EcoRI Recognition Site Research Articles

Proviral unit II of endogenous mouse mammary tumour virus is selectively amplified and expressed in C57B1/10 mammary tumours induced by non-viral carcinogens.

Restriction enzyme analysis and molecular hybridization assay of DNA isolated from C57B1/10 mammary adenocarcinomas induced by a combination of dimethylbenzanthracene, oestrogen, and prolactin, revealed the presence of four extra copies of endogenous mouse mammary tumour virus (MMTV). PstI restriction pattern of the amplified proviral sequences indicated their identity with the proviral Unit II of endogenous MMTV. The amplified proviruses are hypomethylated and expressed in a hormone-dependent fashion. Their internal structure is slightly modified, since an additional EcoRI recognition site is present within the proviral genomic DNA. Selective amplification of Unit II MMTV provirus in the course of mammary tumourigenesis initiated by chemical carcinogens and hormones is compatible with the accepted multifactorial nature of this process, and is interpreted in terms of the insertional mutagenesis model for MMTV-induced oncogenesis. However, sequences of cellular DNA, adjacent to the amplified Unit II proviruses, show no homology to the integration domains int-1 and int-2 common to exogenous MMTV.

Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences.

In an investigation to determine how proteins are localized within the nucleus of a cell, we demonstrate that the restriction endonuclease EcoRI is able to enter and function within the nucleus of Saccharomyces cerevisiae when this prokaryotic protein is synthesized in vivo. The EcoRI endonuclease was produced in yeast under the transcriptional control of a regulated yeast promoter by ligating a DNA fragment containing only coding sequences for the endonuclease to the promoter element of the yeast GAL1 gene (the structural gene for galactokinase, EC 2.7.1.6). Yeast cells harboring a plasmid containing this promoter-gene fusion are able to grow under conditions that repress transcription from the GAL1 promoter. However, under inducing conditions, these yeast cells are unable to grow. Moreover, rad52 mutants, which are deficient in the repair of double-strand breaks, are more sensitive to the presence of the promoter-gene fusion plasmid than are wild-type cells. We demonstrate that the EcoRI endonuclease activity is present in lysates prepared from yeast transformants grown under conditions that induce transcription of GAL1, but this activity is not detectable in cells grown under conditions that repress transcription from the promoter. Furthermore, analysis of yeast chromosomal DNA shows that the endonuclease enters the yeast nucleus and cleaves DNA specifically at EcoRI recognition sites.

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. VI. The binding and cleavage of substrates containing nucleotide analogs

The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT-containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT-containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes.

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg2+, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5′..AATT..3′ sites (with exceptions, the 5′..AATT..3′ sites may function as one type of EcoRI* sites) along the DNAs, indicating unspecific binding. In the absence of Mg2+, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5′..AATT..3′ was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.

GENETIC LINKAGE STUDIES IN THE MARFAN SYNDROME

In order to define a single major gene defect which could account for the Marfan syndrome (MS) phenotype we used a high frequency EcoRI restriction fragment length polymorphism in the pro 2(I) gene of type I collagen as a marker for genetic linkage studies. The presence or absence of one EcoRI recognition site creates a two allele system observed after hybridization with a 6.75kb pro 2(I) genomic probe (Tsipouras et al. JCI 72:1262, 1983). We studied affected and unaffected individuals in three families with MS. In family A the segregation of the clinical phenotype was concordant with the segregation of the marker allele, although random cosegregation could not be excluded due to the small number of informative matings. In family B the segregation of the clinical phenotype and marker allele was discordant. Family C was not informative due to lack of heterozygosity for the marker allele. Our data suggest the some variants of MS may be linked to mutations in or near the pro 2(I) collagen gene. This is in agreement with the observation that a structural alteration in the pro 2(I) chain of type I collagen results in the MS in one patient (PNAS 78:7745, 1981). Our data further suggest that MS is biochemically heterogeneous and that defects in more than one gene may result in the phenotype.

