Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

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.

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 fluorometric assay for DNA cleavage reactions characterized with BamHI restriction endonuclease.

Fluorescently labeled oligonucleotides and DNA fragments have promise in nucleic acid research with applications that include DNA hybridization, automated DNA sequencing, fluorescence anisotropy, and resonance energy transfer studies. Past concerns with fluorescent-labeled DNA arose from interactions between fluorophores and DNA that result in quenched fluorescence. This quenching phenomenon is most problematic in fluorescence resonance energy transfer studies because quenching of the donor fluorescence could result from either resonance energy transfer or nontransfer effects. In the present study, relief of nontransfer quenching of a 14-mer fluorescein 5-isothiocyanate (FITC)-labeled oligonucleotide containing the BamHI restriction site was characterized with both steady-state and time-resolved fluorescence techniques. The FITC-labeled single strand was best fit by a triexponential decay with lifetimes of 0.5, 2.7, and 4.2 ns. The 4.2-ns component was found to contribute more than 80% of the total steady-state intensity. Upon annealing with an unmodified complementary strand, the contribution from the 4.2-ns component was significantly decreased, resulting in twofold quenching of total fluorescence. We reasoned that this quenching phenomenon should be a reversible process and could be employed to study strand separation processes in molecular biology. Hence, cleavage of the fluorescently labeled substrate was examined using DNAase I and BamHI restriction endonuclease. Our results show that the quenched fluorescence is totally recovered upon cleavage (compared to that of the single strand). The extent of cleavage measured by fluorescence was confirmed by nondenaturing polyacrylamide gel electrophoresis analysis. We believe this fluorescence "dequenching" technique may be used to quantify the kinetics of other DNA strand separation and cleavage processes in molecular biology.

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease.

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens.

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E.coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E.coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.

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.

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.

Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulvar carcinoma.

Human papillomavirus type 6 (HPV-6) DNA was detected in a rapidly growing vulvar verrucous carcinoma and two recurrent tumor samples. The viral DNA (HPV-6vc) was molecularly cloned and found to have a high degree of DNA sequence homology to HPV-6b DNA. Comparison of restriction endonuclease cleavage patterns between HPV-6b and HPV-6vc genomes and DNA sequencing analysis demonstrated an additional 106 bases in the HPV-6vc genome. These additional nucleotides were located in the noncoding region of the viral genome which contains the putative viral DNA replication and early gene transcriptional control elements. Seventy-four of the additional 106 nucleotides were found as one insert in the purine-thymidine-rich region 3' to the end of the L1 open reading frame. This 74-base-pair addition had homology with viral sequences immediately upstream to it and to poly(dG-dT) sequences found in the human genome including the conserved repeated sequences in human DNA (EC1) and in the human cardiac muscle actin gene. Two smaller inserts, 19 and 15 nucleotides, were found upstream from the transcriptional control elements and demonstrate homology with regions of human alpha and gamma interferon genes.

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.

Nucleotide sequence analysis of the long terminal repeat of avian myeloblastosis virus and adjacent host sequences.

The nucleotide sequence of the integrated avian myeloblastosis virus long terminal repeat has been determined. The sequence is 385 base pairs long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a 6-base-pair direct repeat of cell DNA next to the inverted repeat of viral DNA. The long terminal repeat also contains promoter-like sequences, an mRNA capping site, and polyadenylation signals. Several features of this long terminal repeat suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with long terminal repeats of other avian retroviruses indicates that there is a great variation in the 3' unique sequence (U3), whereas the 5' specific sequences (U5) and the R region are highly conserved.

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.

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.

Distribution of proviral sequences in chromatin of embryonic fibroblasts infected by Rous sarcoma virus.

Sheared chromatin prepared from chicken embryo fibroblasts and fibroblasts transformed by exogenous Rous sarcoma virus (Schmidt--Ruppin strain D) was separated by rate sedimentation on glycerol gradients into two components: fast-migrating (heavy chromatin fraction) and slow-migrating (light chromatin fraction). DNAs extracted from these fractions were assayed for proviral sequences by molecular hybridization using DNA complementary to the viral sequences. In uninfected cells, the endogenous complementary sequences were found to be equally distributed between heavy and light fractions. However, the newly integrated exogenous proviral sequences were found mostly in the light chromatin fraction in the transformed cells. Additionally, the light fraction was more sensitive to DNase I digestion and contains more material melting at low temperatures when compared with the heavy fraction. The results show that (i) distribution of endogenous proviral sequences is independent of chromatin conformation, and (ii) most of the newly acquired exogenous sequences are integrated within the host's chromatin fraction that exhibits properties of euchromatin. Because chromatin fragmentation and fractionation is accomplished without digestion with degrading enzymes, the chromatin fractions enriched in exogenous sequences remain intact and thus are suitable for further studies.

Analysis of avian myeloblastosis viral RNA and in vitro synthesis of proviral DNA.

Two virus-specific RNA species of 7.5 and 7.0 kilobases have been identified in avian myeloblastosis virus (AMV) by denaturing gel electrophoresis and blot hybridization analysis, and they were found to be in a 10:1 ratio. The individual RNAs direccted the cell-free synthesis of the 76,000-dalton "gag" protein and the 180,000-dalton "gal-pol" protein, thereby demonstrating 5' sequence homology of approximately 4.9 kilobases between the two species. Synthesis of these two precursor proteins by the AMV genome indicates structural differences between AMV and other avian acute leukemia viruses. The two viral RNAs were transcribed into complete cDNA copies with AMV DNA polymerase. Linear proviruses were found to be 90--100% resistant to S1 nuclease. Analysis of single-stranded transcripts demonstrated two distinct species of 2.6 and 2.3 x 10(6) daltons, and analysis of duplexes formed from the single-stranded transcripts demonstrated species of 5.2 and 4.0 x 10(6) daltons.

DNA topoisomerase from Agrobacterium tumefaciens: purification and catalytic properties.

The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. The enzyme is a single polypeptide of about 100,000 in molecular weight. No apparent separation of the nicking and sealing activities could be obtained in attempts to separate the two activities by a variety of methods, including limited protease digestion, thermal denaturation, and differential inhibition. Monoclonal antibodies obtained from hybridomas likewise did not preferentially inhibit one of the two activities. These results suggest that the two catalytic functions are carried by the same essential residues of the active enzyme site.