Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects

Lambda duplication phages grown for several rounds on Escherichia coli strains containing arl mutations were recombined at elevated frequencies (3 to 6-fold higher) in subsequent test infections. Enhanced recombination of Arl^? phages (grown on arl bacteria) was demonstrable by assays for altered genetic linkages as well as by the standard assay, which measures the conversion of duplication phages (EDTA-sensitive) to single-copy phages (EDTA-resistant). The accumulated potential for enhanced recombination was lost during subsequent growth of the phages on arl⁺ bacteria. Arl^? phages had the same mutation frequencies, at a variety of loci, as control phages; arl bacteria themselves exhibited normal mutation rates. Arl^? phages had normal plating efficiencies and buoyant densities. DNA extracted from Arl^? phages exhibited the same frequency of strand interruption, the same superhelical density (when circularized in vivo), and the same thermal denaturation profile as DNA from phages grown on arl⁺ bacteria. Recombination of Arl^? phages in the presence of λ repressor was very low, as is the case for normal phages. The recombination frequency of ultraviolet light irradiated (80 J/m²) Arl^? phages was more than twice the sum of the frequencies for unirradiated Arl^? phages and irradiated control phages. Substantially increased recombination of Arl^? phages was observed when either the E. coli RecBC, or RecE (but not RecF) pathway was active.     

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. I. Identification and mapping of arl mutations

Hyper-rec mutants of Escherichia coli were originally identified as lac-diploid strains whose colonies exhibited unusually high numbers of Lac⁺ papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl. Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl⁺ bacteria, but not in recA cells. Both the high frequency of Lac⁺ papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT, do not confer a hyper-rec phenotype (by the lac-diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

Hotspots for generalized recombination in the Escherichia coli chromosome.

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λbio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA⁺ function, the site-specific stimulation of recombination required recB⁺ function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology.The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 10³ bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.     

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli.

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec⁺ function is not required. In Rec⁺ strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.     

An amber dna mutant of Escherichia coli K12 affecting DNA ligase

We have isolated an amber mutant (dnaL321) of Escherichia coli K12, which affects DNA ligase and which is lethal unless it is suppressed. DNA is degraded under the restrictive conditions. The mutation also affects the sensitivity of the cell to ultraviolet light irradiation, and the capacity to support the growth of phage λ that is deficient in general recombination. This pleiotropy is considered to be due to a single mutation, and is suppressed by supD^?_Isu+ and by supF^?su_III⁺). The mutation is cotransducible with dapE(2%), and with ptsI(85%), by phage Plvir.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

The induction of protein X in DNA repair and cell division mutants of Escherichia coli

Certain temperature-sensitive Escherichia coli cell division mutants and DNA repair mutants were treated in several ways to alter DNA synthesis or cell division. The bacteria were pulsed with [³⁵S]methionine; then membrane proteins were prepared and examined using sodium dodecyl sulfate/polyacrylamide slab gels. Autoradiography was performed on the slab gels so that the rate of synthesis of protein X could be determined by microdensitometry.Several changes in the rate of synthesis of the 40,000 molecular weight protein X were found in the different mutants. The wild-type (rec⁺ and lex⁺) strains synthesized protein X in response to DNA synthesis inhibition. However, neither recA^? strains nor lex^? strains synthesized protein X.Both the filament forming, temperature-sensitive mutants tif^? and tsl^? (which was derived from lex^?) synthesized protein X when DNA synthesis was inhibited, but at rates different from the wild-type strains. Moreover, these strains also produced protein X at their non-permissive temperature, even though DNA synthesis was not inhibited. In the tif^? mutant, the rate of synthesis of protein X was influenced by the addition of nucleic acid precursors.A double mutant tsl^?recA^? produced protein X when DNA synthesis was inhibited, or at the non-permissive temperature (although DNA synthesis was normal). This was the only strain carrying a recA^? mutation capable of synthesizing protein X.From these results it is suggested that the genes lex, recA and tif comprise a system that controls DNA repair and limits DNA degradation by the recBC nuclease. The inducer of this control system might be a DNA degradation product.     

Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO₄ nicks in duplex DNA via reaction of LigA with NAD⁺ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO₄ to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO₄ nicks, k_step2 was fast (6.8–27 s⁻¹) and similar to k_step3 (8.3–42 s⁻¹). Absent ammonium, k_step2 and k_step3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in k_step2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA''s potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions.     

Disco-ordinate expression of the Escherichia coli gal operon after prophage lambda induction.

