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 of these related (17) and of recombinant pBR322 clone with transposon TnS, a high copy the rpoD/rom region (11) are highly conserved. number 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 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 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 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 /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 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 (. r- w m N It contains part of the coding region of the CS gene of P. -< m mmmmaccco-Gmm m02 knowlesi that was isolated from a cDNA library of sporozoite II mRNA by G-C tailing using terminal transferase and inserted into the Pst I site ofpBR322 (28). TnS mutagenesis ofpEG81 bp was performed as described by de Bruijn and Lupski (45). E. 17,300 - 9,600 - coli strain HB101 cells harboring pEG81 were infected with 6,600- a X::TnS transducing phage and plated on LB broth supple- 4,7 _- mented with Ap, to select for pEG81, and Km, to select for 4,450' - bp transposition of TnS from X::TnS. Plasmid DNA was isolated and retransformed into HB101 and again Apr, Kmr colonies 2,300 - -2,527 were selected that therefore now contained Tn5 insertions 1,950 - into the plasmid pEG81, pEG81::TnS (24). Of the 144 TnS 1 r0i - - 1,?336 insertions into pEG81 that were isolated, only one insertion, TnS-88, resulted in a high copy number phenotype (Fig. 1). This plasmid was designated pLO88. The location ofthe TnS insertion in pLO88 was determined by analysis and shown to be close to the Pvu II site of pBR322 (Fig. 2). The orientation of TnS-88 was determined utilizing FIG. 1. Random TnS insertions into pEG81: a 0.5% agarose gel the stained with ethidium bromide to visualize the DNA of 10 recombi- asymmetric restriction sites BamHI and Sma I in TnS (47) nant plasmids containing TnS insertions into pEG81. Plasmid DNAs and the EcoRI and BamHI sites of pBR322. were prepared from a miniprep procedure (34) starting with equal To determine the nucleotide sequence of the TnS numbers ofcells grown at 37°C. The far left and right lanes are marker insertion-host junction, plasmid pLO88 was digested with DNAs, phage X digested with HindIII and Kpn I (far left) and Pvu II plus Hpa I and a restriction fragment was subcloned M13mp8 cut with Hae III (far right), with the appropriate size, in base into M13mp8. Restriction endonuclease Hpa I cuts 185 bp pairs, of the generated fragments shown. Individual pEG81::TnS from the end of Tn5 in the inverted repeat (47) and does not plasmids, pLO83 through pLO92, digested with HindIII are shown cut in pBR322. Pvu II has a restriction site in pBR322 and cuts from left to right. A HindIII digest of a pEG81::TnS plasmid yields TnS four times. A Hpa I plus Pvu II digest of pLO88 yields three restriction fragments because there is one HindIII site in seven pBR322 (27) from which pEG81 is derived (see Table 1) and TnS fragments, the smallest of which was isolated and contains two HindIII sites, one in each inverted repeat. The constant ligated into Sma I-cut M13mp8 (37) and sequenced by the 3400-bp fragment is the internal HindIII fragment of TnS. The other Sanger (38) dideoxy methods. To isolate the other end of the two fragments vary in size, depending on the relative position ofTn5 TnS insertion, pLO88 was digested with EcoRI plus Hpa I in pEG81. Lightly staining higher molecular weight bands are due to and ligated into EcoRI plus HincII-cut M13mp8 and Tcr partial digestion. Downloaded by guest on September 30, 2021 Genetics: Lupski et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7383

3' A C G T A C G T T A I_" T G T EIgo A A ms"-- - _p 5 ___0GC - - --i _-_o- -l __..b -

-- S. S soX _ _ A mo_ - am- _ :- A __ T _ G -n5l T G - _b A -Tr.5 _ W A G _ _ r1~~~~ A _ _ D~~~~~ T .5. 5

a- ~~~~~~~~PvuI[ -- Satia SmaI CTGTGAATCGICTTCACGACCACGCTGATGAGCTTTACCGCAGCTG6CCTCG GACACTTAGCGAAGTGCTG6GTGCGACTACTCGAAATGGCGTCGAC GAGC

