An optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors

This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.     

The Arrangement of Information in DNA Molecules

The anatomy of DNA molecules isolated from mature bacteriophage is reviewed. These molecules are linear, duplex DNA consisting mainly of uninterrupted polynucleotide chains. Certain phage (T5 and PB) contain four specifically located interruptions. While the nucleotide sequence of most of these molecules is unique (T5, T3, T7, λ), some are circular permutations of each other (T2, T4, P22). Partial degradation of these DNA molecules by exonuclease III predisposes some of them to form circles upon annealing, but indicating they are terminally redundant.     

Some substrate properties of analogues of oligothymidylates with p-s-C5'' bonds.

The action of T4 polynucleotide kinase, T4 DNA polymerase, E. coli DNA polymerase I, snake venom phosphodiesterase (VPDE) and S1 nuclease on analogues of oligothymidilates with p-s-C5' bonds and the ability of these analogues to prime the replication of poly (dA) by T4 DNA polymerase were studied. These analogues were shown to be substrates for all these enzymes. Substitution of these analogues for corresponding oligothymidilates in the reaction mixtures resulted in drop in rates of enzymic reactions. This drop in reactions rates was not significant when these oligonucleotides were phosphorylated with T4 polynucleotide kinase or used as a primers, however in comparison with oligothymidilates these analogues were found to be considerably more resistant to nucleolytic hydrolysis. Some possible applications of these analogues are discussed.     

The effect of antibiotics on the T4 polynucleotide ligase catalyzed template dependent polymerization of oligodeoxythymidylates.

The poly(dA) dependent T4 polynucleotide ligase catalyzed polymerization of oligodeoxythymidylates is dependent upon duplex stability. The antibiotics ethidium bromide, netropsin and Hoechst 33258 stabilize the duplex poly(dA) . P(dT)n (n = 6-10) to thermal denaturation. Ethidium bromide to DNA ratio of 1.25 and netropsin or Hoechst 33258 to DNA ratio of 0.1 the Tm of d(pT) 10 . poly (dA) was increased by 10 degrees and 25 degrees C respectively. The T4 polynucleotide ligase activity was not inhibited under these conditions and temperature optimum of joining of d(pT) 10 . poly(dA) was increased 5 degrees to 10 degrees by the binding of the antibiotics. Duplexes containing shorter oligodeoxythymidylates required lower concentrations of the antibiotics netropsin or Hoechst 33258 to show no inhibition of T4 polynucleotide ligase. The temperature optima of joining the duplexes d(pT)6 . POLY(DA) and d(pT) 8 . poly(dA) were increased by 5 degrees C upon binding of the antibiotics. Polyacrylamide gel analysis of the T4 polynucleotide ligase catalyzed joining of the oligodeoxythymidylates showed that the presence of antibiotics affected the product distribution of the polymerized oligomers.     

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.     

Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.     

Enzymic in vitro repair and chemical nature of DNA chain breaks induced by incorporated phosphorus-32P decay.

In vitro repair of single strand breaks in T4 and phage DNA caused by 32p decay was studied. Zone centrifugation procedure showed that polynucleotide kinase, ligase enzyme system failed to repair 32P-damages. It was found that damaged DNA contained gaps and could be repaired by DNA-polymerase I, polynucleotide ligase treatment.     

Transfection of Escherichia coli Spheroplasts IV. Transfection of rec+ and rec? Spheroplasts by Native,Denatured, and Renatured T5 Bacteriophage DNA After Repair of Single-Strand Breaks by Polynucleotide Ligase

Transfection of Escherichia coli spheroplasts by native T5 phage DNA was not affected by treatment with polynucleotide ligase. Denatured T5 phage DNA infectivity, only 0.1% of the native DNA level, was increased slightly by polynucleotide ligase treatment. Renatured T5 phage DNA infectivity was also increased slightly by polynucleotide ligase treatment. To form an infective center with rec(+) spheroplasts, 1.6 to 2.1 native T5 phage DNA molecules were required; however, 1.4 T5 phage DNA molecules were required to form an infective center with recA(-)B(-) spheroplasts, and one molecule was sometimes sufficient for rec B(-) spheroplasts. Polynucleotide ligase treatment of T5 phage DNA had no effect on these parameters. Thus, the single-strand interruptions of T5 phage DNA are probably not essential to the survival of the parental T5 phage DNA, and T5 phage DNA, especially the denatured form, is highly sensitive to some nucleases in E. coli spheroplasts.     

The nucleotide sequences at the termini of adenovirus-2 DNA.

The nucleotide sequence of the first 156 residues from the left end and the first 134 residues from the right end of adenovirus-2 DNA have been determined by direct DNA sequencing techniques. The inverted terminal repetition is 102 nucleotide pairs long. The 5′-ends of the intact DNA are resistant to the action of T4 polynucleotide kinase and the 5′ → 3′ exonucleases from phages lambda and T7. This resistance is most likely due to the covalent attachment of the 5′-terminal C residue to the terminal protein. No significant self-complementarity exists within the inverted terminal repetition, making terminal initiation of DNA replication via a self-priming mechanism unlikely. However, the terminal A + T-rich region followed immediately by a very G + C-rich region is consistent with other schemes for adenovirus-2 DNA replication. The left end of adenovirus-2 DNA contains extensive sequence repetition.     

