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A Temperature-Dependent Pbr322 Copy Number Mutant Resulting from a Tn5 Position Effect JAMES R

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A Temperature-Dependent Pbr322 Copy Number Mutant Resulting from a Tn5 Position Effect JAMES R Proc. Nadl. Acad. Sci. USA Vol. 83, pp. 7381-7385, October 1986 Genetics A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect JAMES R. LUPSKI*tf, STEVEN J. PROJAN§, Luiz S. OZAKI*¶, AND G. NIGEL GODSON* *Biochemistry Department, New York University Medical Center, 550 First Avenue, New York, NY 10016; and §The Public Health Research Institute, 455 First Avenue, New York, NY 10016 Communicated by Sarah Ratner, June 12, 1986 ABSTRACT In the process of randomly mutagenizing a the origin of replication of these related plasmids (17) and of recombinant pBR322 clone with transposon TnS, a high copy the rpoD/rom gene region (11) are highly conserved. number plasmid mutant, pLO88, has been isolated. The copy Most studies ofplasmid copy number control have utilized number phenotype of pLO88 is observed only at elevated mutants that display a high copy number phenotype. Struc- temperatures, >37C, and is due to the precise position of a tural studies of these mutants and comparison to the wild- TnS insertion. Nucleotide sequence of the TnS-pBR322 junc- type plasmid DNA in most cases reveal point mutations in the tion reveals that TnS-88 has inserted into an open reading RNA I and RNA II region. Few studies have analyzed frame that codes for a 63 amino acid protein previously shown plasmid copy number mutants resulting from random inser- to negatively regulate pBR322 plasmid copy number. By tion of pieces of DNA (18, 19). In both of these studies the deleting portions of the TnS it is shown that the copy number exact position of the insertion mutation was not ascertained. phenotype is due not only to the insertion of TnS in pBR322 but Temperature-sensitive plasmid copy number mutants have also to the requirement that some TnS sequences remain intact. been reported (20-22), but in each case they were due to point It appears that an outwardly directed TnS promoter inlitiates mutations in which the temperature-sensitive phenotype of the synthesis of a transcript (RNA X) that interferes with the these mutants appeared to be a consequence of a thermo- normal repressor RNA (RNA I)-primer RNA (RNA II) inter- sensitive secondary structure within RNA II. action at elevated temperatures. In this paper we report the isolation of a plasmid copy number mutant, pLO88, resulting from a TnS insertion. This Initiation of DNA replication for ColEl plasmids depends on insertion is 500 bp downstream from the origin of DNA the formation of an RNA primer by the action of RNA replication in pBR322 and produces a copy number pheno- polymerase and RNase H. Synthesis of the primer precursor type that is 10-fold higher than any other TnS insertion into transcript (RNA II) is initiated 555 base pairs (bp) upstream this plasmid. The high copy number phenotype is due to more of the origin of DNA replication and this then forms a than just mere insertion of foreign DNA, as deletion of most persistent hybrid with the template DNA near the origin of of the Tn5 sequences, leaving behind 185 bp, restores copy DNA replication. RNase H cleaves the hybridized transcript number to the wild-type phenotype. Of further interest, the at the origin and this processed RNA II is used as a primer copy number phenotype of plasmid pLO88 is temperature for DNA synthesis by DNA polymerase 1 (1-4). Regulation dependent. ofthe promoter for RNA II appears to be sufficient to control plasmid copy number (5). Primer formation is inhibited by a MATERIALS AND METHODS plasmid-specified 108-nucleotide repressor RNA, RNA I Bacterial Strains, Plasmids, and Bacteriophage. pEG81 is a (6-8). RNA I synthesis starts 445 bp upstream from the origin pBR322 recombinant plasmid that contains a cDNA copy of of replication and proceeds in the opposite direction from a portion of the Plasmodium knowlesi sporozoite gene (23); RNA II synthesis. RNA I terminates near the site where pLO84 contains a TnS insertion into the cDNA portion of RNA II synthesis starts and is therefore a complement of pEG81 (24, 25). Other plasmids are described in Table 1. All RNA II. When RNA I binds to RNA II, primer formation is plasmids were maintained in Escherichia coli HB101 (32). To inhibited (7, 9). Point mutations that affect formation and test the heat shock response of pLO88 the htpR- recA- E. structure of RNA I will also affect RNA II and these have coli strain CAG456 was used (a gift of Carol Gross). X467 effects on ColEl replication (4). [X::Tn5, where:: signifies a noveljoint)] was used to introduce A third aspect of ColEl plasmid copy number control TnS into E. coli HB101 cells harboring