Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication

Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas). Based on sequence homology, two structural groups are evident. Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase. The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.     

Selective cloning of a DNA single-strand initiation determinant from phi X174 replicative-form DNA.

An M13 phage deletion mutant, M13 delta E101, developed as a vector for selecting DNA sequences that direct DNA strand initiation on a single-stranded template, has been used for cloning restriction enzyme digests of phi X174 replicative-form DNA. Initiation determinants, detected on the basis of clear-plaque formation by the chimeric phage, were found only in restriction fragments containing the unique effector site in phi X174 DNA for the Escherichia coli protein n' dATPase (ATPase). Furthermore, these sequences were functional only when cloned in the orientation in which the phi X174 viral strand was joined to the M13 viral strand. A 181-nucleotide viral strand fragment containing this initiation determinant confers a phi X174-type complementary-strand replication mechanism on M13 chimeras. The chimeric phage is converted to the parental replicative form in vivo by a mechanism resistant to rifampin, a specific inhibitor of the normal RNA polymerase-dependent mechanism of M13. In vitro, the chimeric single-stranded DNA promotes the assembly of a functional multiprotein priming complex, or primosome, identical to that utilized by intact phi X174 viral strand DNA. Chimeric phage containing the sequence complementary to the 181-nucleotide viral strand sequence shows no initiation capability, either in vivo or in vitro.     

Genes and regulatory sequences of bacteriophage phi X174

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.     

DNA sequences which support activities of the bacteriophage phi X174 gene A protein

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.     

Formation of rolling-circle molecules during phi X174 complementary strand DNA replication

The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n"). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.     

Initiation of lagging-strand synthesis for pBR322 plasmid DNA replication in vitro is dependent on primosomal protein i encoded by dnaT

The role of the primosome assembly and protein n' recognition site in replication of pBR322 plasmid was examined. The following evidence indicates that the primosome is involved in lagging-strand synthesis of pBR322 plasmid replication in vitro. Early replicative intermediates with newly synthesized leading strand, approximately 1 kilobase pair long, immediately downstream of the replication origin accumulate in products synthesized in extracts from a dnaT strain that lacks primosomal protein i or in wild-type extracts supplemented with anti-protein i antibody. These intermediates are converted efficiently into full-length DNA by addition of purified protein i. Consistent with the previously proposed role of the primosome (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73), an n' site on the lagging strand, but not on the leading strand, is required for efficient replication of the plasmid in vitro. Plasmids lacking an n' site on the lagging strand replicate only to a limited extent in vitro and early replicative intermediates carrying nascent leading strands are accumulated, although a portion of the intermediates complete replication to yield full-length DNA. The latter reaction is completely inhibited by addition of anti-protein i antibody. Insertion of the n' site of phage phi X174 into pBR322 plasmids lacking lagging-strand n' sites restores the replicative ability of the mutant plasmid comparable to that of the wild-type plasmid. These results indicate that protein i is essential for lagging-strand synthesis of pBR322 plasmid in vitro and that it may play an important role in the priming events as a part of either an n' site-dependent primosome or an n' site-independent, as yet unidentified, priming complex.     

Mechanism of replication of bacteriophage phi X174 XIX. Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome.

Bacteriophage phi X174 viral strand DNA molecules shorter than genome length found late in the infectious cycle in Escherichia coli were 5' end labeled with 32P. Hybridization of the 32P-labeled molecules to restriction enzyme fragments of phi X replicative form DNA revealed an excess of phi X molecules whose 5' ends mapped in HaeIII fragments Z3 and Z4 in comparison with fragments Z1 and Z2. This suggests that initiation of phi X174 viral strand DNA synthesis may occur at internal sites on the complementary strand. There are several appropriately located sequences that might serve as n' (factor Y) recognition sequences and thereby facilitate discontinuous synthesis of the viral strand.     

Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis

Recombinant RF (replicate form) I DNAs containing the bacteriophage phi X174 gene A protein-recognition sequence are cleaved by the phi X A protein yielding a phi X RF II X A protein complex (Zipursky, S.L., Reinberg, D., and Hurwitz, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5182-5186). Such complexes support DNA synthesis in both RF I leads to SS(c) and RF I leads to RF I phi X DNA replication reactions in vitro. Two phi X A protein-recognition sequences were inserted into plasmid pBR322. Both sequences were contiguous with the same strand of the vector DNA and separated by 667 and 4275 base pairs. This recombinant plasmid (G27-4) was cleaved by the phi X A protein at either insert and both inserts support the initiation of RF leads to SS(c) DNA synthesis. This was verified by the finding that replication products were circular molecules of 667 and 4275 nucleotides. This finding is in keeping with the multifunctional activities associated with the phi X A protein; these include the site-specific nicking of RF I DNA which initiates DNA synthesis and site-specific termination resulting in the circularization of the displaced DNA strand. The phi X A protein and the Escherichia coli rep and SSb proteins catalyze the unwinding of phi X RF I DNA in vitro (Scott, J.F., Eisenberg, S., Bertsch, L.L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 193-197). Recombinant plasmid G27-4 RF I DNA was also unwound in vitro by this enzyme system; in this case, both circular and linear single-stranded DNA molecules of 667 and 4275 nucleotides, as well as full length circular single-stranded DNA were formed. Full length linear DNA was not detected. The two single-stranded circular DNA products formed as leading strands in RF leads to SS(c) reaction mixtures containing G27-4 RF I DNA differed in their ability to support lagging strand DNA synthesis. It was shown that the large single-stranded circular product included DNA sequences homologous to a replication factor Y effector sequence required for RF leads to RF and SS(c) leads to RF replication (Zipursky, S.L., and Marians, K.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6521-6525). The 4275-nucleotide, but not the 667-nucleotide, single-stranded circular DNA product was converted to a duplex structure.     

Transposon 10 promoted deletions and inversions in the transfer genes of R100-1

Summary The replication of the ColE1 plasmid was studied in extracts from E. coli dnaG mutants. It was found that the synthesis of the complementary strands of ColE1 DNA can be carried out in these extracts in two consecutive steps: (1) synthesis of the leading L strand independent of the dnaG function, and (2) synthesis of the lagging H strand depending upon addition of wild-type dnaG protein. In contrast to L strand synthesis, the latter reaction is insensitive to rifampicin and novobiocin. Both synthetic pathways are however blocked by antiserum directed against dnaB protein. This indicates an additional role of the dnaB protein in duplex DNA replication besides assisting the dnaG protein in the priming of lagging strand synthesis. The T7 gene-4 protein acting in conjunction with T7 DNA polymerase can substitute for both the function of the dnaB and dnaG protein. It is concluded that plasmid replication proceeds by a semi-discontinuous mechanism.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Synthesis of bacteriophage phi X174 in vitro: mechanism of switch from DNA replication to DNA packaging

Replication of a replicative form DNA of bacteriophage phi X174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA. Gene C protein of phi X174, which is involved in DNA packaging, inhibits the rolling-circle DNA synthesis by binding to the initiation complex in vitro. The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with phi X174 gene J protein and the prohead. Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex. These results indicate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage capsid, which occurs in the late stage of infection.     

Initiation of Escherichia coli minichromosome replication at oriC and at protein n'' recognition sites. Two modes for initiating DNA synthesis in vitro.

The start sites for leading and lagging DNA strands were determined in vitro with minichromosomes as templates. Fragments from replication intermediates were analyzed by hybridization to single-stranded probes. Leading strand synthesis in the counterclockwise direction was found to originate in or close to (position 248 to -44) the minimal origin. Complementary lagging strand synthesis started several positions to the left outside of oriC. The results suggest in addition a concerted synthesis of leading and lagging strands following the dnaA directed assembly of initiation proteins at double-stranded oricC DNA (pre-replisome). In addition, DNA synthesis could initiate at protein n' recognition sequences located within and clockwise to the asnA gene. Initiation at n' sites was dependent on protein i activity, whereas leading and lagging strand initiation in the oriC region was not affected by protein i. Our results argue against an involvement of the phi X174-type primosome in the initiation of discontinuous DNA synthesis at oriC. An alternative function is suggested.     

Initiation of DNA synthesis on the isolated strands of bacteriophage f1 replicative-form DNA.