Gene assembly based on blunt-ended double-stranded DNA-modules

Gene synthesis of leucine zipper genes based on blunt end ligation of ds DNA modules was performed in several ligation steps on magnetic beads. A 3'-phosphate, which was removed by T4 polynucleotide kinase after each ligation step, was used to guarantee orientation and single addition of newly added modules. Not depending on sequence complementarity the method appears well suited for the generation of combinatorial libraries of heterologous molecules.     

The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.     

Joining of simian virus 40 DNA molecules at endonuclease R Eco Ri sites by polynucleotide ligase and analysis of the products by agarose gel electrophoresis.

DNA molecules cut with endonuclease R Eco Ri can be joined at Eco Ri cleavage sites by incubation with polynucleotide ligase. In order to define the optimum conditions for this reaction, linear Simian Virus 40 DNA molecules (SV40(Lri)) produced by endonuclease R Eco Ri cleavage of SV40 form i DNA were joined using polynucleotide ligases specified by bacteriophage T4 and Escherichia coli. We have determined that the concentration of the substrate DNA molecules is the most important factor determining the distribution of covalently joined product molecules into a variety of circular and linear monomeric and oligomeric species.     

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.     

Purification and electrophoretic assay of T4-induced polynucleotide ligase for the in vitro construction of recombinant DNA molecules

A facile method for the determination of bacteriophage T4-induced polynucleotide ligase joining activity is described. The assay is based on the ability of polynucleotide ligase to join the cohesive termini of bacteriophage λ DNA covalently. The observance of this activity is greatly facilitated if λ DNA is previously cleaved with the restriction endonuclease EcoRI and the reaction products subsequently analyzed by electrophoresis in ethidium bromide-agarose gel. A purification scheme is presented which offers a reduction in the number of steps required to purify polynucleotide ligase compared to a previously published procedure and yields an enzyme preparation which is suitable for use in in vitro construction of recombinant DNA molecules.     

In vitro 'sexual' evolution through the PCR-based staggered extension process (StEP)

This protocol describes a directed evolution method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) is essentially a modified PCR that uses highly abbreviated annealing and extension steps to generate staggered DNA fragments and promote crossover events along the full length of the template sequence(s). The resulting library of chimeric polynucleotide sequence(s) is subjected to subsequent high-throughput functional analysis. The recombination efficiency of the StEP method is comparable to that of the most widely used in vitro DNA recombination method, DNA shuffling. However, the StEP method does not require DNA fragmentation and can be carried out in a single tube. This protocol can be completed in 4-6 h.     

Determination of the sequences of 18 nucleotides from the 5''-end of the 1-strand and 15 nucleotides from the 5''-end of the r-strand of T7 DNA.

The sequences of 18 nucleotides from the 5'-end of the 1-strand and 15 nucleotides from the 5'-end of the r-strand of T7 bacteriophage DNA have been determined to be pT-C-T-C-A-C-A-G-T-G-T-A-C-G-T-C-C-C (1-strand) and pA-G-G-G-A-C-A-C-A-G-C-G-C-T-C (r-strand). The 5'-termini of whole DNA or separated strands were kinased using polynucleotide kinase and (gamma-32-P) rATP. The DNA was partially digested with pancreatic DNase and the fragments were separated by two dimensional electrophoresis and homochromatography. To complete the sequence, snake venom phosphodiesterase digestions of these fragments were carried out. The relationship of these sequences to the proposed cleavage of concatemeric DNA during DNA replication is discussed.     

Genetic and physiological studies of an Escherichia coli locus that restricts polynucleotide kinase- and RNA ligase-deficient mutants of bacteriophage T4.

The RNA ligase and polynucleotide kinase of bacteriophage T4 are nonessential enzymes in most laboratory Escherichia coli strains. However, T4 mutants which do not induce the enzymes are severely restricted in E. coli CTr5X, a strain derived from a clinical E. coli isolate. We have mapped the restricting locus in E. coli CTr5X and have transduced it into other E. coli strains. The restrictive locus seems to be a gene, or genes, unique to CTr5X or to be an altered form of a nonessential gene, since deleting the locus seems to cause loss of the phenotypes. In addition to restricting RNA ligase- and polynucleotide kinase-deficient T4, the locus also restricts bacteriophages lambda and T4 with cytosine DNA. When lambda or T4 with cytosine DNA infect strains with the prr locus, the phage DNA is injected, but phage genes are not expressed and the host cells survive. These phenotypes are unlike anything yet described for a phage-host interaction.     

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.     

The Arrangements of Nucleotide Sequences in T2 and T5 Bacteriophage DNA Molecules

The genetic map of T4 (and T2) bacteriophage is circular but the DNA molecule that is liberated by phenol extraction is a linear duplex of polynucleotide chains. If the genetic map is related to the physical structure of the DNA molecule, the problem arises as to how a linear molecule can give rise to a circular map. An explanation can be made on the basis that the bacteriophage liberate molecules which have nucleotide sequences which are circular permutations of each other. Thus, markers which are most distant on one molecules are closest together on another. To test this hypothesis, the middles of T2 and T5 DNA molecules were mechanically deleted and the absence of certain nucleotide sequences was tested by “renaturation” or “reannealing” experiments using columns containing denatured DNA immobilized in agar beads. The results indicate that when the middles are deleted from the T5 DNA molecule, some special sequences are removed; whereas, when the middles are deleted from the T2 DNA molecule, no special group of sequences is removed. This would indicate that T2 molecules begin at different points in their nucleotide sequence, while T5 molecules all begin at the same point. It is likely that this permutation of sequences of T2(T4) molecules is related to the circularity of their genetic map.