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At least 19 recordsLinked to original sources
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↗
PubMed1998

Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme.

The type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA target sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, ATP hydrolysis, which is believed to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA. We have examined the DNA binding affinity and exonuclease III footprint of the EcoKI type IA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence. The influence of the cofactors, S-adenosyl methionine and ATP, on binding to DNA of different methylation states has been assessed. EcoKI in the absence of ATP, with or without S-adenosyl methionine, binds tightly even to DNA lacking the target site and the exonuclease footprint is large, approximately 45 base-pairs. The protection is weaker on DNA lacking the target site. Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site. The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and the footprint shrinks to 30 base-pairs, almost identical to that of the modification enzyme which makes up the core of EcoKI. The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATPgammaS are substituted for ATP. It is proposed that the DNA binding surface of EcoKI comprises three regions: a "core" region which recognises the target sequence and which is present on the modification enzyme, and a region on each DNA cleavage subunit. The cleavage subunits make tight contacts to any DNA molecule in the absence of cofactors, but this contact is weakened in the presence of cofactors to allow the protein conformational changes required for DNA translocation when a target site is recognised by the core modification enzyme. This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint.

Adenosine Triphosphate↗
PubMed1992

Ovine mitochondrial DNA: restriction enzyme analysis, mapping and sequencing data.

Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.

Adenosine Triphosphatases↗
PubMed1985

Comparison of neutralization and DNA restriction enzyme methods for typing clinical isolates of human adenovirus.

Sixty-five adenovirus isolates collected over a 3.5-year period were typed by both standard microneutralization techniques and restriction endonuclease digestion of viral DNA. Of the 65 isolates, 47 (72.3%) representing six adenovirus types could be typed by microneutralization. Eighteen isolates demonstrated partial neutralization with standard antisera to two or more adenovirus serotypes and thus could not be definitively typed. DNA analysis permitted typing of 64 of the 65 isolates (98.5%) (including four isolates which contained mixtures of two adenovirus types), and 12 different types were identified. Neutralization and DNA typing disagreed for five isolates, and in each case, digestion with multiple restriction endonucleases and DNA hybridization studies were consistent with the type assigned by DNA analysis. In addition, the DNA analysis method allowed the identification of genomic variants (genome types) of five adenovirus types. We conclude that typing clinical isolates of adenovirus by restriction endonuclease digestion of viral DNA can be done rapidly, provides additional epidemiological and typing information, and provides fewer ambiguous results than does typing by neutralization.

Adenoviruses, Human↗
PubMed1991

Microflora and bacterial DNA restriction enzyme analysis in young adults with periodontitis.

As a result of an epidemiological survey on the periodontal status of young adults, a school was found with a relatively high number of subjects showing loss of attachment (18 out of 87). In 10 of these subjects the microflora of one pocket and of the dorsum of the tongue was investigated with special reference to black-pigmented species and Actinobacillus actinomycetemcomitans. Prevotella intermedia was the most prevalent species, being found in 9 patients. A. actinomycetemcomitans was isolated from 3 subjects and Porphyromonas gingivalis from 2 subjects. To determine whether cross-infection had occurred, the P. intermedia, P. gingivalis, and A. actinomycetemcomitans isolates were examined by restriction endonuclease analysis of the DNA after digesting with one or a combination of the endonucleases Pst I, Bam HI, and Hind III. Evaluation of the restriction endonuclease typing method showed it to be sensitive and useful for the typing of P. intermedia, P. gingivalis, and A. actinomycetemcomitans. Using this restriction endonuclease typing method, all isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia obtained from single individuals showed different digest patterns. We conclude that in the present study bacterial cross-infection did not occur among subjects in this school.

Actinobacillus↗
PubMedDate not supplied

[Changes induced by low doses of ionizing radiation in the accessibility to DNA restriction enzymes of the ribosomal gene system of human lymphocytes].

Human lymphocytes were X-irradiated at doses 0-10(-2) Gy and allowed to repair for some time. Nuclei were prepared and digested with restriction endonuclease Rsa I to selectively release 28S RNA gene fragment fraction, containing the DNA-binding protein, Hpa II digestion of nuclei was used to investigate the methylation of the 28S RNA gene fragment. There was a differential enrichment of 28S RNA gene binding protein for different X-ray doses with maximum enrichment for dose 2-3 x 10(-2) Gy with following diminish to 10 x 10(-2). The enrichment of less methylated fractions of 28S RNA gene was observed during X-irradiation. This might be explained by a different X-ray-induced changes of methylated and unmethylated rDNA binding with nuclear proteins. The possible mechanism for this phenomena are discussed.

Blood Proteins↗
PubMed2000

[Identification of an introduced bovine herpesvirus type 1 strain in a closed dairy herd by experimental virus reactivation followed by DNA restriction enzyme analysis].