EcoRI endonuclease crystallizes in space group C2 with unit cell parameters $\text{a = 209}\text{A}\text{?}\text{, b = 129}\text{A}\text{?}\text{, c = 50}\text{A}\text{?}\text{and}\text{β = 98.4 °}$. Four 29,000 molecular weight subunits per asymmetric unit would give a reasonable V_m value of 2.87 ų/dalton. EcoRI endonuclease is the first protein which recognizes a specific sequence of bases in DNA to be crystallized in a form suitable for high resolution structure analysis.     

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.     

Cloning of gene lon (capR) of Escherichia coli K-12 and identification of polypeptides specified by the cloned deoxyribonucleic acid fragment

A mutation in the lon (capR) gene of Escherichia coli K-12 results in overproduction of capsular polysaccharide and increased sensitivity to ultraviolet and ionizing radiations. The lon (capR) gene deoxyribonucleic acid was cloned from a new F′ factor. The new plasmids, designated pBZ201 and pBZ203, (i) contained an additional 8.2-megadalton (Md) EcoRI fragment that had the same mobility as one of the EcoRI fragments of the F′, and (ii) conferred repression of capsular polysaccharide synthesis and repression of sensitivity to ultraviolet radiation in a bacterial transformation experiment with capR mutant recipient strains. A capR9 mutant plasmid, pBZ201M9, was also isolated and conferred expression of mucoidy and ultraviolet sensitivity to a capR⁺ (lon⁺) strain, indicating that the capR9 allele was dominant. Plasmids pBZ201M80, pBZ201M9-INSA, and pBZ201M9-INSB were characterized by transformation as containing recessive capR mutant alleles. Heteroduplex analyses and agarose gel electrophoresis of restriction endonuclease digests of plasmid DNA preparations revealed that (i) pBZ201M9-INSA and pBZ201M9-INSB each contains a 0.5-Md insertion (probably IS1) in the cloned DNA fragment at the same site, and (ii) pBZ201 and pBZ203, both capR⁺ plasmids, contain the same 8.2-Md fragment cloned in opposite orientations with respect to the cloning vehicle, pSC101. Plasmid-specified polypeptides were determined by using strain CSR603 maxicells containing each plasmid. Two new polypeptides were coded by the lon⁺ (capR⁺) 8.2-Md DNA fragment: Z1, 94 kilodaltons (94K), and Z2, 67K. The maxicells containing recessive capR mutant plasmids were deficient only in synthesis of the 94K polypeptide, and the dominant (capR9) mutant plasmid specified 5 to 10 times more of the 94K polypeptide than the maxicells containing the capR⁺ plasmid. Other data indicated that the capR9-specified “94K polypeptide” was not identical to the capR⁺-specified “94K polypeptide.” Thus the altered mutant polypeptide was synthesized in increased quantities, suggesting a defective mode of autogenous regulation for the capR9 polypeptide and effective autogenous regulation of the capR⁺ polypeptide.     

Involvement of Recombination Genes in Growth and Viability of Escherichia coli K-12

We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec⁻ cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and “reckless” deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and “cautious” DNA breakdown. (iii) Indirectly suppressed (sbcA⁻) recombination proficient (Rec⁺) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec⁻ ancestors (but not as viable as rec⁺ cells). We suggest the following hypothesis to account for the low viability of Rec⁻E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec⁺ cell. Failure of Rec⁻ cells to repair this normal DNA damage may lead to the observed loss of viability.     

In vivo intermolecular recombination in Escherichia coli: application to plasmid constructions

Repair of a double-strand break (DSB) was investigated by intermolecular recombination in Escherichia coli (Ec) recBC sbcBC cells with restriction enzyme-cleaved model plasmids. Circular plasmids were generated when a linearized plasmid (vector) containing an origin of replication was co-transformed with a DNA fragment (template) containing a homologous sequence. The influence of the position of the DSB in the vector was analyzed using templates which contain various genetic markers, non-homologous sequences and/or deletions relative to the vector. In all cases, when a DSB occurs within a marker, this marker is lost in the resulting plasmid, whereas markers flanked by homologous regions located in the vicinity of a DSB are transmitted. Insertions (deletions), substitutions and shuffling of genetic markers are possible by in vivo recombination using Ec and can be applied to plasmid constructions. It is shown that recombination can occur from both template ends or from one vector and one template end. A D-loop nuclease is suggested to participate in the resolution of the recombination intermediates     

Expression of the phage λ recombination genes exo and bet under lacPO control on a multi-copy plasmid

