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PubMed1999

On the structure and operation of type I DNA restriction enzymes.

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.

Adenosine Triphosphate↗

PubMed2001

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.

Acinetobacter↗

PubMed1984

Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA.

The alleviation of K-specific DNA restriction after treatment of cells by UV or nalidixic acid has been studied in mutants with various alleles of recA and lexA and combinations of these alleles and with recB and recF mutations. The studies show that induction of restriction alleviation by UV or nalidixic acid is abolished in mutants in which the recA protein is defective (recA13, recA56), its protease activity is altered (recA430) or in which it cannot be efficiently activated (recA142). Thermoinduction of restriction alleviation was observed in tif mutant (recA441). In lexA amber mutants restriction alleviation is not constitutive but is still inducible. In a lexA3 mutant restriction alleviation is inducible by nalidixic acid provided that recA protein is overproduced as a result of a recA operator mutation. Induction by UV depends on the recF function and an unidentified function (Y) which is controlled by the lexA protein. The recBC enzyme is necessary for induction by UV or by nalidixic acid. Temperature shift experiments with a thermosensitive recB mutant indicate that the recBC enzyme functions in an early step during UV-induction. It is concluded that the damage-inducible function which alleviates restriction is similar to other damage inducible repair (SOS) functions in the dependence on activated recA protease for induction, but that it differs from these functions by the absence of a direct control through the lexA repressor.

Bacterial Proteins↗

PubMed1989

Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12.

The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that it encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methyl-cytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.

5-Methylcytosine↗

PubMed1988

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation.

The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.

Bacillus subtilis↗

PubMed1976

Restriction in Myxococcus virescens.

1. The plating efficiency of bacteriophage MX-1 on Myxococcus xanthus strains A and B and M. virescens V2 were compared. Comparison of strains V2 and A suggest that V2 is restrictive and A is not (restriction coefficient was approximately 8). A derivative of M. virescens V2 (strain V2-9) was obtained by repeated exposure of strain V2 to ultraviolet radiation. Strain V2-9 was also unrestrictive. Strain B is apparently unrestrictive too but analysis of phenotypic changes in phage derived from hosts V2, B and A suggested that some of the host-cell processes differ from orthodox restriction and modification. 2. Cell-free extracts from M. virescens V2 were fractionated by ion-exchange chromatography and two restriction endonucleases, R. MviV2I and R. MviV2II were identified. Nuclease I was found to hydrolyse coliphage lambdaDNA at apparently one site only and MX-1 DNA at approximatley 10 sites; nuclease II was found to hydrolyse MX-1 DNA at a very large number of sites and its restriction sequence was of comparable frequency with that of R. EcoRII. "Modified MX-1 DNA", obtained from phage whose last host was M. virescens V2 was hydrolysed by nuclease II but not by nuclease I. The significance of these findings for restriction in myxococci is discussed.

Bacteriophages↗

PubMed1987

The aryl hydrocarbon hydroxylase (Ah) locus and a novel restriction-fragment length polymorphism (RFLP) are located on mouse chromosome 12.

The aryl hydrocarbon hydroxylase (Ah) locus that controls the induction of chemical carcinogen-metabolizing enzymes in mice has been found to be linked to a new restriction-fragment length polymorphism (RFLP). Only C57BL/6 and closely related inbred strains displayed a 7.6-kb HindIII restriction fragment, while all other inbred strains tested displayed an 11.2-kb HindIII restriction fragment when using plasmid pRC2.3 as the hybridization probe. Polymorphisms in this region can also be detected with two other restriction enzymes: SacI and EcoRV. Linkage of Ah and the restriction-fragment length polymorphism was first detected using the BXD (C57BL/6 x DBA/2) recombinant inbred strains and was confirmed by a backcross. Both the restriction-fragment length polymorphism and Ah were not linked to the standard genetic markers Hba, Hbb, b, d, C-3, and W. However, comparison of the RFLP strain distribution pattern in the BXD recombinant inbred set with the strain distribution pattern of another RFLP, known to be located on chromosome 12, shows complete concordance in 24 of 24 strains, thereby locating Ah on chromosome 12.

Animals↗

PubMed1986

Restriction fragment length polymorphism (RFLP) of the human insulin receptor gene in Japanese: its possible usefulness as a genetic marker.

Restriction fragment length polymorphism of the human insulin receptor gene was analyzed with a 4.2 Kb cDNA probe in Japanese normal subjects and Type 2 (non-insulin-dependent) diabetic patients. Restriction endonuclease Rsa I digestion showed polymorphism of the human insulin receptor gene, with a band at 6.7 Kb, 6.2 Kb or 3.6 Kb. The frequency of the 6.7 Kb band was less than that in Caucasians. Furthermore, 15% of all the Japanese subjects examined lacked a 3.6 Kb band, which is commonly found in Caucasians. We have also detected restriction fragment length polymorphism in the human insulin receptor gene by Pvu II or Stu I digestion. Although no significant association of restriction fragment length polymorphism with Type 2 diabetes was found in the present study, our results suggest that the restriction fragment length polymorphism in the human insulin receptor gene varies among ethnic groups, and that the restriction fragment length polymorphism linked to the human insulin receptor gene might be a useful marker for the linkage study of the genes located close to the human insulin receptor gene on chromosome 19.

