Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5500-5504, September 1983 Biochemistry

Release of initiation control by a mutational alteration in the R6K w required for plasmid DNA replication (antibiotic resistance/negative replication control/nucleotide sequence/initiation protein/amino acid substitution) DAVID M. STALKER*, MARCIN FILUTOWICZ, AND DONALD R. HELINSKI Department of Biology, University of California at San Diego, La Jolla, California 92093 Contributed by Donald R. Helinski, June 6, 1983

ABSTRACT Plasmid pRK419, a derivative of the naturally has allowed the concentration of, r protein to be varied in vivo. occurring antibiotic resistance plasmid R6K, contains the pir Results from these experiments indicate that although ir is re- that codes for the vT initiation protein and the 13 and y replication quired for the initiation of DNA replication within the y-origin origins of R6K. A mutation in plasmid pRK419, designated cos4051 region, increasing levels of wr do not affect a corresponding results in an elevated plasmid copy number in Escherichia coli change in the copy number of y-origin-containing plasmids (8). growing at 420C and an even greater increase in copy number when In the same study it was shown that the Xrprotein autoregulates the cells are shifted to 30'C. This mutation was assigned to the its expression. Recently, it was shown that a fusion protein of structural gene for the v protein on the basis of suppression of the mutant phenotype in E. coli when the wild-type X protein is Xand E. coli f3-galactosidase enzyme binds to the seven-direct- supplied in trans. Nucleotide sequence analysis of the cos405 mu- repeat region (10). tant confirmed the pir gene location of the mutation and showed Plasmid pRK419 is a 4.35-kb autonomously replicating de- that this mutation results in a single amino acid substitution (gly- rivative of R6K that contains the pir gene and the R6K 3 and cine to aspartic acid) at the 81st position of the 305-amino acid ir y origins of replication. Mutants of pRK419 were screened for protein. The properties of this mutant suggest that the 1T protein an alteration in the frequency of initiation of replication in E. plays a role in.the negative control of the frequency of R6K ini- coli, as reflected by an altered copy number. This report de- tiation in addition to its requirement for the initiation of plasmid scribes the isolation and characterization of a cold-sensitive (cos) replication. mutant of pRK419, designated cos405. This plasmid exhibits an elevated copy'number at 420C in comparison to wild-type Plasmid R6K is 38 kilobases (kb) in size, specifies resistance to pRK419. Shifting cells containing pRK419 cos4O5 to 30'C re- the antibiotics ampicillin and streptomycin, and is maintained sults in an increase in copy number from that observed at 420C. at a copy number of 12-18 per equivalent in Esch- The mutation is located within the-pir gene and results in a sin- erichia coli (1). Electron microscopic analysis of in vivo- and in gle amino acid substitution in the wr protein. The properties of vitro-derived replication intermediates of R6K and its deriv- this mutant suggest that the X protein is involved in the neg- atives has defined a 4-kb replication segment of the plasmid ative regulation of R6K copy number in addition to its absolute that contains three origins of replication (designated a, f3, and requirement for the initiation of R6K replication. y) and an asymmetric terminus of replication (2-4). In addition, this DNA segment contains a structural gene, pir, which en-. codes a 35,000-dalton polypeptide (ir) required for the initiation MATERIALS AND METHODS of R6K DNA replication in vivo and in vitro (5, 6). The pir gene Strains. Strains used in this study were MX399 (metB leu trp is located between the (3 and y origins of replication. Small, lacZ galK galE sueC tsx relA supD43ts str), C600 (thr eu thi autonomously replicating derivatives of R6K have been con- lacY tonA supE44), C2110 (polA his rha), YS1 (thr'leu.thi