The bacteriophage λ genes exo and bet, whose products (λ exonuclease and β protein, respectively; Red phenotype) mediate homologous recombination of λ phages, have been cloned under lacPOlacI^q control on multi-copy plasmids. Induction of recA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in λ exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of λ Red^? phages in vivo was similarly inducible. Only one out of 25 bet^Δ plasmids (constructed by a variety of in vitro techniques) expressed λ exonuclease, a result consistent with the polarity of several known phage bet mutations. A general method for transferring phage exo and bet mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar (bet113) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. When λbet3 phages (Exo^?Bet^?) infected IPTG-induced recA3 bacteria containing exo⁺bet⁺ plasmids, recombination frequencies were no more than twice those typical for infection of plasmid-free recA3 cells with exo⁺bet⁺ phages, even in the case of IPTG induction sufficient to elevate the production of λ exonuclease about 100-fold. Even when plasmid induction was delayed till as late as 50 min after infection, recombination was significant. Preliminary experiments suggest that these plasmids encode a polypeptide with Gam activity that corresponds to the 98-amino acid “shorter” open reading frame assigned to gam by Sanger et al.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ^?transposable element was constructed to insert the lacZ ^?(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14?000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ^?→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ^?and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10^?3 to 10^?2, indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome.     

Changing the target base specificity of the EcoRV DNA methyltransferase by rational de novo protein-design

The EcoRV DNA-(adenine-N⁶)-methyltransferase (M.EcoRV) specifically modifies the first adenine residue within GATATC sequences. During catalysis, the enzyme flips its target base out of the DNA helix and binds it into a target base binding pocket which is formed in part by Lys16 and Tyr196. A cytosine residue is accepted by wild-type M.EcoRV as a substrate at a 31-fold reduced efficiency with respect to the k_cat/K_M values if it is located in a CT mismatch substrate (GCTATC/GATATC). Cytosine residues positioned in a CG base pair (GCTATC/GATAGC) are modified at much more reduced rates, because flipping out the target base is much more difficult in this case. We intended to change the target base specificity of M.EcoRV from adenine-N⁶ to cytosine-N⁴. To this end we generated, purified and characterized 15 variants of the enzyme, containing single, double and triple amino acid exchanges following different design approaches. One concept was to reduce the size of the target base binding pocket by site-directed mutagenesis. The K16R variant showed an altered specificity, with a 22-fold preference for cytosine as the target base in a mismatch substrate. This corresponds to a 680-fold change in specificity, which was accompanied by only a small loss in catalytic activity with the cytosine substrate. The K16R/Y196W variant no longer methylated adenine residues at all and its activity towards cytosine was reduced only 17-fold. Therefore, we have changed the target base specificity of M.EcoRV from adenine to cytosine by rational protein design. Because there are no natural paragons for the variants described here, a change of the target base specificity of a DNA interacting enzyme was possible by rational de novo design of its active site.     

Genetic analysis of the recF pathway to genetic recombination in Escherichia coli k12: Isolation and characterization of mutants

A recombination proficient strain ofEscherichia coli which is recB^? recC^? sbcB^? has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB⁺, three were recA^～ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by F$\text{\$}\text{?}$studies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB⁺recC⁺sbcB^? strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF^?recB⁺ and recF⁺recB^? single mutants are sensitive to u.v. irradiation while the recF^?recB^? double mutant is more sensitive than either single mutant. The sensitivity of the recB^?recC^?sbcB^?recF^? strain approaches the sensitivity of a recA^? single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.     

Influence of phage T3 and T7 gene functions on a type III(EcoP1) DNA restriction-modification system in vivo

Summary The ocr ⁺ gene function (gp 0.3) of bacteriophages T3 and T7 not only counteracts type I (EcoB, EcoK) but also type III restriction endonucleases (EcoP1). Despite the presence of recognition sites, phage DNA as well as simultaneously introduced plasmid DNA are protected by ocr ⁺ expression against both the endonucleolytic and the methylating activities of the EcoP1 enzyme. Nevertheless, the EcoP1 protein causes the exclusion of T3 and T7 in P1-lysogenic cells, apparently by exerting a repressor-like effect on phage gene expression. T3 which induces an S-adenosylmethionine hydrolase is less susceptible to the repressor effect of the SAM-stimulated EcoP1 enzyme. The abundance of EcoP1 recognition sites in the T7 genome is explained by their near identity with the T7 DNA primase recognition site.Abbreviations d.p.m. decompositions per min - EcoB, EcoK, EcoP1, EcoP15, EcoRII, EcoR124, HinfIII restriction endonucleases coded by Escherichia coli strains B or K, E. coli plasmids P1, P15, RII or R124, and Haemophilus influenzae Rf 232, resp. - e.o.p. efficiency of plating - gp gene product (in the sense of protein) - m.o.i. multiplicity of infection (phage/cell) - ocr ⁺ gene function which overcomes classical restriction - p.f.u. plaque-forming units - SAM S-adenosylmethionine - sam ⁺ gene function with S-adenosylmethionine-cleaving enzyme (SAMase) activity - UV ultraviolet light Dedicated to Professor Konstantin Spies on the occasion of his sixtieth birthday