Adult↗

PubMed1990

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation.

A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.

Alkaline Phosphatase↗

PubMed2002

Short-term moderate sodium restriction induces relative hyperfiltration in normotensive normoalbuminuric Type I diabetes mellitus.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is associated with an increased extracellular volume. Sodium restriction might seem a logical form of treatment but data on its renal effects is conflicting. We therefore studied the effects of sodium restriction on renal haemodynamics in uncomplicated Type I diabetes mellitus. METHODS: Uncomplicated Type I diabetic patients ( n = 24) and matched control subjects ( n = 24) were studied twice in random order: after a week of 50 mmol or after 200 mmol sodium intake, respectively. The diabetic patients were studied under normoglycaemic clamp conditions. Glomerular filtration rate and effective renal plasma flow were measured as the clearances of iothalamate and hippuran, respectively. RESULTS: During liberal sodium intake, glomerular filtration, effective renal plasma flow and filtration fraction were similar between the diabetic patients and the control subjects. Sodium restriction decreased the effective renal plasma flow in both groups, whereas glomerular filtration rate only decreased in the control subjects. Consequently, in the diabetic patients, the filtration fraction was increased on low sodium (4.1 +/- 8.4 %, p < 0.05 vs liberal sodium). As a consequence, filtration fraction (24.0 +/- 2.6 vs 22.1 +/- 2.0 %, p < 0.05) and glomerular filtration (119 +/- 14 vs 110 +/- 13 ml/min, p < 0.05) were higher in the diabetic patients than in the control subjects during sodium restriction. CONCLUSION/INTERPRETATION: Short-term moderate sodium restriction induces relative hyperfiltration in uncomplicated Type I diabetes. This could indicate an increased intraglomerular pressure. Sodium restriction could be an unfavourable preventive approach in diabetes mellitus but its long-term effects are not known.

Adult↗

PubMed1997

Alteration of the counterregulatory hormones in the conscious rat after protein-energy restriction.

We have recently reported that in rats submitted to protein-energy restriction early in life, an increased insulin efficiency upon the whole-body glucose utilization rate may be one reason for their chronic mild basal hypoglycaemia. However, the basis for their low plasma glucose level may also lie in the impaired activation of one or several of the counterregulatory hormones that prevent or correct hypoglycaemia. Our study was therefore designed to compare glucose counterregulatory mechanisms in restricted and control rats, both in the basal postabsorptive state and at controlled high plasma insulin level and standardized low glycaemic level (hypoglycaemic-hyperinsulinaemic glucose clamps performed in conscious rats). When tested in the basal postabsorptive state, the restricted rats exhibited prominent increases in the plasma levels of epinephrine (4.5 fold), norepinephrine (3.4 fold) and glucagon (1.7 fold). This was in the presence of significant decreases of plasma growth hormone and corticosterone levels (by 59 and 32%, respectively). With respect to the responses to acute severe hypoglycaemia (2.5 mmol/l), the glucagon, epinephrine and norepinephrine plasma levels in the restricted rats increased to values similar to those in controls. Also, the corticosterone level increased but remained significantly lower (p < 0.001) compared to the control response. The plasma growth hormone level was not significantly affected by acute hypoglycaemia in the restricted or in the control groups. We conclude that protein-energy restriction, starting early in life in the rat, severely impairs the release of counterregulatory hormones that defend against hypoglycaemia.

Acute Disease↗

PubMed2006

Health span extension by later-life caloric or dietary restriction: a view based on rodent studies.

In spite of the potential benefit of lifelong food restriction to retard aging and extend life span, it is unrealistic in human. The restriction late in life may be more practical. There are, however, only limited studies on the effect of late onset caloric or dietary restriction. We and other investigators have shown that the late life restriction rejuvenates some parameters that decline with age in rats and mice. Although such studies may provide a basis for human application of late-life caloric or dietary restriction, the prolongation of maximum life span would not be expected in view of the current status of the long-lived population in which maximum life span potential appears to have already been achieved. The late life caloric restriction, however, could extend the health span if the extent were appropriate.

Aging↗

PubMed1989

Characterization of fastidious adenovirus types 40 and 41 by DNA restriction enzyme analysis and by neutralizing monoclonal antibodies.