FIG. 2. Restriction map of pLO88. Plasmid pLO88 was derived 2,025 b 2,075 by Tn5 insertion into pEG81. The position ofthe origin ofreplication, pBR322 primer RNA (RNA II), and repressor RNA (RNA 1) are shown. Tn5-88 maps close to the Pvu II site but 500 bp downstream from the FIG. 3. Nucleotide sequence of pBR322-Tn5-88 junction. The origin of replication. The orientation of Tn5-88 was determined to be pBR322 nucleotide sequence from bp 2025 to 2075 (27) is shown at with insertion sequence 50L (IS5OL) (46) closer to the origin of the bottom. The Pvu II restriction site is boxed. Above are shown replication. Deletion derivatives ofpLO88, constructed to determine autoradiograms (a and b) of DNA sequencing gels generated from the effects on copy number, are demarcated by appropriate symbols Sanger (38) dideoxy sequencing of the TnS-pBR322 junction of and explained in Table 1: pLO88-1 = ABa I (o), pLO88-2 = A(Bal pLO88. At the top are shown the individual dideoxy lanes A, C, G, I-Hpa 1) (e), pLO88-3 = ABamHI (A), pL0884 = APvu 11(A), and T and to the right of each autoradiogram is the interpretation of pLO88-5 = A(Pvu II-Hpa I) (o), pLO88-6 = ASal I (n). the sequencing gel. (a) From ISSOL into pBR322. (b) From IS50R reading into pBR322 (see Fig. 2). Boxed with dotted lines are the 9 Cells harboring the individual plasmids shown in Table 1 bp of pBR322 duplicated during TnS insertion. were grown at 370C to stationary phase, and DNA was prepared according to Projan et al. (40) and run on an agarose pLO88 Displays a Temperature-Dependent Copy Number gel. Quantitative measurements of plasmid copy number per Phenotype. When cells harboring pLO88 are grown at 37TC chromosome equivalent for all of the plasmids used in this the copy number for pLO88 is significantly greater than a study are shown in Table 1. control, pLO84, grown at the same temperature (Table 1). It Plasmids pBR322 and pEG81 have, within experimental was noted that when the same cells harboring pLO88 were error, the same copy number. This demonstrates that inser- grown at 30TC, the plasmid copy number for pLO88 did not tion of a cDNA at the Pst I site of pBR322 does not alter differ significantly from the control. Therefore, cells harbor- plasmid copy number. The copy number ofpLO84 is slightly ing either pLO84 or pLO88 were grown at either 30'C or 40TC lower than pEG81. This may be due to the increased plasmid and the plasmid copy number was quantitatively determined size, >2-fold, from the insertion of TnS. Insertion TnS-84 is (see Fig. 4). The copy number for pLO88 from cells grown at in the cDNA portion of pEG81 and as the copy number of 40TC is even higher than that determined when cells were pLO84 is not significantly different from pEG81, when grown at 370C (Table 1), whereas the copy number of the plasmid size is accounted for, this demonstrates that TnS control, pLO84, remains the same whether cells were grown insertion does not significantly alter plasmid copy number. at 30TC, 37TC, or 40'C. The high copy number phenotype of Plasmid pLO88 has an 8- to 10-fold greater copy number than pLO88 at >37TC also occurs in the htpR- recA- E. coli strain the control pLO84. This was also demonstrated by radio- background CAG456 (data not shown), suggesting that the immunoassay of the product' encoded by the cDNA portion temperature-dependent copy number phenotype is not the of pLO88, which shows 8- to 10-fold increased expression of result of a heat shock promoter within Tn5. Interestingly, the the CS surface antigen, the cDNA gene product, over that of doubling time increases 2-fold for strains grown at 420C the control pEG81 (data not shown). When most of the Tn5 versus 30'C that harbor pLO88 (data not shown). This is deleted, up to the Bal I site in pLO88-1, and only 369 bp of bacteriostatic effect was consistently seen in our experi- TnS sequences remain intact, the plasmid still displays the ments. of RNA I and RNA II in and its Deletion copy number But deletion of an additional Synthesis pLO88 high phenotype. Derivatives. To investigate the RNA and the 184 bp, to the Hpa I site, yielding pLO88-2, completely primer (RNA II) repressor RNA (RNA I) of pLO88 and