str structed that contain only the ir structural gene and the adja- minA endl), and 411 (C2110 polA pir R6K). cent y-origin region (5). Insertion of the pir gene into a com- Plasmids and Plasmid Manipulations. The properties of patible multicopy plasmid or into the E. coli chromosome has plasmids pRK419 and pRK526 have been described (5, 7). led to the demonstration that -the iT protein can act in trans to pAS751 and pAS752 are pBR322 derivatives containing the ir support replication in vivo of small plasmids containing only the structural gene under the control of different promoters (8). y-origin region (7). The ir protein, which autoregulates its Plasmid pMF16 is described in the text. In vitro mutagenesis expression, acts at.or before a transcription step required for of pRK419 with hydroxylamine was as described previously (7). the initiation of R6K DNA replication (6, 8). Nucleotide se- All strainsharboringplasmids were grown in 10-mlcultures with quence analysis of the pir structural gene and the adjacent y or without the appropriate antibiotics to a Klett 540 of 160-180. origin has revealed the presence of eight 22-base-pair direct Clonal analysis was performed by the Triton X-100 lysis method repeats. Seven of the direct repeats are arranged in tandem of Kahn et al. (11), after normalization of cell density between within the y-origin region, whereas the eighth repeat resides cultures. Nucleic acid pellets were resuspended in 50 jul of buffer near a putative promoter for the si structural gene (9). prior to restriction analysis and agarose gel electrophoresis. It has been postulated that the binding of ir protein to the Copy Number Estimation, Plasmid copy numbers were es- seven direct repeats. within the y-origin region and the eighth timated by growing 2.5-ml cultures in M9 Casamino acids me- repeat near the Ir gene promoter are required for the initiation dium supplemented with 10 tkCi of [3H]dThd (1 Ci = 3.7 x of R6K replication and autoregulation of ir expression, re- 1010 Bq) to a density of 5 X 108 cells per ml. Cells were lysed spectively. Fusion of the I7rstructural gene to various promoters with Sarkosyl and the lysates were centrifuged in CsCl/ethid- The publication costs of this article were defrayed in part by page charge Abbreviation: kb, kilobase(s). payment. This article must therefore be hereby marked "advertise- * Present address: Galgene, Inc., 1910 Fifth Street, Suite F, Davis, CA ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 95616. 5500 Downloaded by guest on September 30, 2021 Biochemistry: Stalker et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5501

ium bromide gradients (12). The gradients were fractionated, 0 acid-precipitable radioactivity in each fraction was determined, 80 and the ratio of radioactivity appearing in the supercoiled DNA 70 peak to that in the chromosomal DNA peak was used to esti- 60 mate plasmid copy number based on a molecular size of 3,750 5C kbfor the E. coli chromosome. In addition, lysates were elec- 40 trophoresed in 0.7% agarose gels, the gels were dried, and the labeled bands of plasmid and chromosomal DNAwere cut from 30 / o the gel and assayed for radioactivity. This latter method in- cluded open-circular plasmid forms in the copy number esti- 201 mate. 0 Plasmid copy number was also estimated by comparison of o the mass ratio of two plasmids maintained in the same cell (8). /0 Plasmid DNA was obtained by the clonal 0~ analysis procedure, 010 linearized with restriction enzymes, electrophoresed on 0.7% 9 agarose gels, and stained with ethidium bromide. The linear- ized plasmid bands were recorded from a film negative taken o7 AA1 of the gel by using a Joyce-Loebl microdensitometer. Relative 6 amounts of DNA were determined by weighing the relevant 5 plasmid peaks obtained from densitometric plots and the weight 4 _S~~~~~~~~,_ ratio between the two plasmid peaks was used to determine copy number. For derivatives of pBR322, a copy number value 3 of 40 copies per chromosome equivalent was used (8). A