The DNA of 48 strains of adenovirus type 40 (Ad40) and of 128 strains of adenovirus type 41 (Ad41), isolated between 1971 and 1986 from various countries, was characterized by restriction enzyme analysis using nine and ten restriction endonucleases respectively. Five new DNA variants of Ad40 and 18 new DNA variants of Ad41 were detected. Most of the restriction sites which differed among the various DNA variants appeared to be distributed at random over the entire length of the viral genomes of the two serotypes. The number of restriction sites by which two DNA variants differed from each other was used as a measure of their relatedness. Several clusters of closely related DNA variants were observed for each of the two serotypes. The 35 DNA variants of Ad40 and Ad41 were used to test monoclonal antibody preparations for their range of reactivity in a neutralization assay. One monoclonal antibody (5-8), raised against Ad40 strain Dugan, showed type-specific neutralization of all 11 Ad40 DNA variants tested. Six monoclonal antibodies, raised against Ad41 strain Tak, neutralized different proportions of the variants of Ad41. Two of these preparations (1-21 and 3-19) neutralized all 24 Ad41 DNA variants, while a third (1-23) reacted with only 12 Ad41 variants. Three other monoclonal antibody preparations (3-10, 3-18, 7-14) reacted specifically with only 6 of these 12 variants. The patterns of reactivity with the monoclonal antibody preparations correlated with the presence or absence of a HindIII restriction site at 56 map units and of an EcoRI restriction site at 52 map units on the Ad41 DNA. This region of the adenovirus DNA codes for the hexon protein, which is known to contain the type-specific neutralizing antigenic determinants.

Adenovirus Infections, Human↗

PubMed1992

Characterization of Serpulina (Treponema) hyodysenteriae and related intestinal spirochetes by ribosomal RNA gene restriction patterns.

Serpulina (Treponema) hyodysenteriae strain A-1 partially purified rRNA, labelled with photobiotin, was used as a non-radioactive probe to identify the rRNA gene restriction patterns of S. hyodysenteriae strains and other spirochetes. Sau3A restriction enzyme digests resulted in similar rRNA gene restriction patterns in S. hyodysenteriae strains from five different countries. Some S. hyodysenteriae strains could be differentiated by variations in their rRNA gene restriction patterns after cleavage of DNA by restriction enzymes SspI or BglII. S. innocens and Treponema succinifaciens, non-pathogenic pig intestinal spirochetes, had rRNA gene restriction patterns that differed markedly from the S. hyodysenteriae patterns, and from each other.

Animals↗

PubMed1977

HindII, HindIII, and HpaI restriction fragment maps of the left arm of bacteriophage lambda DNA.

The sites on the left arm of bacteriophage lambda DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were localized on the lambda physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis. The restriction sites within the b2 region of lambda were mapped by analysis of the digestion profiles of deletion and substitution derivatives of lambda, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. The restriction sites of the lambda genome between the left vegetative end and the b2 region were mapped entirely by succesive digestion experiments. The restriction fragment map for the right arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977).

Chromosome Mapping↗

PubMed1978

Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus.

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R-PstI site, (formula: see text), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3'-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3'-OH termini, thus regenerating the PstI cleavage site sequences. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r-m-recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage lambdacI-0). A single phage-resistant clone was found. The recombinant plasmid, pD110, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.

DNA Restriction Enzymes↗

PubMed1991

Direct cloning of a long restriction fragment aided with a jumping clone.

Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes. Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig. 1a), facilitates the physical mapping [Poustka et al., Nature 325 (1987) 353-355]. A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest. For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material. The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease. The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands. In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast. We describe the protocol of this method with Escherichia coli DNA as a model experiment. Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available. The instability of inserts in the pYAC vector is also discussed.

Base Sequence↗

PubMed1996

The distinction of serologically related ruminant alphaherpesviruses by the polymerase chain reaction (PCR) and restriction endonuclease analysis.

The amplification and analysis of a 468bp fragment from the gB gene of the serologically related ruminant alphaherpesviruses bovine herpesvirus-1.1 and 1.2 (BHV-1.1 and BHV-1.2), caprine herpesvirus-1 (CapHV-1), cervine herpesvirus-1 (CerHV-1) and rangiferine herpesvirus-1 (RanHV-1) by PCR and restriction endonuclease analysis is described. As primers, 22bp oligomers selected from the BHV-1 gB gene sequences were used for the amplification of the DNA from the five viruses. The amplification product from each virus was analysed by the restriction endonuclease enzymes BglI, HinfI, SmaI and AvaI. The specific amplification obtained demonstrate the existence of the gB gene sequences for each of the five alphaherpesviruses. However, sequences from some of the fragments were found to be different from those predicted from the gB gene following restriction endonuclease analysis. All five amplification products generated the same number of fragments after digestion with HinfI except for two additional bands evident in CapHV-1. The CerHV-1 and RanHV-1 fragments contained slightly different BglI restriction sites from those of the other three. While BHV-1.1, BHV-1.2, CapHV-1 and CerHV-1 contained SmaI and AvaI restriction sites, the RanHV-1 amplification product lacked both SmaI and AvaI restriction sites.

Animals↗