its deletion deriva- destroys the high copy number phenotype and restores the tives, total RNA was isolated from HB101 cells harboring the number to less than control levels. Other that copy deletions respective plasmids and an RNA transfer blot was performed do not contain the Bal I-Hpa I restriction fragment of IS50L using the Msp I B fragment (bp 2154-2681) (27) from pBR322 (pLO88-4, pLO88-5) have copy numbers closer to the wild- as the probe. This fragment spans the region ofthe replication type levels, whereas one that does contain it (pLO88-3) origin ofpBR322 and, by using it as a probe, RNA I and RNA maintains a high copy number phenotype. II are readily visualized. As shown in Fig. SA, pLO88-5, a low The variance in copy number determination of pLO88-1 copy number deletion derivative of pLO88, makes two (280 + 109) may be due to instability ofthis plasmid. This may transcripts corresponding to RNA I and RNA II, respective- also explain the relatively low copy number of pLO88-6 and ly. These two transcripts are found in all of the plasmids the reason for a satellite band in pLO88-3. shown in Table 1 (data not shown). In plasmid pLO88-1, a Downloaded by guest on September 30, 2021 7384 Genetics: Lupski et al. Proc. Natl. Acad. Sci. USA 83 (1986)

o 0 0 0 A B o 0 0 0 rl( ";j rl qt .a) 0D O C CO COD OD OD 'IZ- ';j- OD 00 00 00 O0 OD arCL CL 0 0 0 0 -j lrg~ C. I

23S rRNA -

- Origin 16 S rRNA - IV - RNA X RNA IL - f Chromosomal DNA RNA I - X ^ 4 -Plasmid DNA

Aps RNAI ORI IS5OL ftr IS5OR TCr Plasmid 300C 400C . _ O4j~zzffrn pL088 U I pL084 42± 10 44±11 IRNA -a t I -aX pL088 44 ± 9 333±70 A- FIG. 4. Temperature dependence of pLO88 high copy number phenotype. E. coli HB101 cells harboring the individual plasmids B pnnKIUNA shown and described in Table 1 were grown to stationary phase at the Copy Number indicated temperature, and DNA was prepared according to Projan et al. (40). Quantitative determinations of plasmid copy number are pL088-1 i I 280 ± 109 shown at the bottom for pLO84 and pLO88 from cells grown at BalI BalI different temperatures. As shown, the copy number for pLO88 is dramatically increased at 40'C compared to 30TC. At 30'C the copy pL088-5 i i 80± 35 number for pLO88 is the same as the control pLO84. Pvu 11 Hpa I FIG. 5. RNA transfer blot analysis of transcripts involved in high copy number deletion derivative of pLO88, a third plasmid copy number control: transfer analysis ofRNA isolated from transcript is visualized that migrates at a higher molecular HB101 cells harboring a high copy number plasmid (pLO88-1) and a weight than RNA I and RNA II. This third transcript, RNA low copy number plasmid (pLO88-5) derivative of the Tn5 mutant X, is not seen in the control low copy number plasmid pLO88. Blots of the agarose denaturing gels and the positions of pLO88-5. By using a single-strand probe it was demonstrated migration of 16S and 23S RNA, as markers, are shown at the top. The map of pLO88 and the portions deleted in pLO88-1 and pLO88-5 are that RNA X is synthesized from the same strand as RNA I. shown at the bottom. InA, the probe DNA was a nick-translated Msp a The size of RNA X corresponds to length of =800-900 I restriction fragment (Msp I B fragment) that spans the origin of nucleotides, which suggests that its point of initiation is from replication (ORI). In B, the 1455-bp Pvu II restriction fragment from within TnS. To unequivocally demonstrate that the point of pL088, which contains 1423 bp of ISSOL, was used as a probe. The initiation of RNA X is within TnS, the gel from Fig. 5A was position of migration of RNA I and RNA II was determined by blotted with a Tn5-specific probe. The probe used was the running total RNA isolated from HB101 cells harboring pBR322 and 1455-bp Pvu II restriction fragment that contains 1423 bp of blotting with the Msp I B fragment as in A (data not shown). Note the a ISSOL purified from a Pvu II digest of pLO88. As shown in existence of higher molecular weight RNA (RNA X) that blots to Fig. SB, only the higher molecular weight transcript, RNA X, probe A and probe B. The