value of two to five copies per chromosome was used for plasmid 2L pRK248 (13), which was introduced into all strains carrying wild- type pRK419 and pRK419cos4O5. RESULTS 1 Isolation and Properties of the cos4O5 Mutant. pRK419 is 1 2 3 4 a 4.35-kb kanamycin-resistant derivative of R6K containing the Hours and of and the that codes for 13 y origins replication pir gene FIG. 1. Growth rate of E. coli strain C2110. harboring either plas- the protein (5, 9) located between these two origins. A num- mid pRK419 or pRK419cos4O5. Cells containing either plasmid were ber of temperature-sensitive mutants of pRK419 were screened maintained overnight at 42°C and diluted into 10 ml of fresh L broth for variation in plasmid copy number by plating E. coli cells con- to a density of 107 cells per ml. Time points were monitored every 20 taining in vitro-mutagenized pRK419 DNA at 30°C, followed min. e, pRK419 grown at 42°C; o, pRK419cos4O5 grown at 420C; A, by a shift to 42°C. Mutants of this plasmid with properties sim- pRK419 grown at 30°C; A, pRK419cos4O5 grown at 300C. ilar to a mutant previously described (pRK419ts68) that was temperature-sensitive for replication at 42°C and exhibited a (pRK248) containing plasmids.pRK419 and pRK419cos405 were decreased copy number at the permissive temperature (7) were grown at 42°C. A parallel set of cultures was shifted to 30°C for obtained at a frequency of 102. All of-these ts mutants were 4 hr after incubation at 42°C. Cell densities between the cul- complemented by a wild-type protein supplied in trans at the tures were normalized and plasmid DNA was prepared as de- nonpermissive temperature. scribed in the Materials and Methods. Plasmid pRK248 was Plating of E. coli strain MX399 containing mutagenized present in the strains as an internal standard for copy number pRK419 DNA at 42°C also resulted in the identification of col- estimation. Xho I- and Sal I-linearized plasmid DNA was sub- onies that exhibited a decreased growth rate when shifted to jected to electrophoresis in 0.7% agarose gels for copy number 30°C. One of the colonies (designated pRK419cos4O5) grew estimation. As shown in Fig. 2, pRK419cos405 exhibited a higher poorly at 30°C, even in the absence of any antibiotic selection. copy number with respect to wild-type pRK419 in both strains To determine whether or not this growth inhibition was due to at 42°C (lanes b and f) and a substantially increased number of an altered plasmid or the result of a spontaneous host mutation, copies after the cultures were shifted from 42°C to 30°C (lanes DNA was isolated from this colony and transformed into E. coli d and h). Wild-type pRK419 was maintained at a constant copy strains YS1 and C2110. Both strains exhibited similar growth number at both temperatures as was the apparent copy number characteristics in the presence and absence of kanamycin, in- of the marker plasmid pRK248 (two to five copies per cell). A dicating that a mutant plasmid is responsible for the observed more exact copy number measurement for pRK419cos4O5 was inhibition of host cell growth. The extent of the reduction in calculated from dye/CsCl gradient analysis and agarose gel growth rate of cells harboring pRK419cos4O5 was measured. E. analysis as described in the Materials and Methods. The data coli C2110 containing either wild-type pRK419 or cos4O5 was are summarized in Table 1. A copy number of =15 per chro- diluted from overnight cultures maintained at 42°C to 107 cells mosome equivalent was calculated for wild-type pRK419 at both per ml and then was incubated at 30°C or 42°C. As shown in temperatures, whereas copy numbers of 90, 185, and 253 were Fig. 1, cells containing pRK419cos4O5 showed a decreased observed for pRK419cos4O5 immediately after a shift of the growth rate at 42°C compared to cells without plasmid and an culture from 42°C to 30°C and after incubation for 0, 2, and 4 almost total inhibition of growth when shifted to 30°C. Viability hr, respectively. No additional increase in copy number of of the cells when