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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.

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 dosedependent 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 Bam HI cleavage of plasmid DNA isolated from cells cotransformed by two compatible plasmids, one carrying the inducible amh gene, the other M· Bam HI methyltransferase encoding gene. The induction of amh decreased the intracellular pool of AdoMet which M· Bam HII requires as a cofactor. Under these conditions, there is a decrease in DNA methylation. The unmethylated DNA is assayed by Bam HI cleavage. This system will be useful for studying transcription, DNA replication, gene repair and other cellular phenomena affected by methylation.

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.

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.

We have inserted a genomic clone of the human EJ bladder oncogene into a murine retrovirus shuttle vector. Cotransfection of this shuttle vector DNA containing the activated ras oncogene with molecularly cloned Moloney murine leukemia virus into NIH/3T3 cells was able to rescue a replicating and transforming retrovirus. The viral DNA from infected cells was excised by fusion to mouse COP-5 cells and recloned into Escherichia coli as plasmid DNA. Analysis of the recloned plasmids by size, restriction enzyme mapping, and DNA sequence indicated that approximately 5% of the recloned plasmids contained the intronless EJ ras oncogene.

Publisher Summary This chapter discusses the preparation and properties of immobilized sequence specific endonucleases. The type II restriction endonucleases have become valuable reagents for research in molecular biology. This is primarily due to their unique property of cleaving DNAs at a limited number of specific sites. These enzymes have been employed for use in physical DNA mapping, plasmid construction, and gene cloning. Most of these enzymes recognize and cleave DNA at specific sequences, which are usually in the form of a palindrome. There is evidence implying that these endonucleases are membrane bound, therefore, one approach to study them is to determine what effect in-solubilization (that is, mimicking membrane binding) has on their activity. The immobilized enzymes can be packed in the form of a column or rapidly pelleted from solutions by centrifugation. These procedures allow quick removal of restriction endonucleases from reaction mixtures. Such recovered enzymes are reusable several times over, permitting cleavage of large amounts of DNA with relatively few units of enzyme. Furthermore, the chapter describes the catalytic and functional characterization of covalently bound restriction endonucleases.

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.

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.

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.