higher molecular weight smear is probably due to poor transcription termination at the 3' end of RNA I. pDNA, hybridizes with the TnS-specific probe. Thus, RNA X ap- plasmid DNA. pears to originate from an outward-directed TnS promoter. rom from a ColEl derivative plasmid in another work did not DISCUSSION alter plasmid copy number significantly (48). One ofthe most interesting aspects ofthe pLO88 high copy This paper reports the isolation of a high copy number number phenotype is its temperature dependence. At 30'C plasmid, pLO88, resulting from insertion of the transposable the copy number ofpLO88 is 44 ± 9, whereas at 37CC it is 242 element Tn5, which displays its high copy number phenotype ± 41, and at 40'C is 333 ± 70 (Fig. 4). This cannot be due to only at elevated temperatures. The resulting phenotype a temperature effect resulting from TnS insertion, as a control results from a position effect of TnS, as only 1 of 144 plasmid with a TnS insertion located elsewhere (pLO84) has individual TnS insertions isolated into this plasmid gives the the same copy number, within experimental error, at 30'C phenotype. TnS-88 inserts into the rop or rom gene ofpEG81, and at 40'C (Fig. 4). Nor can this be due to a heat shock which is a gene implicated in the negative control of plasmid promoter (49, 50) within TnS-88, as the pLO88 high copy copy number. But the high copy number phenotype ofpLO88 number phenotype remains the same at 40'C in an htpR- cannot be due to mere insertional inactivation of rop or rom background (data not shown). since deletion derivative pLO88-2, which, in effect, has a The only measurable difference between a high copy 185-bp inserted piece of TnS in rop, has the lowest copy number deletion derivative of pLQ88 (pLO88-1) and a low number (18 + 4) ofall ofthe pLO88 deletion derivatives. This copy number deletion derivative of pLO88 (pLO88-S) is the result, in fact, suggests that rop or rom plays a minor, if any, presence of an additional transcription RNA X, with RNA role in copy number control of pBR322 derivatives. In transfer analysis of transcripts derived from cells harboring addition, deletion of a restriction fragment containing rop or these plasmids. By the use of different probes it was dem- Downloaded by guest on September 30, 2021 Genetics: Lupski et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7385 onstrated that RNA X originates from within TnS and spans 14. Som, T. & Tomizawa, J. (1983) Proc. Natl. Acad. Sci. USA 80, the origin of replication. To our knowledge, identification of 3232-3236. 15. Tomizawa, J. & Som, T. (1984) Cell 38, 871-878. an outwardly directed TnS transcript, has not been reported 16. Cesarini, G. & Banner, D. W. (1985) Trends Biochem. Sci. 10, previously, although literature on lack of polarity in the lac 235-237. operon with certain TnS insertions suggested its existence 17. Seizer, G., Som, T., Itoh, T. & Tomizawa, J. (1983) Cell 33, (51). Perhaps the existence of RNA X is detected because it 1119-1129. is properly terminated by the terminator for RNA I. From the 18. Heffron, F., So, M. & McCarthy, B. J. (1978) Proc. Natl. Acad. deletion data and the size of RNA X one would predict that Sci. USA 75, 6012-6016. this transcript is initiated from within the Hpa I-Bal I 19. Bergquist, P. L., Downard, R. A., Caughey, P. A., Gardner, R. C. & Lane, H. E. D. (1981) J. Bacteriol. 147, 888-899. restriction fragment of ISSOL, but no obvious promoter 20. Grindley, N. D. G., Grindley, J. N. & Kelley, W. S. (1978) in sequence (52) exists within this restriction fragment. S1 Microbiology, ed. Schlessinger, D. (Am. Soc. Microbiol., Wash- nuclease mapping of the 5' end of RNA X will be required to ington, DC), pp. 71-73. determine the precise point of its initiation. RNA transfer 21. Wong, E. M., Muesing, M. A. & Polisky. B. (1982) Proc. Natl. analysis demonstrates that RNA X is synthesized in Acad. Sci. USA 79, 3570-3574. 22. Moser, D. R., Moser, C. D., Sinn, E. & Campbell, J. L. (1983) equimolar amounts at 30'C and at 40'C (data not shown). Mol. Gen. Genet. 