maintained at 30°C was tested by dilution and pRK419cos405 was observed if the shifted culture was main- replating at 420C..Even after prolonged incubation at 300C, 100% tained at 30°C for longer then 4 hr. The maximal copy number viability was observed after replating at 42°C. of pRK419cos4O5 at 30°C [20Q-300 copies per chromosome To determine whether or not pRK419cos4O5 exhibited an equivalent (14)] is-similar to that exhibited by cop- mutants of altered copy number, cultures of YS1 (pRK248) and C2110 ColEl. Downloaded by guest on September 30, 2021 5502 Biochemistry: Stalker et al. Proc. Natl. Acad. Sci. USA 80 (1983) a b c d e f g h Table 1. Effect of the cos4O5 mutation on the copy number of pRK419 Time of incubation after shift to nonpermissive Copy number* conditions, hr pRK419 pRK419cos4O5 0 15 90 2 ND 185 rnmm~~~~~~*,.,,,-,WIP 4 ND 253 6 15 221 Cultures ofE. coli strain P678-54 harboring the wild-type and mu- tantplasmids were maintained at4200 andthen shifted to 300C forthe times indicated. ND, not determined. * Copy number values (number of copies of plasmid DNA per chro- mosome equivalent) were obtained by the agarose gel electrophoresis procedures described in the text. Similarvalueswere obtainedby CsCI/ FIG. 2. Copy number ofpRK419 and pRK419cos4O5 inE. coli strains ethidium bromide gradient centrifugation (data not shown). YS1 (pRK248) and C2110 (pRK248). Ten-milliliter cultures harboring either pRK419 or pRK419cos4O5 were grown and DNA was prepared. derivatives in vivo and in vitro (7). Plasmids pAS751 and pAS752 The DNA was digested with Sal I and Xho I to linearize pRK248 and consist of a Hinfl restriction fragment of the ir structural gene pRK419 (pRK419cos4O5), respectively. Digested DNA samples were electrophoresedin 0.7% agarose gels and stained with 5 tg ofethidium that does not contain the 1T promoter sequence inserted into bromide perml. In each lanetheupperbandis linearizedpRK248 DNA. the pBR322 derivative pAS620 (8). pAS751 produces an NH2- Lanes: a, YS1, pRK419 at 42°C; b, YS1, pRK419cos4O5 at 42°C; c, YS1, terminal fusion of X. with the severn NH2-terminal amino acid pRK419 at 42°C shifted to 300C for 4 hr; d, YS1, pRK419cos4O5 at residues of the trpE gene product, whereas pAS752 encodes 42°C shifted to 30°C for 4 hr; e, C2110, pRK419 at 42°C; f, C2110, a 1T polypeptide that is most likely missing three or four NH2- pRK419cos4O5 at 42°C; g, C2110, pRK419 at 42°C shifted to 30°C for terminal amino acids (8). Both of these iT protein molecules are 4 hr; h, C2110, pRK419cos405 at 42°C shifted to 30°C for 4 hr. functional in trans in supporting replication of y-origin plas- mids in vivo (8). Plasmids pRK419 and pRK419cos4O5 were in- Localization of the cos405 Mutation. To evaluate whether or troduced into strains 451 and 4)11 (containing pRK248 as an not the cos4O5 phenotype is specified by sequences within the internal standard) and into E. coli strain C600 harboring pAS751 IT structural gene, wild-type and altered pir sequences coding or pAS752. Cultures were grown as described for previous ex- for the Ir protein were supplied in trans and the copy number periments, DNA was prepared, and linearized plasmid DNA of pRK419cos4O5 was determined. Strains 051 and 411 contain was electrophoresed in 0.7% agarose gels. The copy numbers the 1T structural gene inserted into the E. coli chromosome (ref. of pRK419 and pRK419cos4O5 were determined in relation to 7; R. Kolter, personal communication). Strain 451 codes for a the internal marker plasmid from densitometric plots. The re- full-length 35,000-dalton protein, whereas strain 411 codes a sults are displayed in Fig. 3. Both the wild-type XT protein ex- truncated IT polypeptide missing 30 COOH-terminal amino acid pressed in strain 451 and the possibly truncated X protein residues (7, 9). This 32,000-dalton truncated protein is fully specified by pAS752 gave full suppression of the cos4O5 phe- functional in the initiation of replication of R6K self-replicating notype relative to the wild-type copy number of pRK419. Al-

