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The recognition sequence 5'-CC(A/T)GG for EcoRII in the bacteriophage T7 genome is refractory to this restriction endonuclease, despite not bearing the specific (protective) methylation. Following the integration of this site as part of a 219 bp fragment (in which the recognition sequence is flanked by about 100 bp of T7 origin) into the EcoRII-sensitive vector pUC18, the T7 site becomes susceptible to cleavage, too. The same is true of recombinant pBR322 plasmids containing the T7-derived recognition site. The results show that the flanking sequences are not immediately responsible for the refractory behaviour of EcoRII sites and are in agreement with data according to which EcoRII requires the coordinated presence of at least two recognition sites in its DNA substrate.

When restriction-active Escherichia coli cells (R+P1m+P1) are transformed with the pSF2124 plasmid, a common vector in experimental gene transfer, the efficiency of transformation (e.o.t.) is lowered by 2 orders of magnitude compared with restriction-negative (r-P1m-P1 or r-P1m+P1) recipient cells due to restriction of the pSF2124 DNA by endoR.EcoP1. Preinfection of r+P1m+P1 cells with UV-inactivated ocr+ phages (T3, T7, T3sam-) still able to express their early genes protects the plasmid DNA against restriction by endoR.EcoP1: The e.o.t. of r+P1m+P1 recipient cells with pSF2124 attains the same high value as that of r-m- cells. The specific role of the ocr+ gene function was demonstrated by the use of ocr- mutants (T3/R7, T7/D111): Preinfection with such phage mutants does not increase the e.o.t. of r+P1m+P1 cells. An unspecific e.o.t. alteration of restriction-negative (r-m-) recipient cells by ocr+ or ocr- phages was excluded. The ocr+ gene function can be exploited to protect pSF2124 against DNA restriction. The recipient cells survive the process of phage preinfection and transformation and stably replicate themselves as well as the plasmid DNA.

Bacterial virus T3 undergoes host-controlled modification which is not based on "classical" processes of DNA modification and restriction. The adsorption and thus the growth of T3 on Escherichia coli W cells (E. coli K12 derivative) decisively depends on the host strain on which the virus was previously propagated. Depending on the modification conferred to the virus by its last host, its efficiency of plating (e.o.p.) on E. coli W varies by six orders of magnitude between 10(-7) and 10(-1). This does not reflect the appearance of T3 host-range mutants, but a fully reversible modification of genotypically unchanged T3 wild-type phage. The behaviour of T3 in the described host system constitutes a second case of so-called non-classical modification and restriction (KRUGER et al. 1977, Molec. gen. Genet. 153, 107-110) of bacteriophages. Non-classical modification (protein modification) is additive to and independent of DNA modification and restriction as demonstrated with the ocr- phage T3/R7. - Furthermore, our results suggest that the adsorption specificity of T3 is determined by at least two independent genetic factors; in both of these factors T3 differs from T7.

In host cells containing the Salmonella typhimurium DNA restriction-modification systems SA(+) and SB(+), replication of the ocr(+) bacteriophages T3 and T7 is not impaired. However, ocr (gene 0.3) mutants of these phages are susceptible to DNA restriction and modification by the SA(+) and SB(+) systems.

The influence of the T3 gene functions ocr+ and sam+ on the extent of phage DNA methylation in Escherichia coli K12 cells was studied by determining the proportion of 6- methylaminopurin to adenine in the purified DNA of T3 wild-type, sam- and ocr- sam- phage strains. We demonstrate that the DNA of T3 ocr- sam- mutants carries 12 methyl groups as a result of the action of the host-specificity methylase EcoK . In contrast to this the DNA of ocr+ strains is not EcoK -specifically methylated.

In order to test the influence of the restriction endonucleases of Haemophilus species on in vivo DNA restriction of phage T7 and its close relative T3 as well as to determine whether the anti-DNA restriction effect of the T7 and T3 ocr+ gene is also expressed in Haemophilus cells, we studied the interaction of T7 and T3 with Haemophilus influenzae serotypes Rd and Rc and Haemophilus parainfluenzae. The results show that T7 and T3 are unable to infect these bacterial species because they do not absorb to the cells.

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.

The ocr+ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated. These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5'-CG) is also true for animal DNA viruses. Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzyme EcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonuclease EcoRII.

Some DNA species are resistant towards the restriction endonuclease EcoRII despite the presence of unmodified recognition sites. We show that 14 base-pair oligonucleotide duplexes containing the EcoRII recognition site 5'-CC(A/T)GG are cleaved by this enzyme and are able to stimulate EcoRII cleavage of such resistant DNA molecules (e.g. DNA of bacterial virus T3). A direct correlation between the concentration of oligonucleotide duplex molecules and the degree of EcoRII digestion of the primarily resistant DNA is observed. This indicates a stoichiometric rather than a catalytic mode of enzyme activation. An excess of DNA devoid of EcoRII sites ('non-site' DNA, e.g. MvaI-digested T7 DNA) does not interfere with the activity of EcoRII.

