeded by translation termination codons in all three possible reading frames. Therefore, it is very likely that this sequence is a functional terminator of transcription. It remains unclear whether or not the possible hairpin structure preceeding the terminator has any biological significance. It displays the typical attenuator arrangement; however, we did not find possible alternative structures typical of attenuators [17]. The DNA sequence on the 1275 bp Hinc II fragment plus 165 nucleotides from the Hp_a I site on the 675 bp Hpa I - Hind III fragment contains only one open reading frame which encodes a protein of 43.3 kDa. Translation of this sequence terminates downstream of the Hpa I site at position 1266. This agrees with the observation that insertion of the 1275 bp DNA into a plasraid leads to the synthesis of a fusion protein [1] and is consistent with the mapping of mutants affecting tetracycline resistance [26]. The molecular weight of 43.3 kDa, however, does not agree with the apparent molecular weight of 36 kDa found in minicell assays [1,2,5]. This discrepancy may be due to the high content of hydrophobic amino acids in the protein which is known to reduce the apparent molecular weight on Laemmli gels in many cases 535 Nucleic Acids Research A T A T A T A T B A T B A T B A T B A T B A T B Met Lys Ser Asn Asn Met Asn Ser Asp Leu Ala Val Glv Leu Asp Ala Met Gly Leu Leu Arq Asp H e Leu Leu Arq Glu Phe Glv Val Leu Leu Ala Glv Val Leu Leu Ala Ala Leu H e Val Ser Thr Lys H e H e Glv Leu, Val H e Gly Leu H e Val His Ser Asp Tie Ala Sen r,iu Leu Tvr Ala Leu Leu Tvr Ala Leu Pro Val Leu G.1Y Ala Leu Ser Pro Trp Leu Glv Lys Met Ser Thr Arg Ser Arg Ser Thr Val Leu Leu Ala Ser Leu Leu Gly Leu Leu Leu S«r Leu H e Glv Pro Ala Arg Phe Ala Thr Trp Ala Thr Thr Pro Val Leu Trp Ala Phe Ser Ser Ala Leu Trp Gly Asp His Thr Arg Pro Val He Ala Met Met Ser Asp Met Met Ara Phe Glv Arq Arq Arq Phe Glv Arq Arq Arg Pro Leu Trp Pro Pro Ala Thr He Asp Tvr Ala Ala Ser Leu A S P Tvr Leu Ser His Tyr Arg Leu Arg He Leu Tvr Ala Gly Arq Met Leu Tvr Leu Gly Arq Asp Pro Leu Arg Arg Thr ASP ASP A T B Ala Glv lie Thr Glv Ala Thr Glv Ala Val Ser Glv lie Thr Glv Ala Thr Glv Ala Val Gly Arg His His Arg Arg His Arg Cys Gly Ala A S D H e Thr Asp Gly Glu Asp Ara Ala Ala A S P Thr Thr Ser Ala Ser Gin Arq Val Arg Arg His His Arg Trp Gly Arg Ser Gly Met Ser Ala Cvs Phe Gly Val GlY Met Val Leu Glv Ala Ser Phe GlY Leu Glv Leu H e His Glu Arg Leu Phe Arg Arg Gly Tyr Gly Glv Asp Cys Trp Ala Pro Ser Pro Cys Met Glv GlY Phe Ala GlY Glu He Ser Pro His Glv GIY Leu Leu GlY Ala H e Ser Leu His Arg Arg Cys Met Thr Ala Ser Thr Tyr Tyr Ala Ala Leu Leu Asn Tip Val _Thr Phe Leu Ala Ala Val Leu Asn Gly Leu Asn Leu Leu T B T B T B T B Phe Met Gly Ala Thr He Glv Gly Arq Gin Val Phe Met Val Gin Gin Glu Glu Glu Asn Pro Ala He Val T B T B T Phe Phe Gly Gly Ala Thr Gly Arq Leu He Thr Lys Trp Trp Leu Leu Lys Arg A T B A T B 3 536 Thr Ser Thr Pro He Ala Pro Pro Lys His Gin Val T.en Leu Ala Ala Asn Lys Ser Ser Leu Met Thr Ala Asn Ser Met Ser Ala Thr His Ser Val His Ala Leu Trp Gly Glu Phe Glv Glu Thr Gly Asn Ser He Thr Val Leu Met Met Phe Ala Lys Lys Leu Gly Thr Val Thr Leu Val H e Thr Leu Pro Ye* Leu Pro ,Gly Pro Va.1, Leu Pro Thr H e Ala Ser HJs Tvr Ile- Ala Asft H i * Phe Gln Phe Leu Cys Ala Gin Val H e Phe Ala Met Arg Arg Glu Ser Phe He Val Trp Trp Val H e Gin Gin Thr Gin Asp Arg Val Arg Tyr Phe Val Val Glv Glv Ala Ala Ala Ala Pro Val Pro Val Ser H e Met Leu Leu Asp His He Val Leu Leu His Arq Ala Gly Ala Tyr He Ala Ala Ser Val H e Cys Trp Arq Leu Tyr Arg His Phe Glv. Leu Lys Trc Phe Glv Trp Ser Pro Leu Arg Ala Ala QlY Pro Trp Pro Ala GlY Pro . H e H e Gly Arg Pro .Val Ala His His Ser Leu Arg Ser Pro Phe Phe He Ala Pro Phe Leu Ala Trp Ala Ala Ser term Val Val Met Phe Trp Leu Gly Cys Phe Leu Asn Thr Asp Thr Glu Val Arg Pro Met Pro Leu Arq Tyr H e Thr Leu Phe Lys Trp Ala Arg Gly Met Thr Phe. Ser Ala G3n Leu H e Phe H e Met Gin Leu Val Leu Phe Thr Glu Asn Arq H e Phe Gly Gin Asp Arq Phe Ser Leu Ala Gly Leu Leu Ser Leu Ala Val Phe Phe Val Ala Gly Arq H e Phe Val Thr Glv Pro Ala Val Leu Leu