Copy number 9 15 2 of pRK419cos405 I ori-- egi 11 8911- III I ori- Bgi 42° 300 .-I I Tl-r..l l,...- l, l I r-l- I I I PW1rf 82 221 COON NH v

i W 1 38 110

-j IQ51 1 8 21

j pAS 751 59 109

j pAS752 17 22

FIG. 3. Copy number of pRK419cos4O5 in the presence of wild-type and altered 7r supplied in trans. Relevant restriction sites are in- dicated within the pir structural gene. III and f refer to Hindu and Hinfl replication sites, respectively. The HindI fragments of R6K present in pRK419 and cos4O5 plasmids are designated 9, 15, and 2*. The location ofthe irpromoter-is designated p,,, whereas the eight 22-base-pairrepeats are indicated by boxes. 4)11 and 451 are icoding sequences integrated into the host chromosome. pAS751 and pAS752 contain ircoding sequences inserted into the pBR322 derivative pAS620 (8). TrpE refers to the seven NH2-terminal amino acid residues of the trpE. gene and the tryptophan operon promoter. The copy number of pRK419cos4O5 was measured in 10-ml cultures grown at 42°C and in cultures that have been shifted from 42°C to 30°C for 4 hr. Linearized pRK248 was the internal standard in strains 011 and 451. Downloaded by guest on September 30, 2021 Biochemistry: Stalker et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5503 tered ir proteins specified by strain 411 and plasmid pAS751 per chromosome equivalent) is unchanged at both 42°C and 30°C suppress partially the overproduction of the plasmid containing (lanes c and d) in the presence of pRK666, which specifies the the cos4O5 mutation. These experiments suggest that the al- wild-type ir protein. The copy number of the helper plasmids teration in pRK419cos4O5 resides in the iX structural gene and pRK666 and pMF16 remained unchanged at both tempera- that the mutation is recessive. When the growth properties of tures. Thus, a functionally altered product of the i structural strain 451 carrying pRK419cos4O5 were examined, it was ob- gene acts in trans to facilitate high copy replication of the y- served that the cells retained their normal rate of growth at origin plasmid pRK526. both 420C and 300C (data not shown). Identification of the cos405 Mutation. To determine whether If a functionally altered v protein is solely responsible for the or not the cos4O5 phenotype was the result of a single mutation, cos405 phenotype, insertion of the genetically altered Vr coding the DNA segment of pRK419cos4O5 containing the ir coding region into another compatible plasmid should result in the over- region was subjected to nucleotide sequence analysis and com- initiation of the y-origin plasmid pRK526 when both plasmids pared to the DNA sequence previously obtained for the wild- are present in an E. coli host. Plasmid pRK419cos4O5 DNA was type ir structural gene (9). The sequence obtained included the digested partially with Bgl II and the resulting fragments were entire ir structural gene and the region including the pir gene cloned into the unique BamHI site of the ampicillin resistance promoter. Of the 1,185 base pairs sequenced, a single base sub- plasmid pBR322. Clones containing the 0.52-kb and 1.0-kb Bgl stitution was found at position 696 of the wild-type II DNA fragments (indicated on the map in Fig. 3) in the ap- DNA sequence (9). This change involves a G-C to A-T transition propriate orientation were selected. The resultant 5.8-kb plas- in the 81st codon of the structural gene, which corresponds to mid, designated pMF16, contains the intact cos4O5 structural the mutagenic event produced by hydroxylamine. This alter- gene and the ir promoter sequences. Plasmid pRK666, a 10.8- ation is located in the second position of the codon (GGT) for kb plasmid containing the two Bgl II DNA fragments encoding the amino acid glycine. The specific change produces a GAT the wild-type iX protein cloned into the BamHI site of pBR322, codon that codes for the amino acid aspartic acid. No base sub- was used as a control. (pRK666 also contains a 5.0-kb TrpE stitutions were found in the regulatory region controlling