Gene, 114 (1992) 81-83 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

81

GENE 06437

Short Communications

Corrected nucleotide sequence of Ml3mpl8 gene !II (Epitope vectors; peptide libraries; coat protein; sequence correction)

Richard Ebright, Qianping Dong and Joachim Messing Waksman bastitute, Rutgers, The State University of New Jersey. Piscataway. NJ 08855-0759 (U.S.A.) Received by A.J. Podhajska: 13 December 1991 Revised/Accepted: 4 February/10 February 1992 Received at publishers: 13 February 1992

SUMMARY

There are seven differences between the actual nucleotide (nt) sequence of bac~eriophage M13mpl8 gene I11 and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M 13 gene m).

INTRODUCTION

Bacteriophage M 13mp 18 is a widely used cloning vehicle (Yanisch-Perron et al., 1985; review in Messing, 1991). Bacteriophage M13mpl8 was constructed from bacteriophage M I3 by introduction of lac and polylinker sequences and by elimination of three restriction endonuclease cleavage sites outside the polylinker sequence (by random chemical mutagenesis followed by selection for resistance to restriction endonuclease digestion) (Messing et al., 1977; Gronenborn and Messing, 1978; Messing, 1979; 1981; Messing et al., 1981; Yanisch-Perron etal., 1985). The complete nt sequence of M 13mpl 8 has been reported (Yanisch-Perron et al., 1985). EXPERIMENTAL AND DISCUSSION

In the course of constructing a series of epitope display vectors (cf. Scott and Smith, 1990; Cwirla et al., 1990;

C¢,rrespondence to: Dr. J, Messing, Waksman Institute, Rutgers, State University of New Jersey, P.O. Box 759, Piscataway, NJ 08855-0759 (U.S.A.) Tei. (908)932-4256: Fax (908)932-0072. Abbreviations: aa, amino acid(s): lac, lactose operon; nt, nucleotide(s): PCR, polymerase chain reaction.

Devlin et al., 1990; IV/cCafferty et al., 1990; Bass et al., 1990; Breitling et ai., 1991; Hoogenboom et al., 1991; Barbas et al., 1991), we have redetermined the nt sequence ofM13mpl8 gene III (Fi~;. 1). We find that there are seven differences between ~he a~aual nt sequence of Ml3mpl8 gene 111 and the previously reported nt sequence (YanischPerron et al., 1985)." i.e., T at nt 1663, C at nt 1737, G at nt 2220, 1" at nt 2224, T at nt 2225, T at nt 2229 and G at nt 2710. These seven differences in the nt sequence result in three differences in the inferred aa sequence: the correct aa are Ser 29, Leu216 and Gly 378. The previously reported nt sequence of M13mpl8 gene 111 was not determined directly, but was 'compiled and reprinted' (Yanisch-Perron et al., 1985). It was assumed that the nt sequence of M 13mp 18 gene III was identical to the nt sequence of M I3 gene I11 (Van Wezenbeek et al., 1980), except for the elimination of the BamHl site at nt 2220. Furthermore, it was assumed that elimination of the BamHI site at nt 2220 involved a single-bp substitution that: (I)was a G:C-~A:T transition (based on the specificity of the chemical mutagen used; Gronenborn and Messing, 1978), and (2)was located at the third nt positionofa codon (based on retention ofwild-type phenotype). The present results show that these assumptions were not correct. There are tbur differences between the nt sequence ofM 13mp18 gene lIl and the nt sequence of M 13

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1720 1740 1760 1780 1800 GACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTT -. ......... + ......... * ......... * ...... T--* ......... ÷ ......... * ......... * ......... ÷ ......... * ......... ÷ ......... ÷ ........ AspAspLysThrLeuAspArgTyrA•aAsnTyrG•uG•yCysLeuTrpAsnA•aThrG•yVa•va••a••ysThrG•yAspG•uThrG•n•ysTyrG•yThrTrpVa••r•I•eG•yLeu 50 60 70 80 1840 1860 1880 1900 1920 GCTATCCCTGAAAATGAGGGTGGTGGC TCTGAGGGTGGCGGTTC TGAGGGTGGCGGTTCTGAGGGTGGCGGTAC TAAACCTC CTGAGTA CGGTGATACAC CTATTC C GGGCTA TACTTAT

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AATAGG~AGGGGG~ATTAACTGTTTATACGGGCAcTGTTAcTcAAGGcACTGA~C~GTTAAAAcTTATTAcCAGTACACTCCTGTATCATCAAAAG:~CATGTATGACGCTTACTGGAAC

170

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2200 2220 2240 2260 2280 GGTAAATTCAGAGACT~CGCTTTCCATTCTGGCTTTAATGAGGATTTATTTGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCC%GTCAATG~TGGCGGCGGCTCTGGT . . . . . . . . . . .

