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Initiation of Protein Synthesis in Bacteria at a Translational Start

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Initiation of Protein Synthesis in Bacteria at a Translational Start Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp. 1442-1446, March 1980 Biochemistry Initiation of protein synthesis in bacteria at a translational start codon of mammalian cDNA: Effects of the preceding nucleotide sequence (genetic expression/dihydrofolate reductase/plasmid/DNA cloning/ribosome binding) ANNIE C. Y. CHANG*tt, HENRY A. ERLICHt, ROBERT P. GUNSALUS§, JACK H. NUNBERG§, RANDAL J. KAUFMAN1, ROBERT T. SCHIMKE§, AND STANLEY N. COHEN*tll Departments of *Genetics, tMedicine, §Biological Sciences, tMedical Microbiology, and IPharmacology, Stanford University, Stanford, California 94305 Contributed by Stanley N. Cohen, December 31, 1979 ABSTRACT Plasmids containing a mouse cDNA sequence Thus, these bacterial plasmids were a potentially useful source encoding the enzyme dihydrofolate reductase (DHFR; te- of information about the effects of DNA sequence variation in trahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ the translational control region on the efficiency of hetero- oxidoreductase, EC 1.5.1.3) have been used to study the effi- ciency of initiation of protein synthesis at an ATG (AUG) specific gene expression. The results of studies on these effects translational start codon indigenous to the eukaryotic cDNA. are reported here. Differences in DHFR production assayed phenotypically, en- zymatically, and immunologically were correlated with the MATERIALS AND METHODS primary structure of the DNA segment that precedes the Bacterial Strains and Plasmids. The construction, isolation, translational start codon. Our results indicate that initiation of and endonuclease mapping of chimeric plasmids that contain a structurally discrete and biologically functional eukaryotic encoding for mouse DHFR (pDHFR protein can occur in bacteria on a fused mRNA molecule, and double-stranded cDNA that the efficiency of expression is strongly affected by: (i) the plasmids) have been described (6). Escherichia coli strain extent of homology of the translational control region with the X2282, a thy + derivative of X1776 (7), was used as the bacterial 3'-OH end of 16S ribosomal RNA, and (ii) the distance between host under P2 containment conditions, as specified in the NIH the protein start codon and the ribosome-binding sequence on Guidelines for Recombinant DNA Research of Dec. 22, 1978. the mRNA. Experimental Procedures. Restriction endonucleases were purchased from either Bethesda Research Labs (Rockville, MD) Introduction of double-stranded complementary DNA (cDNA) or New England BioLabs and used according to the suppliers' or chemically synthesized DNA into an endonuclease cleavage recommendations. The 5' end of endonuclease-generated DNA site in a bacterial gene has enabled the synthesis of hybrid fragments was labeled with ['y-32P]ATP by using polynucleotide peptides that react with antibodies against their eukaryotic kinase (8) (specific activity 3000 Ci/mmol; 1 Ci = 3.7 X 1010 component (1-5). Although these hybrid proteins have not been becquerels). DNA nucleotide sequences were determined as shown to be biologically functional, biological activity has been described by Maxam and Gilbert (8); chemically cleaved DNA found for a discrete (i.e., nonhybrid) protein encoded in bacteria fragments were run on 0.35-mm gels containing 8% (9) or 20% by a mouse cDNA sequence for the enzyme dihydrofolate re- (8) acrylamide. ductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-te- Enzyme assays for activity of mouse DHFR in extracts of E. trahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) (6); the high coli cells were described previously (6). Filter affinity transfer level of resistance to the antimetabolic drug trimethoprim (Tp) assays (6, 10) and in vitro synthesis of proteins were carried out specified by the mouse enzyme allowed isolation of bacterial as described (11). The [asS]methionine-labeled translation cells that phenotypically expressed the eukaryotic genetic se- products from in vitro or in vivo synthesis were immunopre- quence. These bacteria were found to synthesize a protein that cipitated (12) with rabbit anti-mouse DHFR or rabbit anti-E. has the enzymatic properties, immunological reactivity, and coli f-lactamase serum (the gift of R. B. Sykes of Squibb) and molecular size of the mouse DHFR. Moreover, the cDNA Staphylococcus aureus protein A. The direct chemical transfer segment in such clones was in a different translational reading of proteins from gels to a reactive diazo filter paper has been frame from the bacterial f3-lactamase gene into which it had described by Renart et al. (13). been inserted, suggesting that the DHFR was not part of a fused protein. Together, these findings implied that initiation of RESULTS translation was occurring at the translational start codon nor- Relationship of Structure at Vector-cDNA