Vr gene marker cloned into the EcoRI site of pBR322 and therefore is expression. From the sequence information, we conclude that 5.0 kb larger than pMF16. In all other respects, pMF16 and a single amino acid substitution (glycine to aspartic acid) pro- pRK666 are identical.) Plasmids pRK666 and pMF16 were in- duces a functionally altered ir polypeptide that results in a re- dependently maintained in E. coli C600 and the strains were laxation of plasmid R6K replication control. transformed to kanamycin resistance with the 2.7-kb y-origin plasmid pRK526 (y-origin region of R6K joined to a kanamycin resistance fragment). Ten-milliliter cultures were grown at 420C DISCUSSION or shifted to 30°C for 4 hr after growth at 42°C and DNA was We have identified and characterized an alteration in plasmid prepared as described above. pRK526 and pRK666/pMF16 were pRK419 (= 15 copies per chromosome equivalent) obtained by linearized by digestion with Xho I and Sal I, respectively, and in vitro mutagenesis with hydroxylamine. This mutant plas- the DNA was electrophoresed in 0.7% agarose gels. As shown mid, pRK419cos4O5, is maintained at a copy number of 90 in Fig. 4, pRK526 exhibited an increased copy number at 42°C per chromosome equivalent at 42°C. When cells containing (lane a) and a further elevation in the number of copies when pRK419cos4O5 are shifted from 42°C to 30°C, the number of the cells were shifted to 30°C (lane b) in response to the altered copies per chromosome equivalent increases to -250. Cells ir protein supplied in trans by pMF16. In contrast, the copy containing the mutant plasmid also exhibit a decreased growth number of pRK526 (previously determined to be 18 copies rate at both temperatures. Previously constructed plasmids containing wild-type and altered sequences coding for the ir a b c d protein allowed us to determine the effect of ir protein sup- plied in trans on pRK419cos4O5. It was observed that the 7r protein produced in strain 451 and specified by plasmid pAS752 suppresses the pRK419cos4O5 mutation because wild-type copy .-z" number levels are observed when these r coding sequences .."11"NO are provided in trans. r proteins encoded by strain 011 and *-_W. by plasmid pAS751, containing COOH-terminal and NH2-ter- minal alterations in ir, respectively, only partially suppress the cos4O5 phenotype with respect to the copy number of pRK419cos4O5. These four overlapping sequences supplying ir protein in trans exclude the possibility that sequences adjacent to the ir structural gene are providing a product responsible for suppression of the cos4O5 phenotype (Fig. 3). DNA sequence analysis of the altered wr coding region identified a single base change corresponding to a single amino acid substitution (gly- cine to aspartic acid) in the r protein. From these data, we con- FIG. 4. Effect in trans of the cos4O5 structural gene on the repli- cation of the y-origin plasmid pRK526. Construction of the ampicillin- clude that a structurally altered i protein is responsible for the resistant plasmids pRK666 and pMF16 is described in the text. Cul- high copy phenotype of pRK419cos4O5 and that the cos4O5 mu- tures (10 ml) ofE. coli strain C600 were grown in the presence of am- tation is suppressed by a wild-type i protein when supplied in picillin and kanamycin andDNA was prepared. pRK526 was linearized trans. with Xho I, whereas pRK666 and pMF16 were linearized with Sal I. It has previously been shown that replication of small R6K Electrophoresis was carried out on 0.7% agarose gels. In each lane the derivatives containing the y origin are dependent solely on Tr lower band consists of plasmid pRK526. Lanes: a, pMF16 and pRK526 maintained at 420C; b, pMF16 and pRK526 maintained at 420C and then protein supplied in trans (7). As described in Fig. 4, cloning of shifted to 30°C for 4 hr; c, pRK666 and pRK526 maintained at 420C; d, the sequences specifying the cos405 phenotype into the pBR322 pRK666 and pRK526 maintained at 420C and then shifted to 300C for derivative pMF16 