A method is presented allowing a clear distinction between bacterial viruses T3 and T7 by plating on selectively permissive host cells. The indicator strains are Escherichia coli cells containing either cloned pif genes (exclusively permissive for T3) or the EcoRV DNA restriction system (permissive only for T7): The efficiencies of plating of the two phages on these hosts differ by more than 8 orders of magnitude. This method was applied to reinvestigate the controversial question of mutual exclusion between T3 and T7. Under single-burst conditions, about 50% of coinfected cells (permissive for both viruses) produced T3 and T7 progeny while about 25% reproduced only T3 and about 25% only T7. The burst size of co-infected cells was slightly reduced, compared to controls infected with only one virus type. Homologous exclusion among T3 phages was also not seen; rather, there was a gene dosage effect: T3-encoded RNA polymerase activity as well as T3-specific RNA synthesis increased proportionally to the multiplicity of infection (2.5-20 plaque-forming units/cell).

Phage T7 infects male (F-plasmid-carrying) Escherichia coli cells abortively, whereas the closely related phage T3 grows normally. The inability or ability of phage to replicate in male host cells depends on whether the right end of gene 1 (coding for the phage-specific RNA polymerase) consists of T7 or T3 DNA base sequences.

The ability of the bacteriophage T3 to adsorb on the host cells of Escherichia coli W1655 changes depending on the host strain in which the phage was propagated before. This phenomenon is termed "non-classical" host-controlled modification in contrast to "classical" DNA modification. We demonstrate here that T3 phages with various non-classical modifications as well as the host range mutant T3hW differ from each other in the antigenic determinants of the phage adsorption protein.

The use of different precisely mapped T3/T7 recombinants strengthens the conclusion that abortive infection by T7 of F plasmid-carrying cells is due to the nucleotide sequence at the end of the T7 gene 1. Furthermore, we demonstrate that the exclusion requires suppression of ochre stop codons, a phenomenon that occurs with low frequency in wild-type cells due to ribosomal misreading. The introduction of rspL mutations in which ribosomal misreading is reduced alleviates the exclusion and the presence of ochre tRNA suppressors increases its severity.

The closely related phages T3 and T7 exhibit different growth patterns on Escherichia coli W hosts cells (E. coli K12 derivatives). T7 grows normally while T3 does not adsorb. T3hw mutants displaying a T7-like host range were isolated and described.

The EcoRII recognition sites 5'-CCATGG-3' which occur three times in T3 DNA are refractory to this restriction endonuclease. However, these sites are specifically cleaved by EcoRII when a second, susceptible DNA species (pBR 322, phage lambda) is present.

We have investigated the susceptibility of the genomes of the related bacteriophages T3 and T7 to the three major DNA methyltransferases (EcoK, dam, dcm) of their host, Escherichia coli K12. In vivo the EcoK host specificity enzyme only methylates the DNA of ocr- phages. This is due to an inhibition of the enzyme by the phage ocr+ gene product, which had previously been shown to be an inhibitor of the restriction endonuclease. EcoK-specific DNA methylation protects the ocr- viruses after one growth cycle on these host cells against the action of corresponding restriction endonuclease EcoK. Owing to the unique S-adenosyl-L-methionine hydrolase (sam+) activity of the T3-coded ocr+ protein, the T3 DNA is absolutely devoid of the methylated bases 6-methylaminopurine and 5-methylcytosine. In contrast to this, T7 derivatives and sam- derivatives of T3 carry a small number of about 2-4 molecules 6-methylaminopurine and 5-methylcytosine per genome. The presence of 6-methylaminopurine is due to dam methylation, though the majority of dam sites remain unmethylated. In vivo as well as in vitro the ocr+ protein has no influence on the activities of the dam and dcm methylase. The experiments gave some evidence for the existence of a second cytosine methylase in E. coli K12. Besides dam and dcm recognition sites being undermethylated, their absolute number in T3 and T7 DNAs is far below the expected value. Moreover, one of the two dcm sites present in T7 (Studier strain) is missing in our T7 strain owing to a 1300-base-pair deletion in gene 0.7.

When E. coli cells carrying the plasmid pLG13 (coding for the newly discovered type II restriction endonuclease EcoRV) are infected with phage T3 or T7, only T7 is able to replicate normally. T3 wild-type as well as its ocr- mutants are subject to DNA restriction in vivo and in vitro. The EcoRV enzyme cuts T3 DNA at 5 sites. T7 and its ocr- mutants have no EcoRV sites in their DNA. In contrast to the anti-restriction activity of the T3 and T7 ocr+ gene function against type I and III restriction enzymes, the ocr+ protein is unable to inactivate the type II restriction endonuclease EcoRV.

Selected and counterselected oligodeoxynucleotide sequences were identified in the total sequence of bacteriophage T7 DNA using a statistical criterion derived for a probability model of the Markov chain type. All extremely rare tetra- and pentadeoxynucleotides are (or contain) recognition sequences for the Escherichia coli DNA methylases dam or dcm. Most of the 37 hexadeoxynucleotides absent from T7 DNA are recognition sequences for type II modification/restriction enzymes of E. coli or related species. In contrast to most restriction sites counterselected during evolution, the EcoP1 site GGTCT occurs 126 times in the T7 genome, and phage T7 replication is severely repressed in P1-lysogenic host cells. We demonstrate that the frequency of the EcoP1 site is determined by that of the overlapping recognition sites for T7 primase, an essential phage enzyme. The recognition site of a type III enzyme, EcoP15, is also not counterselected. In T7 DNA all 36 EcoP15 sites are arranged in such a manner that the sequence CAGCAG is confined to the H strand, the complementary sequence CTGCTG to the L strand. This "strand bias" is highly significant and, therefore, very probably selected. A functional relation between this strand bias and the refractive behaviour of phage T7 to EcoP15 restriction is suspected.