Viral and complementary strand circular DNA molecules were isolated from intracellular bacteriophage f1 replicative-form DNA. Soluble protein extracts of Escherichia coli were used to examine the initiation of DNA synthesis on these DNA templates. The initiation of DNA synthesis on f1 viral strand DNA was catalyzed by E. coli DNA-dependent RNA polymerase, as was initiation of f1 viral strand DNA isolated from mature phage particles. The site of initiation was the same as that used in vivo. In contrast, no de novo initiation of DNA synthesis was detected on f1 complementary strand DNA. Control experiments demonstrated that the E. coli dnaB, dnaC, and dnaG initiation proteins were active under the conditions employed. The results suggest that the viral strand of the f1 replicative-form DNA molecule carries the same DNA synthesis initiation site as the viral strand packaged in mature phage, whereas the complementary strand of the replicative-form DNA molecule carries no site for de novo primer synthesis. These in vitro observations are consistent with the simple rolling circle model for f1 DNA replication in vivo proposed by Horiuchi and Zinder.     

Initiation and termination of the bacteriophage phi X174 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage phi X174 coats.

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with two phi X (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (greater than 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (greater than 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.     

Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides

Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX² DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung^? (uracil N-glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al., 1980a).Using (5′-³²P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA.Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro. Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas &; Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA.Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.     

DNA synthesis in a mitochondrial lysate of Xenopus laevis oocytes. H strand replication in vitro

Conditions for efficient replication in vitro of mitochondrial DNA L strand into H strand products have been established. Gel electrophoresis and hybridization analyses of the products show that neosynthesized H strands are progressively elongated from the D-loop region, and some of them are synthesized as full-length molecules. Evidence for initiation of these H strands de novo is presented. In contrast, there is no detectable L strand synthesis in vitro in this system. This may prove useful for analyzing the distinct molecular mechanisms operating at OH and OL. Use of specific inhibitors indicates that DNA synthesis in the mitochondrial lysate in vitro requires DNA polymerase gamma. These observations support the conclusion that replication in vitro in this system closely resembles the first steps of mitochondrial DNA replication in vivo.     

Initiation signals for complementary strand DNA synthesis in the region of the replication origin of the Escherichia coli chromosome.

We have used an in vivo plasmid-phi X174 packaging system to detect replication initiation signals in the region of the replication origin (oriC) of the Escherichia coli chromosome. The results obtained are summarized as follows: (i) Neither within nor close to oriC effective signals for initiating complementary strand synthesis could be detected. We conclude that initiation mechanisms for leading and lagging strand synthesis at oriC are not identical to any known priming mechanism of DNA synthesis. (ii) At least five signals that can function as complementary strand origins on ss-plasmid DNA are located in a region about 2000-3300 base pairs away from oriC in the clockwise direction on the chromosome. We suggest that these signals are protein n' like recognition sequences since they are dependent for their activity on dnaB protein and show sequence similarities to other putative n' recognition sequences. Surprisingly, some of the signals are located on the template for leading strand synthesis.     

Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.     

The complete 30-base-pair origin region of bacteriophage phi X174 in a plasmid is both required and sufficient for in vivo rolling-circle DNA replication and packaging

The origin of replication of the isometric single-stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling-circle DNA replication and packaging of single-stranded (ss) plasmid DNA into phage coats in phi X174 or G4-phage-infected cells. This system was used to study the nucleotide sequence requirements for rolling-circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30-base-pair (bp) origin region of phi X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30-bp origin region and plasmids carrying the 30-bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of phi X174 replicative-form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30-bp origin region and that no contribution is made by sequences which immediately surround the origin region in the phi X174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30-bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the phi X174 and G4 systems respectively). Previous studies [Fluit, A.C., Baas, P.D., van Boom, J.H., Veeneman, G.H. and Jansz, H.S. (1984) Nucleic Acids Res. 12, 6443--6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by phi X174 gene A protein. Moreover, Brown et al. [Brown, D.R., Schmidt-Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402--8412] have shown that omission of the last 2 bp of the origin region does not interfere with phi X174 rolling-circle DNA replication in vitro. Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for phi X174 phage DNA replication and morphogenesis.