A DNA-unwinding enzyme induced in bacteriophage-T4-infected Escherichia coli cells.

A single-stranded DNA-dependent ATP gamma-phosphohydrolase of Mr 56000 induced after infection of Escherichia coli cells with bacteriophage T4, probably the ATPase dependent on gene dda of the phage, was isolated. Studies on the enzyme show that in the presence of ATP and M2+ ions it is capable of dissociating partially double-stranded fd bacteriophage DNA into the single strands and that some 3000 enzyme copies are required to unwind the 6400-nucleotides-long DNA. Unwinding is inhibited by reducing the length of the single-stranded portion of DNA to two nucleotides. In addition it can be inhibited by sulfhydryl reagents which block the ATPase or by trapping free enzyme molecules in the assay system. The results suggest that unwinding is initiated near the single-stranded portion of the DNA and is driven by the ATPase. It further appears that the enzyme unwinds by adsorbing to the DNA. Affinity of the enzyme for double-standed DNA is not detectable by DNA binding assay.     

M13-oriC cloning vehicles: use for amplification of high copy lethal (HCL) genetic elements

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the M13 viral strand origin requires a functional rep gene product, using oriC these vehicles propagate as low-copy-number plasmids in E. coli rep mutants. This property is exploited to amplify cloned "high copy lethal" (HCL) DNA fragments, those containing genetic elements which kill the E. coli host when present at multiple copies in the cell. Following cloning of such fragments in these vehicles and initial selection in E. coli rep cells, the M13-oriC chimeric plasmid DNA is used to transfect appropriate E. coli rep+ cells. The chimeric DNA propagates as M13 viral DNA, yielding double-stranded and single-stranded DNA products and phage particles prior to killing of the host via expression of the HCL element; these events mimic a lytic phage infection. Such amplification will greatly facilitate both DNA "library" constructions (HCL elements are absent a priori from libraries using high-copy-number cloning vehicles) and studies of HCL elements including restriction mapping, DNA sequencing, and physiological studies.     

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.     

Isolation of mutant genes for human leukocyte alpha2-interferon by a method of localized mutagenesis

An efficient method to obtain the mutant genes for human leucocyte alpha 2-interferon (IFN) has been elaborated.The technique includes the following main stages: cloning of interferon gene in M13mp8 DNA; isolation of double-stranded hybrid DNA complex, containing IFN gene as a single-stranded fragment; selective modification of a single-stranded hybrid DNA by sodium bisulphite; the repair of hybrid DNA by DNA polymerase I from Escherichia coli, transformation of Escherichia coli JN103 cells by double-stranded circular DNA, containing the selectively modified gene IFN. The technique is based on the protection of bacteriophage M13 genome from mutagen induced damage by means of converting phage DNA into the double-stranded structure leaving the single-stranded fragment to be mutagenized prone to mutagen action. This is achieved by reannealing of single-stranded M13mpB DNA hydrolyzed by restriction endonuclease BamHI. The technique preserves the infectiousness of vector DNA under the conditions permitting modification of up to 10% cytosine residues in IFN gene. Every clone resulting from transformation of Escherichia coli by modified DNA carried mutations in IFN gene, identified by sequencing after Sanger.     

Genetic control of the UV-induced SOS mutator effect in single- and double-stranded DNA phages

The SOS hypothesis postulated that the mutator effect on undameged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage λ for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA⁺ and umuDC⁺ genes. In this paper, we show thet (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage λ, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and φX174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage λ and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5′ → 3′, activities of Po1I. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.     

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.     

A simple and rapid nucleotide sequencing strategy and its application in analyzing a rice histone 3 gene

An improved rapid method for sequencing a target DNA is described. A new plasmid, pAA-PZ1, which contains the origin of replication from phage M13 and a portion of the Tn9 transposon was constructed. A long fragment of target DNA cloned into this vector is progressively shortened in vivo from one end by transposon-mediated deletions. The plasmids carrying different lengths of target DNA are then made into single-stranded DNA in the same host upon infection with an M13 phage and their sequence is determined using the dideoxynucleotide chain-termination method. This method bypasses the in vitro enzymatic manipulations for progressive deletions and requires no subcloning. Using this strategy, we sequenced 1.3 kb of rice DNA containing a histone 3 gene within three weeks.     