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2800 2820 2840 TTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAA PheLeuLeuTyrValAlaThrPheMetT~rValPheSerThrPh&AlaAsnileLeuArgAsnLysGluSerEnd 410 420

Fig. 1. The nt sequence of M 13mp18 gene ili and the in~r~d aa sequence. Residues that differ in the previously reported nt sequence and in~rred aa sequence (Yanisch-Perron et al., 1985) are indicated beneath the sequence. Numbering differs by one nt from Yanisch-Perron et al. (1985), due to an insertion at nt 898 of M 13 on p. 18. Last digits of numerals are aligned with eor,esponding nt. GenBank accession No. is VB0018.

gene 111 (Van Wezenbeek et al., 1980) in addition to elimination of the BamHI site at nt 2220. Furthermore, elimination of the BamHl site at at 2220 involves a double-bp substitution at the first and second nt positions of a codon. We suggest that the four differences between the nt sequence of M 13mp 18 gene III and the nt sequence of M 13

gent: 111 in addition to elimination of the BamHl site at nt 2220 relate to the random mutagenesis procedures used to introduce and eliminate restriction sites (N-methyl-Nnitrosurea mutagenesis to introduce the EcoRI site at nt 6231 and to eliminate the BamHI site at nt 2220; hydroxylamine mutagenesis to eliminate the Hincll site at nt 7248

83 and to eliminate the Accl site at nt 6933) (Gronenborn and Messing, 1978; Messing et al., 1981). The present results, together with the results in Zhou et al. (1991), indicate the necessity to re-determine all the nt sequences after chemical mutagenesis or PCR.

ACKNOWLEDGEMENTS

We thank Dr. Yon Ebright for the synthesis of oligodeoxyribonucleotide primers. This work was supported by National Institutes of Health grants GM41376 to R.H.E. and GM43261 to J.M.

REFERENCES Barbas, C., Kang, A., Lerner, R. and Benkovic, S.: Assembly of combinatorial antibody libraries on phage surfaces: the gene !!! site. Proc. Natl. Acad. Sci. USA 88 (1991) 7978-7982. Bass, S., Greene, R. and Wells, J.: Hormone phage: an enrichment method for variant proteins with altered binding properties. Proteins 8 (1990) 309-314. Breitling, F., D0bel, S., Seehaus, T., Klewinghaus, I. and Little, M.: A surface expression vector for antibody screening. Gene 104 (1991) 147-153, Cwirla, S., Peters, E., Barrett, R. and Dower, W.: Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87 (1990) 6378-6382. Devlin, J., Panganiban, L. and Devlin, P.: Random peptide libraries: a source of specific protein binding molecules. Science 249 (I 990) 404406.

Gronenborn, B. and Messing, J.: Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature 272 (1978) 375-377. Hoogenboom, H., Griffiths, A., Johnson, K., Chisweli, D., Hudson, P. and Winter, G.: Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19 (1991)4133-4137. McCafferty, J., Griffiths, A., Winter, G. and Chiswell, D.: Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348 (1990) 552-554. Messing, J.: A multipurpose cloning system based on the single-stranded DNA bacteriophage M13. Recomb. DNA Tech. Bull. (NIH Publ. No. 79-99, 2) 2 (1979) 43-48. Messing, J.: Ml3mp2 and derivatives: a molecular cloning system for DNA sequencing, strand-specific hybridization, and in vitro mutagenesis. In: Walton, A. (Ed.), Proceedings of the Third Cleveland Symposium on Macromolecules. Elsevier, Amsterdam, 1981, pp. 143153. Messing, J.: Cloning in M 13 phage or how to use biology at its best. Gene 100 (1991) 3-12. Messing, J., Gronenborn, B., Mtlller-Hill, B. and Hofschneider, P.H.: Filamentous coliphage M13 as a cloning vehicle: insertion of a Hindlll fragment of the lac regulatory region in M13 replicative form in vitro. Proc. Natl. Acad. Sci. USA 74 (1977)3642-3646. Messing, J., Crea, R. and Seeburg, P.H.: A system for shotgun DNA sequencing. Nucleic Acids Res. 9 (1981) 309-321. Scott, J. and Smith, G.: Searching for peptide ligands with an epitope libraD. Science 249 (1990) 386-390. Van Wezenbeek, P., Huisebos, T. and Schoenmakers, J.: Nucleotide sequence of filamentous bacteriophage M 13 DNA genome: comparison with phage fd. Gene 11 (1980) 129-148. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved MI3 phage cloning vectors and host strains: nucleotide sequences of the Ml3mpl8 and pUCI9 vectors. Gene 33 (1985) 103-119. Zhou, Y., Zhang, X. and Ebright, R.H.: Random mutagenesis of genesized DNA molecules by use of PCR with Taq DNA polymeras~. Nucleic Acids Res. 19 (1991) 6052.

Corrected nucleotide sequence of M13mp18 gene III.

There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (whi...
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