192, 95-100. Recent experiments (53-55) have focused much attention 23. Ellis, J., Ozaki, L. S., Gwadz, R. W., Cochrane, A. H., Nus- on secondary and tertiary RNA structural information that is senzweig, V., Nussenzweig, R. S. & Godson, G. N. (1983) Nature proposed to have importance in repressor RNA (RNA (London) 302, 536-538. )-primer RNA (RNA II) interactions. These studies have 24. Lupski, J. R., Ozaki, L. S., Ellis, J. & Godson, G. N. (1983) Science 220, 1285-1288. demonstrated that RNA I and RNA II have extensive 25. Lupski, J. R., Gershon, P., Ozaki, L. S. & Godson, G. N. (1984) secondary structures and that conformational changes in Gene 30, 99-106. RNA II modulate its interaction with RNA I. 26. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, We propose the following model for the temperature- 77-90. dependent high copy number phenotype ofpLO88. An RNA 27. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., transcript, RNA X, is probably initiated from within the Hpa Heynecker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977) Gene 2, 95-113. I-Bal I restriction fragment of ISSOL on TnS-88. This tran- 28. Godson, G. N., Ellis, J., Svec, P., Schlesinger, D. H. & Nus- script proceeds in a counterclockwise direction through the senzweig, V. (1983) Nature (London) 305, 29-33. origin of replication on the same DNA strand where RNA I 29. Peden, K. (1983) Gene 22, 277-280. is synthesized. RNA X can pair normally with RNA II at 30. Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B. & Schaller, H. 30'C; thus, plasmid DNA copy number is kept at the normal (1982) Gene 19, 327-336. wild-type level. At elevated temperature, due to the changes 31. Mazodier, P., Cossart, P., Giraud, E. & Gasser, F. (1985) Nucleic Acids Res. 13, 195-205. in the secondary structure of RNA X, the RNA X-RNA II 32. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, binding is much less inhibitory, even though it still base pairs, 459-472. and primer formation is not properly repressed, leading to a 33. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold high copy number phenotype. Transcription through the Spring Harbor Laboratory, Cold Spring Harbor, NY). plasmid DNA origin of replication has been shown to inter- 34. Lupski, J. R., Smiley, B. L., Blattner, F. R. & Godson, G. N. (1982) Mol. Gen. Genet. 185, 120-128. fere with copy number control (56) but, in that case, caused 35. Lupski, J. R., Smiley, B. & Godson, G. N. (1983) Mol. Gen. a lower copy number. Genet. 189, 48-57. Further analysis ofpLO88 may reveal important aspects of 36. Lupski, J. R., Ruiz, A. A. & Godson, G. N. (1984) Mol. Gen. in vivo requirements for initiation of pDNA replication. Genet. 195, 391-401. These studies take an added importance in light of recent 37. Messing, J. & Vieira, J. (1982) Gene 19, 269-276. experiments that show that RNase H does not appear to be 38. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Nati. Acad. Sci. USA 74, 5463-5467. required for in vivo replication of ColEl pDNA (57), thus 39. Novick, R. P., Murphy, E., Gryczan, T. J., Baron, E. & Edelman, indicating that in vitro studies may not reflect actual in vivo I. (1979) Plasmid 2, 109-12. occurrences. 40. Projan, S. J., Carleton, S. & Novick, R. P. (1983) Plasmid 9, 182-190. 41. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, We are grateful to JoAnn Monroe for typing the manuscript. This W. J. (1979) Biochemistry 18, 5294-5299. work was funded by National Institutes of Allergy and Infectious 42. Glisin, V., Crkvenjakov, R. & Byus, C. (1974) Biochemistry 13, Diseases Grant 7-1142-996 (to G.N.G.) and Public Health Service 2633-2637. Grant GM33278 (to S.J.P.). J.R.L. is a recipient of an American 43. Lehrach, H., Diamond, D., Wozney, J. M. & Boedtker, H. (1977) Cancer Society postdoctoral fellowship. Biochemistry 16, 4743-4751. 44. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 45. de Bruijn, F. J. & Lupski, J. R. (1984) Gene 27, 131-149. 1. Conrad, S. E. & Campbell, J. L. (1979) Cell 18, 61-71. 46. Berg, D. E. (1983) Proc. Natl. Acad. 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