demonstrated that the y-origin plasmid 4 hr. pRK526 is maintained at high copy number at both 42°C and Downloaded by guest on September 30, 2021 5504 Biochemistry: Stalker et aL Proc. Natl. Acad. Sci. USA 80 (1983) 30'C in the presence of the mutant irprotein. This observation tenance of plasmid R6K at a specific copy number. Thus, it is is consistent with the DNA sequence and suppression data that likely that the role of the irprotein is multifunctional, involving indicate that the altered polypeptide is responsible for the cos405 activities that have both positive and negative effects on the phenotype. initiation of R6K DNA replication. An interesting feature of the cos4O5 phenotype is that cells This work was supported by grants from the National Institute of Al- harboring plasmid pRK419cos4O5 exhibit a marked reduction lergy and Infectious Diseases (Al-07194) and the National Science in their rate of growth at 420C and 30'C (Fig. 1) with no ob- Foundation (PCM77-06533). D.M.S. was supported by a Smith Kline- served loss in cell viability. However, cells containing both Beckman Corporation postdoctoral fellowship. pMF16 (which provides the functionally altered ir protein in 1. Kontomichalou, P., Mitani, M. & Clowes, R. C. (1970) J. Bacte- trans) and pRK526 (maintained at high copy number) exhibit riol. 104, 34-43. essentially normal growth rates at 420C and 300C. This obser- 2. Crosa, J. H. (1980)J. Biol. Chem. 255, 11067-11070. vation indicates that a high DNA content in these cells is not 3. Inuzuka, N., Inuzuka, M. & Helinski, D. R. (1980)J. Biol. Chem. responsible for inhibition of host cell growth. Thus, the cos4O5 255, 11071-11074. mutation is unlike the runaway copy number mutants of plas- 4. Kolter, R. & Helinski, D. R. (1978)J. Mol. Biol. 124, 428-441. 5. Kolter, R. & Helinski, D. R. (1978) Plasmid 1, 571-580. mid RI, where increased DNA content is directly responsible 6. Inuzuka, M. & Helinski, D. R. (1978) Proc. Natl. Acad. Sci. USA for the inhibition of host cell growth (15, 16). It is also worth 75, 5381-5385. noting that cells containing a cold-sensitive mutation in the dnaA 7. Kolter, R., Inuzuka, M. & Helinski, D. R. (1978) Cell 15, 1199- gene (dnaAcos) of E. coli exhibit similar growth properties (un- 1208. der specific nutrient conditions) as cells containing the plasmid 8. Shafferman, A., Kolter, R., Stalker, D. M. & Helinski, D. R. (1982) pRK419cos4O5 (17). J. Mol. Biol. 161, 57-76. 9. Stalker, D. M., Kolter, R. & Helinski, D. R. (1982) J. Mol. Biol. Previous experiments indicate that ir protein functions at or 161, 33-43. before a transcription step required for the initiation of R6K 10. Germino, J. & Bastia, D. (1983) Cell 32, 131-140. DNA replication (6). It will be interesting to determine the 11. Kahn, M., Kolter, R., Thomas, C., Figurski, D., Meyer, R., Re- mechanism of action of the genetically altered X protein spec- maut, E. & Helinski, D. R. (1979) Methods Enzymol. 68, 268-280. ified by the cos4O5 mutant as to whether or not it involves an 12. Bazaral, M. & Helinski, D. R. (1968)J. Mol. Biol. 36, 523-531. 13. Thomas, C. M., Meyer, R. & Helinski, D. R. (1980)J. Bacteriol. alteration in an enzymatic activity, a reduction in binding ef- 141, 213-222. ficiency for the direct repeats of nucleotide sequences within 14. Muesing, M., Tamm, J., Shepard, H. M. & Polisky, B. (1981) Cell the y origin, or an altered interaction with host gene products 24, 235-242. or putative RNA repressor molecules. The observation that the 15. Uhlin, B. E. & Nordstrom, K. (1978) Mol. Gen. Genet. 165, 167- cos4O5 mutation results in the overinitiation of R6K DNA 179. rep- 16. Stougaard, 'P., Molin, S. & Nordstrom, K. (1981) Proc. Natl. Acad. lication and the fact that such a mutation is recessive suggests Sci. USA 78, 6008-6012. that i protein provides a negative function in the control of 17. Kellenberger-Gujer, G., Podhajska, A. J. & Caro, L. (1978) Mol. initiation frequency or an override mechanism for the main- Gen. Genet. 162, 6-9. Downloaded by guest on September 30, 2021