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.     

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.     

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.     

Biology and genome sequence of Streptococcus mutans phage M102AD

M102AD is the new designation for a Streptococcus mutans phage described in 1993 as phage M102. This change was necessitated by the genome analysis of another S. mutans phage named M102, which revealed differences from the genome sequence reported here. Additional host range analyses confirmed that S. mutans phage M102AD infects only a few serotype c strains. Phage M102AD adsorbed very slowly to its host, and it cannot adsorb to serotype e and f strains of S. mutans. M102AD adsorption was blocked by c-specific antiserum. Phage M102AD also adsorbed equally well to heat-treated and trypsin-treated cells, suggesting carbohydrate receptors. Saliva and polysaccharide production did not inhibit plaque formation. The genome of this siphophage consisted of a linear, double-stranded, 30,664-bp DNA molecule, with a GC content of 39.6%. Analysis of the genome extremities indicated the presence of a 3'-overhang cos site that was 11 nucleotides long. Bioinformatic analyses identified 40 open reading frames, all in the same orientation. No lysogeny-related genes were found, indicating that phage M102AD is strictly virulent. No obvious virulence factor gene candidates were found. Twelve proteins were identified in the virion structure by mass spectrometry. Comparative genomic analysis revealed a close relationship between S. mutans phages M102AD and M102 as well as with Streptococcus thermophilus phages. This study also highlights the importance of conducting research with biological materials obtained from recognized microbial collections.     

Replication of Coliphage M-13 II. Intracellular Deoxyribonucleic Acid Forms Associated with M-13 Infection of Mitomycin C-treated Cells

Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F⁺, hcr⁻, thy⁻) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with ³H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.     

SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light

Summary In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mpl8 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts. Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target. When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA. Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvrA fpg-1 double mutant than that seen in uvrA, fpg-1, or umuC single mutants or in the wild-type. Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions. The most frequent base change is the GCTA transversion. MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly GT transversions. Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli. The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

Construction and characterization of new coliphage M13 cloning vectors

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.     

Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase

EcoP1I methyltransferase (M.EcoP1I) belongs to the type III restriction-modification system encoded by prophage P1 that infects Escherichia coli. Binding of M.EcoP1I to double-stranded DNA and single-stranded DNA has been characterized. Binding to both single- and double-stranded DNA could be competed out by unlabeled single-stranded DNA. Metal ions did not influence DNA binding. Interestingly, M.EcoP1I was able to methylate single-stranded DNA. Kinetic parameters were determined for single- and double-stranded DNA methylation. This feature of the enzyme probably functions in protecting the phage genome from restriction by type III restriction enzymes and thus could be considered as an anti-restriction system. This study describing in vitro methylation of single-stranded DNA by the type III methyltransferase EcoP1I allows understanding of the mechanism of action of these enzymes and also their role in the biology of single-stranded phages.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

Preparation of single-stranded deoxyribonucleic acid probes using an immobilized template

A new method for the easy preparation of specific single-stranded DNA fragments is presented. Recombinant M13 DNA containing the strand complementary to the sequence of interest is made partially double-stranded by elongating a conventional M13 sequencing primer. Following linearization by enzymatic digestion downstream from the insert (relative to priming site), this DNA is coupled to diazotized paper through its single-stranded (vector) portion. Subsequent denaturation of the double-stranded region generates an immobilized template strand. Successive runs of primed syntheses of the (desired) complementary strand can be realized using the same template. The copies are easily isolated by release upon denaturation. DNA probes prepared by this method have proven to be valuable tools for gene analysis.