DNA polymerase with characteristics of reverse transcriptase purified from human milk.

A DNA polymerase purified from a particulate fraction of human milk has biochemical and biophysical properties similar to those of viral reverse transcriptases. This enzyme is immunologically distinct from cellular DNA polymerases obtained from a variety of human sources.

Nucleotide sequence of the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.

A novel Tn10 tetracycline regulon system controlling expression of the bacteriophage T3 gene encoding S-adenosyl-L-methionine hydrolase.

To study the effects of in vivo DNA methylation, we have developed an inducible system to control the intracellular concentration of S-adenosyl-L-methionine (AdoMet). The product of the bacteriophage T3 AdoMet hydrolase-encoding gene (amh), which degrades AdoMet to L-homoserine and 5'-methylthioadenosine, was employed to lower AdoMet concentrations in vivo. The amh gene was placed downstream from the inducible tetA promoter of the Tn10 tetracycline regulon substituting for most of the tetA gene. Unlike in the original isolates [Hughes et al., J. Bacteriol. 169 (1987) 3625-2632], this promoter allows controlled expression. These constructs are stable and can be induced in a dose-dependent manner. The system is maximally induced 2-3 h after addition of the inducer, autoclaved chlortetracycline (cTc). DNA methylation in vivo was assessed in this model system by BamHI cleavage of plasmid DNA isolated from cells cotransformed by two compatible plasmids, one carrying the inducible amh gene, the other M.BamHII methyltransferase encoding gene. The induction of amh decreased the intracellular pool of AdoMet which M.BamHII requires as a cofactor. Under these conditions, there is a decrease in DNA methylation. The unmethylated DNA is assayed by BamHI cleavage. This system will be useful for studying transcription, DNA replication, gene repair and other cellular phenomena affected by methylation.

A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment.

We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.

Alternative to polyacrylamide gels improves the electrophoretic mobility shift assay.

In this paper we outline a simplified protocol for the electrophoretic mobility shift assay utilizing TreviGel 500, a nontoxic alternative to polyacrylamide. The TreviGel 500 matrix combines the strength and resolution of polyacrylamide with the simplicity and flexibility of agarose in the casting of gels. Therefore, this method provides a simple, rapid and nontoxic alternative to current protocols for the investigation of protein: DNA interactions.

Biospecific fractionation matrices for sequence specific endonucleases.

Fractionation of several type II specific restriction endonucleases was achieved by separation on two novel biospecific matrices. The matrices are pyran, a copolymer of divinyl ether of maleic anhydride, and Cibacron Blue F3GA, a blue dye commonly used for the calibration of molecular sieves. Both compounds are insolubilized by coupling to sepharose through a cyanogen bromide linkage and in their soluble form inhibit the restriction endonucleases which we have tested. These affinity matrices can be used to obtain restriction endonucleases from crude extracts after removal of nucleic acids. They have also proven to have a high capacity when used as subsequent steps in enzyme purification. Their additional advantage is the rapid development time and reusability of columns packed with the two matrices.Images

Sequence-specific endonuclease Bgl I. Modification of lysine and arginine residues of the homogeneous enzyme.

The sequence-specific endonuclease Bgl I from Bacillus globigii (RUB561) has been purified to homogeneity as determined by denaturing polyacrylamide gel analysis. The active form of the enzyme is a single polypeptide with a molecular weight of 32,000. The enzyme requires Mg2+ in the reaction mixture and displays a broad pH and monovalent cation requirement. Bgl I is not sensitive to sulfhydryl reagents but was affected by reagents that modify lysine and arginine residues. When lysine residues were modified by pyridoxal 5'-phosphate, both binding and catalysis were diminished while modification of arginine residues by 2,3-butanedione inhibited the enzyme activity but had no effect on its binding properties.

Sequence-specific endonuclease BamHI: relaxation of sequence recognition.

The effect of glycerol on the specificity of DNA cleavage by the restriction endonuclease BamHI has been examined. In addition to the canonical G decreases from G-A-T-C-C site, BamHI cuts DNA at several sites that we have named noncanonical BamHI.1 sites. The number of BamHI.1 sites in simian virus 40 and pBR322 was determined to be 13 for each DNA. Cutting sites determined by DNA sequence analysis include G decreases from G-A-A-C-C, G decreases from G-C-T-C-C, G decreases from G-G-T-C-C, and G-A-A-T-C-C with the complementary strand sequence assignments of G-G-T-T-C-C, G-G-A-G-C-C, G-G-A-C-C-C, and G-G-A-T-T-C. The relaxation in specificity was related to hydrogen bond acceptor and donor sites in the recognition sequence, in an attempt to generate a model of BamHI recognition of cognate sites in DNA.

Purification and characterization of the sequence-specific endonuclease Bam HI.

The specific endonuclease Bam HI from Bacillus amyloliquefaciens (RUB 500) has been purified to apparent homogeneity. Two active forms of the enzyme corresponding to the dimeric and tetrameric forms have been isolated. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme dissociated into Mr = 22,000 +/- 500 subunits. Bam HI has a broad pH optimum on the alkaline side and requires Mg2+ which can be partially replaced by Mn2+. The enzyme catalysis appears to be governed by a two-step mechanism.

Structural requirements for binding of bovine tRNATrp with avian myeloblastosis virus DNA polymerase.

Avian DNA polymerases use host tRNATrp as the primer for transcription. Bovine tRNATrp has been previously shown to be a biologic substitute for the avian primer. A bovine tRNATrp fragment has been identified as having a high binding affinity for the polymerase. The fragment is assigned to be 67 nucleotides, and contains most of the elements required to maintain the secondary and tertiary structure of tRNATrp.

