Cohesive Termini Research Articles

Interaction between X-ray- and restriction endonuclease-induced lesions in the formation of chromosomal aberrations

Experiments were performed to analyze the possible interaction between lesions induced by X-rays and restriction endonucleases in the production of chromosome-type exchanges. A stronger interaction was found between X-rays and the AluI-induced ‘blunt termini’ lesions than between X-rays and the BamHI- induced ‘cohesive termini’ lesions

Amplification and detection of lentiviral DNA inside cells.

Visna virus and human immunodeficiency virus are prototypes of animal and human lentiviruses, respectively, that persist and are disseminated despite the host immune response because cells in the tissues and the bloodstream harbor viral genomes in a covert state. To facilitate identification of these latently infected cells, the polymerase chain reaction has been adapted to amplify viral DNA in fixed cells for detection by in situ hybridization. By using a multiple primer set that generates DNA segments with overlapping cohesive termini, visna virus DNA can be amplified, retained, and detected in infected cells with sensitivities that exceed those of existing methods by more than 2 orders of magnitude. This advance in single-cell technology should prove useful in diagnosing and gaining insight into the pathogenesis of viral infections and provide new opportunities to look for viruses in chronic diseases of unknown etiology.

Cloning of PCR products after defined cohesive termini are created with T4 DNA polymerase

Cloning of PCR products after defined cohesive termini are created with T4 DNA polymerase

Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.

33] Expression and secretion vectors for yeast

The chapter focuses on methods used to efficiently express and secrete biologically active proteins from Saccharomyces cereoisiae . The chapter describes yeast expression vectors utilizing episomal vectors. The expression cassettes from these vectors can also be integrated into the yeast chromosome, where they are presented at controlled copy number, exhibiting a high degree of mitotic stability. The chapter discusses extrachromosomal replication vectors, centromere plasmids, and the assembly of expression cassettes. One of the advantages of a secretion system is the production of heterologous proteins with authentic NH 2 termini. The methods utilized to analyze heterologous protein secretion are the same as those used for proteins expressed directly in the cytoplasm (direct expression vectors) The chapter describes several related procedures including the Mung Bean nuclease digestion, the S1 nuclease digestion of cohesive termini, and the analysis of carbohydrate additions to heterologous proteins.

A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus.

The glucocorticoid response element (GRE) of mouse mammary tumor virus (MMTV) was chemically synthesized as two complementary DNA strands bearing cohesive termini. During automated synthesis, random mutations were introduced into the DNA by "doping" each of the four nucleoside phosphoramidites (A, G, C, and T) with a low level of the other three. These preparations were annealed and cloned into an M13 phage vector to produce a library of GRE mutants. Mutations within the synthesized region were identified by sequencing phage isolates at random. All of the chemically distinct classes of transition and transversion mutations have been observed. Statistical considerations indicate that the library contains all of the possible 90 point substitution mutations within a 30-nucleotide mutagenic target. So far 88 of these substitutions have been isolated, 74 as single mutants. At least two of the three possible single mutants at each of the 30 positions have been identified.

A simple method for constructing directly repeated multimeric DNA segments

A simple and efficient procedure is described for the construction of a series of oligomers of a DNA segment in which all repeating units are aligned as head-to-tail tandem repeats. The technique is based on the utilization of a pair of restriction endonucleases that generate identical cohesive termini but whose individual specificity of cutting depends on specific flanking bases adjacent to the central common sequence. Such a pair of enzymes is SalI (G ↓TCGAC) and XhoI (C ↓TCGAG). Ligation, at a high DNA concentration, of a single linear DNA fragment bearing SalI and XhoI termini resulted in a complex mixture of DNA aggregates in which the orientation of adjacent inserts was uncontrolled. Digestion of these ligation products with SalI and XhoI eliminated inverted repeats as these contain reconstituted SalI and XhoI sites. Because direct repeats contain hybrid SalI/ XhoI junctions that are not recognized by either enzyme these were resistant to this redigestion. The use of this method generated a good yield of multimeric arrays ranging from 2 to 5 directly repeated units. The yield of higher-order directly repeated oligomers could be considerably enhanced by including SalI and XhoI in the ligation mixture itself.

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1 c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1 c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5 ' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.

Synthesis of the leu-enkephalin gene.

The synthesis of Leu-enkephalin gene by an alternative approach was described in this paper. The sequence of the synthetic gene, 26 base-pairs in length, was derived from the amino acid sequence of the hormone peptide Leu-enkephalin. It bears single-stranded cohesive termini for the restriction endonucleases EcoR I and BamH I so that it may be inserted into a pBR 322 plasmid. The 4 deoxyoligonucleotide fragments, varying in length from octanucleotide to octadecanucleotide, were synthesized by an improved phosphotriester method developed in our laboratory. All chemically synthetic fragments were pure and had the correct sequences. The assembly of the Leu-enkephalin gene was carried out in a one-step ligation reaction catalysed by T4-DNA ligase with good yield. Finally, the purified products from polyacrylamide gel electrophoresis had the correct joining-points as predicted.