Junction to mally used for the mouse DHFR. pDHFR Expression. Each of the plasmids studied was con- Different DHFR cDNA-containing clones showed greatly structed by insertion of poly(dC)-"tailed" DHFR cDNA at the different levels of Tp resistance. Such differences appeared to poly(dG)-tailed PstI cleavage site of the pBR322 plasmid vector reflect control at the translational level, because all of the (6), and all except pDHFR 23 showed phenotypic expression plasmid constructs used the same 3-lactamase gene promoter of Tp resistance. Sequence analysis of the nucleotides in the and had the same site of cDNA insertion. However, the length vicinity of the vector-cDNA junction was carried out for each of the DNA segment on the 5' side of the structural gene ap- plasmid by 5' end-labeling of Hpa II and Taq I endonuclease- peared to be different in the various plasmids, and gel analysis generated DNA termini according to the scheme shown in Fig. of endonuclease-cleaved plasmid DNA showed differences in 1. This experimental plan enabled determination of the se- the sequence preceding the DHFR translational start codon. quence of both DNA strands within the region of interest. The publication costs of this article were defrayed in part by page Abbreviations: DHFR, dihydrofolate reductase; Tp, trimethoprim; charge payment. This article must therefore be hereby marked "ad- bp, base pair(s); RBS, ribosome-binding sequence; S-D, Shine-Dal- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate garno. this fact. II To whom reprint requests should be addressed. 1442 Downloaded by guest on September 26, 2021 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 77 (1980) 1443 3- Lactamase DH F R resistance to Tp concentrations of 500 gAg/ml or greater; clones A Pstla Taqi Hael l Hpall that expressed lower levels of resistance could not be distin- guished enzymatically, but showed different minimal inhibi- HaelIl Hpall Hinfl the 37561 3658 161 1 tory concentrations of Tp during growth. The sequence of pDHFR 7, 12, 27, 29 DNA strand equivalent to the mRNA is shown. The primary 13, 26, 28 sequencing data for some of the more interesting plasmids are 26 shown in Fig. 2. 28 As shown in Table 1 (Column IV), the A of the translational DHFR ,- Lactamase start codon is located 11 to 14 bases after the center of the 5- B Hpal HaelII Taql Pstlb base-pair (bp) sequence homologous to the C-C-U-C-C of 16S rRNA in all clones that express resistance to Tp, except for HpalI HaellI pDHFR 26, in which the ribosome-binding sequence (RBS) 1361 113548 l3489 (15-17) is located 24 nucleotides before the ATG. In the pDHFR 25 pDHFR 7 plasmid, the sequence formed by a Pst I cleavage FIG. 1. Scheme used to determine the nucleotide sequence ofthe site and the poly(dG) additions show a 5-nucleotide-long seg- DHFR cDNA insert at the vector-cDNA junctions corresponding to 3' OH end of 16S ri- the 5' end of the mRNA pDHFR plasmids. The boxed numbers at the ment of homology with a sequence at the Hae III, Hpa II, and Pst I sites indicate the nucleotide positions in bosomal RNA (Shine-Dalgarno, or S-D sequence) (15, 16). In the pBR322 plasmid as determined by Sutcliffe (14); these sites were the other plasmids, the Pst I site is further from the translational used to orient the structural gene coding for mouse DHFR. The arrows start codon, and a segment homologous with the S-D sequence above the map show the direction of translation of mouse DHFR in is formed entirely by nucleotides within the cDNA or by a the plasmid with respect to the gene coding for f3-lactamase. The as- combination of sequences from the cDNA and the homopoly- terisks indicate the 5'-labeled end of the DNA fragments selected for sequence analysis, and arrows indicate the direction of sequencing. meric "linker." (A) Scheme used for sequencing pDHFR plasmids 7, 12, 13, 26, 27, Comparison of the sequence at the vector-cDNA junction 28, and 29. (B) Scheme used for sequencing the corresponding seg- for the pDHFR 12, 13, and 28 plasmids indicates that minimal ment of pDHFR 25, which contains a cDNA insert in orientation alterations in nucleotide sequence or positional changes in the opposite to that of the f3-lactamase gene of the vector. putative ribosomal binding region can have marked effects on expression. For example, the pDHFR 12 and 28 plasmids contain 28 and 27 base pairs, respectively, between the terminal Table 1 shows the primary structure of the DNA segment nucleotide of the vector DNA and the translational start codon between the Pst I cleavage site and the ATG (AUG) transla- for DHFR, and in both cases the translational reading frame tional start codon in each of the plasmids studied, and it indi- of DHFR is different from that of the f3-lactamase. In pDHFR cates other parameters used to characterize these plasmids. 12, mutation to an A (coincidentally generating an Alu I Expression of DHFR was assayed enzymatically in vitro and cleavage site) has occurred at position -13, which is ordinarily phenotypically.
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