The EcoDXX1 restriction and modification system: Cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and Ecob. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

The effect ofrecA mutation on the expression ofEcoKI andEcoR124Ihsd genes cloned in a multicopy plasmid

Type I restriction-modification (R-M) endonucleases are composed of three subunits—HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of clonedEcoKI andEcoR124Ihsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wthsd genes in a multicopy plasmid inEscherichia coli recA ⁺ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the clonedhsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation inE. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.     

Overproduction of the hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype

The geneshsdM andhsdS for M.EcoKI modification methyltrasferase and the complete set ofhsdR,hsdM andhsdS genes coding for R.EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation inhsdS gene, were cloned in pBR322 plasmid and introduced intoE. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. ThehsdS _ts-1 mutation, mapped previously in the distal variable region of thehsdS gene with C^{1 245}-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42°C. In clones transformed with the wholehsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutanthsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of thehsdS _ts-1 mutation on restriction and modification.     

DNA restriction and modification systems in Salmonella. II. Genetic complementation between the K and B systems of Escherichia coli and the Salmonella typhimurium system SB, with the same chromosomal location

Summary E. coli x S.typhimurium partially diploid hybrids were constructed to investigate the possibility of genetic complementation between the SA and the SB restriction and modification systems of S. typhimurium and the K and B systems of E. coli.An hsdR _K ^- mutation was complemented in a stable hybrid in which the hsd _SA ⁺ -hsd _SB ⁺ -serB ⁺ portion of the S. typhimurium chromosome was integrated at a non-homologous locus. By isolating hsd ^- mutants in that hybrid, it was shown that complementation occured between K and SB, but not between K and SA.Similarly, in a set of F-prime merodiploids bearing the SA, SB and B systems, complementation was observed between B and SB, but not between B and SA.     

Incomplete EcoRI digestion may lead to false diagnosis of fragile X syndrome

Molecular diagnosis of fragile X syndrome is usually performed using Southern blot analysis of DNA digested with EcoRI. In the course of diagnostic studies, we observed that a specific EcoRI restriction site in the fragile X gene (FMR1) is sometimes refractory to digestion, generating additional fragments on a Southern blot suggestive of a full mutation in FMR1. This may lead to a false-positive diagnosis of fragile X syndrome. Such additional bands are avoided by the use of HindIII instead of EcoRI. Therefore, we recommend the use of HindIII for the molecular diagnosis of fragile X syndrome. Received: 11 September 1997 / Accepted: 25 September 1997     

Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli

Summary The host-controlled EcoK-restriction of unmodified phage .O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold. In addition, the EcoK modification activity is substantially decreased in dam ^- strains. We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants. However, no relief of EcoRI restriction (Type II) occurs in dam ^- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed. We interpret the alleviation of the type I restriction in dam ^- strains to be a consequence of induction of the function which interferes with type I restriction systems.     

Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes

Type I restriction enzymes comprise three subunits encoded by genes designated hsdR, hsdM, and hsdS; S confers sequence specificity. Three families of enzymes are known and within families, but not between, hsdM and hsdR are conserved. Consequently, interfamily comparisons of M and R sequences focus on regions of putative functional significance, while both inter- and intrafamily comparisons address the origin, nature and role of diversity of type I restriction systems. We have determined the sequence of the hsdR gene for EcoA, thus making available sequences of all three hsd genes of one representative from each family. The predicted R polypeptide sequences share conserved regions with one superfamily of putative helicases, so-called ‘DEAD box’ proteins; these conserved sequences may be associated with the ATP-dependent translocation of DNA that precedes restriction. We also present hsdM and hsdR sequences for EcoE, a member of the same family as EcoA. The sequences of the M and R genes of EcoA and EcoE are at least as divergent as typical genes from Escherichia coli and Salmonella, perhaps as the result of selection favouring diversity of restriction specificities combined with lateral transfer among different species.     

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     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.     

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

A Temperature Sensitive Suppressor Mutation of Escherichia Coli

Abstract

In an Escherichia coli K 12 strain, with a Pho ^- nonsense mutation and the suppressor gene Su-4, we have induced a mutant with a temperature dependent alkaline phosphatase negative phenotype. This phenotype can be ascribed to a mutation mapping in the Su-4 gene that makes the suppression temperature sensitive.     

Cloning of a restriction fragment of phage Mu DNA coding for early functions

Summary The DNA of an E. coli K12 strain harboring ten wildtype Mu prophages was restricted with endonuclease EcoRI, and the fragments ligated into the plasmid vector pMB9. Upon transformation of a strain carrying a heat inducible (Mu cts62) prophage, one temperature-resistant transformant was isolated. This transformant strain harbors the hybrid plasmid pKN001, containing the EcoRI.C fragment of Mu DNA as shown by restriction and heteroduplex analysis. Stable transformants of pKN001 are immune to superinfection with phage Mu. Transformation of superinfection with phage Mu. Transformation of Mu sensitive bacteria with pKN001 results in killing of the recipients (10^-4 surviving bacteria). The killing function is not expressed upon transformation of Mu-immune (lysogenic) bacteria.This paper is dedicated by EGB to Dr. Luis F. Leloir, on the occasion of his 70th birthday     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Transduction of Plasmid Antibiotic Resistance Determinants with PseudoT-Even Bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 fromEscherichia coli recA ⁺ and recA ^– donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc–segE–uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages , T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limitedin vivo by modification–restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification–restriction systemsEcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification–restriction system.     

An improved method for detecting Y chromosomal DNA

Summary The DNA probe Y97 was derived from a repeat sequence in the human Y centromere, a region which must be present in a mitotically functional Y chromosome. We have demonstrated that Y97, which detects a Y-specific 5.5-kb Eco RI fragment by Southern analysis, is very useful for the molecular detection of small amounts of Y-derived material and represents a significant improvement over previous tests for molecular diagnosis of sex. The male-female difference in hybridization was unequivocal even when only 25 ng of total DNA was used per lane. Furthermore, in mixing experiments the 5.5-kb Eco RI fragment was detectable even when only 5% of the total DNA was male. By increasing hybridization stringency, we have developed a rapid, sensitive, and accurate method to detect Y chromosomal DNA in unrestricted samples.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes

Summary A hybrid phage (Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA ⁺-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979     

Two new mutations detected by single-strand conformation polymorphism analysis in cystic fibrosis from Russia

Single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing of exons containing ATP-binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was performed on 80 Russian DNA samples. Two new alterations — S1196X (exon 19) and W1282R (exon 20) — and two novel polymorphisms — 1525-61 (intron 9) and 1716+12 T-C (intron 10) — were identified. Mutation S1196X changes a TCA codon to TGA and destroys an EcoRI site. Alteration W1282R results from a T-to-C change at position 3976. It was found in one Russian patient and creates an AciI site; however, it is unclear whether this is a disease-causing mutation or a polymorphism. Polymorphism 1525-61 results from an A-to-G change. Alteration 1716+12 T-C was found in a Moldovian patient and creates a new MaeII site. It is not known whether this alteration affects the splicing of the mRNA. The previously described A4002G polymorphism was encountered in approximately 9% of Russian CF chromosomes. In addition, we have found the previously described 3732delA mutation in 7 CF chromosomes, making it the second (after F508) most frequent mutation in the Russian population.