A new site-specific endonuclease Bbei from Bifidobacterium breve.

Abstract A new site-specific endonuclease, BbeI, has been partially purified from the anaerobic bacterium, Bifidobac-terium breve. BbeI recognizes the hexanucleotide sequence and cleaves it at the sites indicated by the arrows, producing 3′-cohesive termini four bases long.

Specific and reversible inhibition of the blunt end joining activity of the T4 DNA ligase.

Specific, complete and reversible inhibition of the joining of blunt ended DNA duplexes catalyzed by the T4 DNA ligase can be obtained by using ATP, the enzyme cofactor, at concentrations of 5 mM and higher. On cohesive DNA ends, 5 mM ATP, which completely inhibits blunt end ligation, brings about only a limited (8%) reduction in the level of joining obtainable under optimal ATP concentration (0,5 mM or lower). A similar but less drastic uncoupling of the two kinds of joining can be achieved at lower ATP concentration (2,5 mM) using 1 mM Mg++. The joining of DNA blunt ends can also be inhibited almost completely by 10 mM spermidine, without noticeable effect on the joining of cohesive termini.

The nucleotide sequence recognized by the BstEII restriction endonuclease

The restriction site for the Bst/EII endonuclease is characterized by the heptamer sequence: ▪ with five nucleotide long cohesive termini.

8] Repair of overlapping DNA termini

This chapter deals with repair of overlapping DNA termini. The natural cohesive termini of bacteriophage DNA, such as γ and the staggered phosphodiester bond cleavages produced by various restriction endonucleases have several possible arrangements. If the arrangement is such that a protruding 5' single-stranded end and an internal 3'-hydroxyl end are formed, then the termini will serve both as primer and template for repair synthesis by a DNA polymerase. Overlapping DNA termini can be repaired using the RNA directed DNA polymerase, which is called reverse transcriptase, from RNA tumor viruses and radioactive nucleoside triphosphates. The incorporation into the termini can be quantified and the sequence analyzed from nearest neighbor data. The chapter further explains the labeling procedure.

Restriction cleavage maps of coliphages 186 and P2

The restriction enzymes BamHI, Bg/II, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, Bg/II-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and Xho-I-1; b) P2, BamHI-3, Bg/II-2, EcoRI-3, HindIII-0, PstI-O, XbaI-1 and XhoI-O. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.

Chemical synthesis of genes for human insulin.

A rapid chemical procedure has been developed and used for the synthesis of 29 oligodeoxyribonucleotides to build synthetic genes for human insulin. The gene for insulin B chain, 104 base pairs, and the one for A chain, 77 base pairs, were designed from the amino acid sequence of human polypeptides. They bear single-stranded cohesive termini for the EcoRI and BamHI restriction endonucleases and are designed to be inserted separately into a pBR322 plasmid. The synthetic fragments, deca- to pentadecanucleotides, were synthesized by a block phosphotriester method with trinucleotides as building blocks. Final purification was by high-performance liquid chromatography. All 29 oligonucleotides were pure and had the correct sequences.

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

Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.

In vivo site-specific genetic recombination promoted by the EcoRI restriction endonuclease.

Site-specific genetic recombinations promoted in vivo by the EcoRI endonuclease has been demonstrated by using constructed hybrid plasmids in which the chloramphenicol resistance gene was inactivated by insertion of DNA fragments at an EcoRI site within the gene. Such recombination can involve either the joining of intracellularly generated cohesive termini of the same DNA fragment or intermolecular ligation of different DNA fragments. DNA cleavage and ligation in vivo are precise: recombinant DNA molecules show functional continuity of the gene sequence cleaved by the enzyme and regeneration of nucleotide recognition sites for both the EcoRI endonuclease and the EcoRI DNA methylase. In other experiments, EcoRI-generated fragments of eukaryotic DNA that had not been modified by the Escherichia coli K methylase were shown to be taken up by bacterial cells and to undergo intracellular ligation to segments of bacterial plasmid DNA.

Viral integration and excision: structure of the lambda att sites.

Viral integration and excision: structure of the lambda att sites.

Chemical synthesis of two deoxyribododecanucleotides for the attachment of restriction termini to an artificial minigene

In order to permit in vivo cloning of an artificial minigene designed to code for a modified S-peptide, the phosphodiester method for the chemical synthesis of two dodecadeoxyribonucleotides is described. Each of the latter possesses antiparallel complementarity one of the wto minigene strands and to the single-stranded EcoRI-generated end. They can thus serve as cohesive termini (“splints”) for polynucleotide ligase joining.