pEG81. Bacterial strains involves a trans-acting (10) 63 amino acid protein that were grown in Luria-Bertani broth (LB broth) (33). negatively regulates copy number. This trans-acting function Biochemical Procedures. Restriction endonucleases were has alternatively been called the Rop or Rom protein. The from New England Biolabs, DNA polymerase I and Klenow Rop protein (11) has been postulated to, in the presence of fragment were from Boehringer Mannheim, and antibiotics RNA I (12), affect the secondary structure of the nascent were from Sigma. Digestion, filling-in sticky ends, ligations, RNA primer, resulting in efficient transcription termination and transformations were as described in refs. 34-36. After (13). The Rom protein has been shown to increase the subcloning fragments of the target DNA in the filamentous inhibitory action of RNA I on in vitro primer formation and phage cloning/sequencing vector M13mp8 (37), the sequence to enhance binding of RNA I to RNA II (14, 15). Therefore, the regulation of plasmid copy number is under tripartite Abbreviations: Apr, ampicillin resistance; bp, base pair(s); CS, control involving RNA I, RNA II, and a trans-acting 63 circumsporozoite; IS, insertion sequence; Kmr, kanamycin resist- amino acid protein (16). Comparative studies of ColEl ance; LB, Luria-Bertani broth; Tcr, tetracycline resistance. plasmids and pBR322 show that nucleotide sequence around tPresent address: Department of Pediatrics and Institute for Molec- ular Genetics, Baylor College of Medicine, Texas Medical Center, Houston, TX 77030. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at new address. payment. This article must therefore be hereby marked "advertisement" Present address: Institut Pasteur, Unite de Parasitologie Experi- in accordance with 18 U.S.C. §1734 solely to indicate this fact. mentale, 25 Rue du Dr. Roux, 75015 Paris, France. 7381 Downloaded by guest on September 30, 2021 7382 Genetics: Lupski et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Plasmids used in this study and the quantitative determinations of plasmid copy number Antibiotic Size, Copy Source or Plasmid marker Derivation and/or description bp number ref. pBR322 Apr, Tcr Multipurpose cloning vector 4,363 57 ± 4 26, 27 pEG81 Tcr cDNA insert of P. knowlesi CS gene by 4,714 60 ± 17 23, 28 poly(G-C) tailing into the Pst I site of pBR322 pLO84 Tcr, Kmr Tn5 insertion into pEG81 pEG81::Tn5-84 10,541 40 ± 8 24, 25 pLO88 Tcr, Kmr High copy number plasmid mutant resulting 10,541 242 ± 41 This work from TnS insertion into pEG81 pLO88-1 Tcr pLO88 = ABal I 4,475 280 + 109 This work pLO88-2 Tcr pLO88 = A(Bal I-Hpa I) 4,291 18 ± 4 This work pLO88-3 Kmr pLO88 = ABamHI 6,094 416 ± 10 This work pLO88-4 Tcr pLO88 = APvu II 6,105 129 ± 46 This work pLO88-5 Tcr pLO88 = A(Pvu II-Hpa I) 4,867 80 ± 35 This work pLO88-6 Kmr pLO88 = ASal I 5,997 20 ± 11 This work Apr, ampicillin resistance; Tcr, tetracycline resistance; Kmr, kanamycin resistance; CS, circumsporozoite. The size for pBR322 is taken from Sutcliffe (26) and Peden (29); pEG81 size is from Godson et al. (28); pLO88 size is from pEG81 plus the size of TnS [5818 bp (26, 30, 31) plus 9 bp duplicated during TnS insertion]. All measurements were performed in at least triplicate and standard deviations were determined. All cells were grown at 37°C. was determined using a synthetic universal primer (P-L recombinants were selected. The nucleotide sequence of the Biochemicals) and the Sanger dideoxy chain-termination Tn5-pBR322 DNAjunctions is shown in Fig. 3. Note Tn5-88 method (38). The Microgenie (Beckman) computer programs has inserted 32 bp from the Pvu II cutting site in pBR322 and were used to organize and analyze the data. Plasmid DNA 9 bp, bp 2035-2044 of the Sutcliffe (26) sequence, have been was prepared and analyzed as described (39, 40). Plasmid duplicated at the TnS insertion site. TnS-88 interrupts an copy numbers were determined by fluorimetric densitometry open reading frame that encodes .a 63 amino acid protein that of ethidium bromide-stained agarose gels of sheared whole- has been demonstrated to be involved in ColEl plasmid copy cell minilysates of exponentially growing cultures (40) using number control (11, 13-15). The insertion leaves 43 of the 63 a Shimadzu dual-wavelength chromatogram scanner (model amino acids intact and is in-frame with this open reading CS-910). Transfer analysis of RNA transcripts was as de- frame. scribed (41-44). Deletions of pLO88 DNA Sequences Alter the Copy Number Phenotype. The plasmids used in this study are described in RESULTS Table 1. The portions of pLO88 that were deleted to give Isolation of TnS-Induced Plasmid Copy Number Mutant. plasmids pLO88-1 through pLO88-6 are shown in Fig. 2. Plasmid pEG81 (23) was used as a for TnS target mutagenesis. N- rnr 1: 0 (.
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