Characterization of exogenous proviral sequences in hamster tumor cell lines transformed by Rous sarcoma virus rescued from XC cells

Alterations in viral structural genes have been studied in five cell lines derived from Syrian hamster tumors which had been induced by the virus rescued from XC cells by transfection. Two cell lines, H-18 and H-20, have all the viral structural genes expressed, but a new EcoRI recognition site appeared in the region of the pol gene sequence. Provirus present in H-12 lacks the 3′ part of the gag gene sequences as well as the pol gene, therefore, it gives rise to an anomalous 1.8 Md EcoRI fragment. This line also does not synthesize viral RNA of genomic size, and none of the subgenomic RNAs found hybridized with the DNA itpol probe. The H-19 cell line harbors only the src gene and LTR sequences, the U 3 part of which seems incomplete or different from that of PR-RSV. The cryptic proviral structure in H-19 is transcribed into src mRNA. The degree of transcription of the src gene is about 25 viral RNA equivalents per cell. The H-9 cells harbor the complete provirus and, in addition, proviral structures having the deletion in gag-pol genes. The possible ways of development of provirus alterations and the role of cryptic proviral sequences in oncogenesis are discussed.

ChemInform Abstract: STUDIES ON DEOXYNUCLEIC ACIDS AND RELATED COMPOUNDS. IV. SYNTHESES OF AN OCTANUCLEOTIDE CONTAINING A RECOGNITION SITE FOR RESTRICTION ENZYME ECO RI AND OF AN ARABINOSYLADENINE ANALOG

AbstractDie Synthese des Octanucleotids (I) und seines Arabinose‐Analogons (II) werden beschrieben.

Studies on deoxynucleic acids and related compounds. IV. Syntheses of an octanucleotide containing a recognition site for restriction enzyme Eco RI and of an arabinosyladenine analog.

An octanucleotide containing a recognition site for Eco RI and its arabinosyladenine (araA) analog, dGGAATTCC and dGGaraAATTCC, were synthesized by the phosphotriester approach with phosphoro-p-anisidate as the protecting group for 3'-phosphodiesters. araA was converted to 5'-dimethoxytrityl-N, 2'-O-benzoyl 3'-p-chlorophenyl phosphate and condensed with N-benzoyldeoxyadenosine 3'-p-chlorophenyl phosphoro-p-anisidate to yield the protected araAdAp. Other deoxynucleotide blocks (dAAp, dGGp) were prepared similarly and condensed with a 5'-deblocked dTTCC block having the 3'-O-benzoyl group after removal of the p-anisidate group with isoamyl nitrite.

Plasmid cointegrates and their resolution mediated by transposon Tn3 mutants

We have generated recombinant plasmids in vivo between pHS1, a temperature-sensitive replication mutant of pSC101 which codes for tetracycline (Tc) resistance, and pMB8, carrying the ampicillin (Amp) transposon Tn3. Recombinants were selected in a recA − Escherichia coli strain at 42°C in the presence of Tc. When the repressor gene tnpR of Tn3 was inactive, all cells examined contained cointegrate recombinants, even in the absence of selective conditions for the recombinant. Tn3's were present at both junctions between the two parental plasmids, in a direct orientation, and a 5-bp pHS1 sequence was directly duplicated at the plasmid junctions. Complementation of the cointegrate with active repressor ( tnpR +) in a recA − background at 30°C, resolved the recombinant into two plasmids, yielding the pMB8:: Tn3 parent and pHS1, which has acquired a copy of the Tn3 mutant. These results indicate that the repressor is responsible for this resolving recombination event. Recombinants were also formed between pHS1 and pMB8 carrying wild-type Tn3 or its mutant No. 25, which is tnpR + and tnpA + but has an EcoRI recognition site inserted at a position corresponding to the amino-terminus of the tnpA-coded transposase. Various sized recombinants were generated at 42°C (+Tc), all smaller than the cointegrates described above, but larger than the parental plasmids. The frequencies of recombination between wild-type Tn 3 and pHS1, and mutant No. 25 and pHS1 were 10 −6–10 −7, i.e., drastically lower than the frequency of formation of cointegrates mediated by tnpR − mutants. Genetic and nucleotide-sequence analysis suggest that the formation of these recombinants is through intermediate cointegrate structures, which subsequently undergo deletions removing all or part of a copy of Tn3, leaving one intact Tn3 in the plasmid. Interstingly, analysis of one recombinant showed that such a deletion occurred by recombination at a region of homology between the left inverted Tn3 repeat and pMB8, in a recA − strain. Tn3 mutants that are defective in either of their inverted end repeats or in the carboxy-terminus of the transposase cannot mediate recombination between two plasmids, or do so at a very low frequency. Based on these results we discuss the role of genes and sites encoded by Tn3 in cointegration and transposition.