In a closed dairy herd in the province of Utrecht in 1995, nine replacement heifers were erroneously intramuscularly vaccinated with Tracherine, a live virus IBR vaccine. More than 18 months later, serology of the herd revealed that a large part of the herd had developed an antibody response towards BHV1 (62 of 87 animals). To investigate whether Tracherine had recirculated on the farm, four BHV1 antibody positive animals, of which two had been vaccinated with Tracherine, were treated with corticosteroids to reactivate latent BHV1. Two virus isolates were obtained and subsequently analysed by resctriction enzyme analysis. Both isolates were identified as BHV1.1 subtypes. One of the isolates was clearly distinct from Tracherine and was most likely a BHV1 field virus. A BHV1 field virus was most likely introduced into the farm even though the herd was closed, the animals had not been in contact with other cattle, and preventive hygienic measures had been implemented. There was no indication that Tracherine had recirculated.

Animals↗
PubMedDate not supplied

Inhibition of DNA restriction enzyme digestion by anthracyclines.

The inhibition of restriction enzyme digestion of lambda phage DNA by anthracyclines (i.e., adriamycin, daunomycin, epirubicin, idarubicin and esorubicin) commonly used in the treatment of human leukemia and cancer has been studied in vitro. The anthracyclines used inhibit DNA digestion by SmaI, AvaII, HaeIII, HhaI and HpaII, which cut DNA at guanine-cytosine (G-C) sequence sites, and by EcoRI, which cut DNA at adenine adenine-thymine thymine (AATT) sequence sites. Adriamycin completely inhibits DNA restriction by the indicated enzymes, daunomycin, epirubicin and idarubicin determine only a partial inhibition, while esorubicin does not inhibit DNA restriction at all. An attempt is made to correlate the extent of the in vitro interaction between anthracyclines and DNA to in vivo cardiotoxicity.

Antibiotics, Antineoplastic↗
PubMed1979

Physical map of bacteriophage BF23 DNA: restriction enzyme analysis.

Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases SalI (3 fragments), BamHI (5 fragments), EcoRI, (8 fragments), BalI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with lambda exonuclease.

Chromosome Mapping↗
PubMed2005

Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis.

Acidophilic iron-oxidizing microorganisms are important both environmentally and in biotechnological applications. Although, as a group, they are readily detected by their ability to generate ferric iron (resulting in a distinctive color change in liquid media), these microbes highly diverse phylogenetically. Various other characteristics, such as optimum growth temperature, response to organic carbon sources, and cellular morphologies, facilitate, in some cases, identification of isolates to a genus or species level, although this approach has limitations and may give erroneous results. In this study, a combined approach of using physiological traits together with amplified ribosomal DNA restriction enzyme analysis (ARDREA) has been successful in identifying all known acidophilic iron-oxidizing bacteria to the species level. Computer-generated maps were used to identify restriction enzymes that allow the differentiation of the acidophiles, and these were confirmed experimentally using authentic bacterial strains. To test further the validity of this approach, six acidophilic moderately thermophilic iron-oxidizing bacteria isolated from Montserrat (West Indies) were analysed using the ARDREA protocol. Three of the isolates were identified as Sulfobacillus acidophilus-like, and one as Sulfobacillus thermosulfidooxidans-like bacteria. The fifth isolate gave DNA digest patterns that were distinct from all known strains of iron-oxidizing acidophiles. Subsequent sequencing of the 16S rRNA genes of these isolates confirmed the identity of the four Sulfobacillus isolates, and also that the fifth isolate was a novel species. Schematic diagrams showing how ARDREA may be used to rapidly identify all known acidophilic iron-oxidizing bacteria are presented.

Base Sequence↗
PubMed1980

Segmental amplification in a satellite DNA: restriction enzyme analysis of the major satellite of Macropus rufogriseus.

The 1.708 g/cc satellite DNA of the red necked wallaby is shown to consist of a number of related families of sequences arranged in tandem arrays. Particular families are subpopulations of other families; their distribution supports a model of successive amplification events during the generation of the satellite. In each amplification episode one 2,500 bp unit is multiplied into a tandemly repeated array of that unit (segmental amplification). The 1.708 g/cc sequences can be detected in related kangaroo species in much reduced amount, and with changes to the long order periodicity of the repeat units.

Animals↗
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↗
PubMed1979

The polypeptide and the DNA restriction enzyme profiles of spontaneous isolates of herpes simplex virus type 1 from explants of human trigeminal, superior cervical and vagus ganglia.

Analysis of the infected cell polypeptides and the DNA restriction profiles of 31 HSV-1 isolates from the trigeminal, superior cervical and vagus ganglia from 17 individuals (12 U.S.A., 2 Japanese, 3 Norwegian) could be classified as 15 different virus strains. With the exception of the three Norwegian isolates which gave identical profiles, virus isolates from the ganglia of different individuals could all be distinguished from one another. In contrast virus isolates from the trigeminal, superior cervical and vagus ganglia of the same individual, or virus isolates from the left and right ganglia of the same individual or multiple isolates from different explants of a single ganglion were indistinguishable. In conclusion, a single virus strain infects each individual initially and virus descended from this event subsequently infects and becomes latent in different cells of the same ganglion as well as in different ganglia.

DNA Restriction Enzymes↗