Glu Phe H e H e H e Ala Gly Met Ala Nucleic Acids Research T B T B T B T B T B T B T B T B Ala Asp Ser Ser Ala Asp Ala Leu Trp Leu Asp Phe Trp Met Ala Phe Ala Leu Pro Ala Gly Met Pro Ala His Asp Ala Leu His Ala Ala Ala Glu His Thr Thr Ser Ser Phe Leu Gin Gin Gly Ser Leu Ala Tyr Tyr Ala Gly Pro Pro Leu Leu Phe Tyr Val He Ala Phe Leu Val Leu Leu Leu H e Leu Met H e Leu Gin Gly Val Met Gin Ala Met Leu Gly Ala Leu Gly Gin Leu Val H e Gly H e Thr Gly Pro H e Trp Ser Thr Trp Gin Gin Pro Pro Asp Asn Cys lie He H e Leu Val Cys Leu Thr Pro Gin Ala Gin Gly Ser Ser Arg Ala Thr Ser Thr term Gly Gly Leu Leu Gly Gly Leu Pro Lys Leu Ser Leu He Trp Leu Leu Ala Gin Ala Ala Leu Leu Ser Ser Phe H e Ser Glu Gly Phe Ala Thr Arq Gly Ala Ala He Arq Gly Gly Gly H e Ser Giy Gly H e Gin Thr Lys Ser Gin Val Asp Asp Val Ala Thr Thr H e Trp Ala Trp Ser Leu Thr Asn Ala Leu Thr Ser Leu Leu Phe Val Val H e Tyr Ala H e Tyr H e H e Gly H e Val Gly Ser Met Thr Phe Met Leu Arg Arg Gly Ala Asn Ala Leu Ala Leu Trp Glu Thr Ser Ala term Figure 8 Comparison of the amino acids sequences of the Tn10(A) encoded 4 3.3 kDa protein (T) with the pBR322 encoded 21 kDa and 40 kDa (B) tetracycline resistance proteins [8] . Homologous amino acids are underlined. [27], Indeed, using a phosphate buffered SDS polyacrylaraide gel system, a molecular weight of 50 kDa has been found for the TET protein instead of 36 kDa as seen in the glycine buffered gel system [5], Therefore, we conclude that the real molecular weight of the TET protein is 4 3.3 kDa. However, it may also be processed resulting in a reduced molecular weight. The existence of two complementation groups for tet resistance has led to the conclusion that there are two genes being carried on this DNA [6]. Complementation may also occur, however, at the protein level, if it is assumed that an oligomer of the 4 3.3 kDa protein is the functional unit. This is supported by the situation of the tet genes on pBR322 where two proteins are encoded in the tet resistance sequence [8]. The existence of homology between pBR322 TET proteins and the Tn10 TET protein favours this interpretation. Interestingly, homology does not occur between the amino terminal end of the second pBR322 protein, which overlaps with the first gene, and the TET protein from Tn10. Taken together, the existence of two functional domains on the TET proteins seems to be possible and may explain the complementation results [6]. Although hybridisation experiments revealed that pBR322 and 537 Nucleic Acids Research Tn10 fall into different classes with respect to their tetracycline resistance determinants due to a lack of DNA homology [7], the amino acid sequences of the resistance proteins show good homology. This apparent contradiction can be explained by differences in the use of wobble bases. The gene start for the 17.6 kDa protein in the same reading frame as the 4 3.3 kDa protein shows a good Shine-Dalgarno sequence [18]. This may be the low molecular weight tetracycline- inducible protein which has been found in other experiments [5]. Within the DNA sequence reported here it is the only possible open reading frame for a protein in the molecular weight range of 15 kDa [5]. ACKNOWLEDGEMENTS We wish to thank Dr. R, Schmitt and Dr, H C G. Gassen for many helpful discussions, Dr. S..H. Waters for sharing his unpublished results with us, M. Luxnat and I t Kaffenberger for their assistance in preparing some of the DNAs for sequencing, and E. R6nnfeldt for typing the manuscript. This work was supported by a grant from the Chemische Fabrik RiJhm GmbH, Darmstadt. REFERENCES [1] Jorgensen, R.A. and Reznikoff, W.S : (1979) J. Bacteriol. 138, 705-714 [2] Wray, L.V. Jr., Jorgensen, R<A t , and Reznikoff, W.S. (1981) J. Bacteriol. 147, 297-304 [3] Bertrand, K.P., Postle, K,, Wray, L.V., and Reznikoff, W.S., manuscript in preparation, [4] Hillen, W., Klock, G,, Kaffenberger, I., Wray, L.V. Jr., and Reznikoff, W.S. (1982) J. Biol. Chem. 257, 6605-6613 [5] Yang, H.L., Zubay, G., and Levy, S.B. (1976) Proc. Natl. Acad. Sci. 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