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10 6 was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with Bam HI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.

Location of a new endonuclease restriction site associated with a temperature-sensitive mutation of vaccinia virus

The novel EcoRI recognition site at the temperature-sensitive locus of ts 9251, derived from vaccinia IHD-W, has been mapped on the poxvirus genome. Using Southern blot hybridization, it was demonstrated that the EcoRI fragment D of vaccinia IHD-W and of rabbit poxvirus were identical in terms of sequence and presumed map position and that the novel restriction site found in ts 9251 occurred at a point 28 megadaltons from the right terminus of the molecule. This study reveals the first identification of a poxvirus mutation on the physical map and provides a potential marker for further, genetic mapping of temperature-sensitive mutations of vaccinia and related poxviruses.

A study of sequence homologies in four satellite DNAs of man

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities. Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10 3 basepair fragment, in addition to a 3.4 × 10 3 base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10 3 base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.

An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.

EcoRI ★ activity: Enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI ∗ activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg 2+ with Mn 2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI ∗ conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI ∗ activity has been found to be 10 mM Tris·HCl buffer (pH 8.8) + 2 mM Mn 2+. Similar activity is induced also by addition to EcoRI solution of 40–50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI ∗ activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2–3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI ∗ have cohesive termini and can be easily ligated. It is suggested that the EcoRI ∗ activity can be due not only (or largely not) to modification of the

Free ribosomal DNA molecules from Tetrahymena pyriformis GL are giant palindromes

Restriction ondonuclease EcoRI was used to study the structure of the free ribosomal DNA molecules from Tetrahymena pyriformis, strain GL. From the following observations we conclude that the free rDNA molecules from Tetrahymena are giant palindromes ‡ ‡ The term palindrome is defined as a word or sentence reading the same in both directions. We apply this term to those double-stranded DNA regions with an axis of 2-fold rotational symmetry, since only such regions will reud (transcribe) identically from bot,h ends of the region, e.g. ▪ , each containing two genes for preribosomal RNA arranged in rotational symmetry as inverted repeating sequences. Analyses of the sizes of products of partial or complete digestion and quantitative analyses of the products of complete digestion of uniformly 32P-labeled rDNA yielded an RI endonucleolytic cleavage map which showed that the EcoRI recognition sites are arranged symmetrically about the center of the rDNA molecule. When heat-denatured rDNA was rapidly cooled under conditions in which no renaturation would occur between separated complementary strands of DNA, molecules of half the size of the original rDNA molecule were produced. These were double-stranded DNA molecules as evidenced by their resistance to digestion with S1 nuclease. Moreover, they could be digested with EcoRI to produce fragments of sizes which would be predicted from the assumption that each single strand of the original rDNA molecule had folded back on itself to form a “hair-pin” double-stranded DNA structure. Hybridization experiments between ribosomal RNA and purified rDNA showed that each rDNA molecule contains two genes for rDNA. Hybridization of the isolated EcoRI fragments of rDNA with 25 S or 17 S rRNA suggested that the two structural genes for 17 S rRNA are located near the center of the rDNA molecule and the two genes for 25 S rRNA are found in distal positions.