Sequence-specific BamHI endonuclease. The proposed role of arginine residues in substrate binding and recognition.

Arginyl residues in BamHI endonuclease were examined because of their alleged role in proteins that contain nucleotide- or phosphate-binding sites. Butanedione, an arginine-specific reagent, inhibited the endonuclease in the presence of sodium borate. The inhibition was decreased by preliminary incubation of the enzyme with DNA or competitive inhibitors which were the 5'-phosphoryl deoxydinucleotide subsets of the BamHI recognition sequence. The dinucleotide pdGpdG protected the enzyme most efficiently against the butanedione modification. Dinucleotides that were unrelated to the recognition sequence failed to protect the enzyme from inactivation. These studies indicate that arginine residues may reside in the enzyme's active site and might function in the sequence-specific recognition of the BamHI palindrome.

Sequence-specific BamHI methylase. Purification and characterization.

BamHI methylase has been purified to apparent homogeneity. The isolated form of the enzyme is a single polypeptide with a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Unlike BamHI endonuclease, which is isolated as a dimer and higher aggregates, the methylase has no apparent higher form. The methylase requires S-adenosyl-L-methionine as the methyl-group donor and is inhibited by Mg2+. The enzyme is also inhibited by 2,3-butanedione and reagents specific for sulfhydryl groups, such as N-ethylmaleimide, which suggests a role for arginine and cysteine residues, respectively. DNA efficiently protects the enzyme against the butanedione modification while S-adenosylmethionine has no effect. In contrast, S-adenosylmethionine protects against cysteine modification while DNA produces only small amounts of protection. Studies on the mechanism of methylation indicate that both strands of the recognition sequence are modified in a single binding event. The sequence specificity of the methylase is relaxed upon the addition of glycerol in the reaction mixture. In the presence of 30% glycerol the enzyme methylates sequences that are also recognized by BamHI endonuclease when acting under conditions of relaxed specificity.

Differences in the kinetic properties of BamHI endonuclease and methylase with linear DNA substrates.

BamHI endonuclease and methylase were found to exhibit a kinetic preference for linear pBR322 DNA substrates containing the recognition site in a central location. The Km values for substrates having the recognition site in a terminal location were approximately 3-fold greater than those with a centrally located site. This phenomenon may be partially due to facilitated transfer of the enzymes to the recognition site over nonspecific flanking sequences. The exploitation of facilitated transfer by these enzymes has been inferred from studies demonstrating kinetic preferences for longer DNA substrates. The reaction rates of the endonuclease were 9-fold greater with full-length pBR322 DNA than with a 74-base pair derivative. The methylase exhibits a kinetic preference for longer substrates but only under conditions of comparatively higher DNA concentrations. In addition, the methylase has the property of increasing long chain preference with increasing salt concentrations up to 120 mM. Increasing salt concentrations decreased the endonuclease's preference for longer substrates. Nonspecific inhibition studies revealed qualitative and quantitative differences between the two enzymes under catalytic conditions. These studies suggest that BamHI endonuclease and methylase interact with nonspecific DNA in different ways.

Affinity chromatography of viral DNA polymerases on pyran-sepharose.

Pyran covalently linked to cyanogen bromide-activated Sepharose has been shown to be an effective affinity matrix for several viral DNA polymerases. Differential salt elution of viral compared with cellular polymerases, as well as substrate elution, suggests the affinity nature for the matrix. Unlike some other affinity systems described, pyran-Sepharose is totally resistant to nuclease digestion and is stable at 4 degrees for several months. DNA polymerases isolated from several viruses by detergent treatment were recovered in good yield. Analysis of iodinated proteins by sodium dodecyl sulfate-gel electrophoresis revealed that the DNA polymerase of avian myeloblastosis virus found in crude preparations of the virus could be purified nearly to homogeneity by a single passage through the column. These results suggest that pyran-Sepharose is an effective affinity column that is potentially adaptable as part of a general purification procedure for viral DNA polymerases.

DNA endonucleases associated with the avian myeloblastosis virus DNA polymerase.

A DNA endonuclease, Endo-I, which cleaves superhelical DNAs, has been isolated from avian myeloblastosis virions stripped of their coats by mild detergent treatment. The enzyme has a broad pH optimum around 7.5-8.0 and requires Mg2+ for activity. A second endonuclease, Endo-II, with a requirement for Mn2+, also present in viral cores, copurified with avian myeloblastosis virus alpha beta DNA polymerase (reverse transcriptase, RNA-dependent DNA nucleotidyltransferase) and similarly cleaved superhelical DNAs. Heat denaturation and sodium fluoride and N-ethylmaleimide inhibition studies were carried out to demonstrate a possible relationship between the two endonucleases and the viral DNA polymerase and RNase H activities. It appears that Endo-II may be an intrinsic activity of the polymerase.

Isolation of a single polypeptide leucyl-tRNA synthetase from bakers' yeast.

A single polypeptide of leucyl-tRNA synthetase (LRS) has been purified from budding bakers' yeast by a modification of the procedure published earlier. On denaturing polyacrylamide gel electrophoresis LRS was one band corresponding to molecular weight of 120,000 +/- 5,000 daltons. Variable amounts of LRS with a similar molecular weight but which dissociated into equal subunits of 58,000 were also isolated. The affinities (KM) for substrates for this form of the enzyme were similar to those previously reported for the dimeric form of the enzyme.