In Z-DNA the sequence G-C-G-C is neither methylated by Hha I methyltransferase nor cleaved by Hha I restriction endonuclease.

Plasmids carrying 24- or 32-base-pair inserts of alternating (dG-dC) residues were used to analyze the level of methylation of the G-C-G-C sites by Hha I DNA methyltransferase and their cleavage by Hha I endonuclease in the B-DNA or Z-DNA conformation. In supercoiled plasmids in which the inserts formed Z-DNA, the extent of methylation at the insert G-C-G-C sites was dramatically lower than the level of methylation at the G-C-G-C sites located outside the insert in the same plasmid. Similarly, cleavage by Hha I endonuclease was sharply lowered when the insert was in the Z-DNA form. In the relaxed plasmid, all its G-C-G-C sites were methylated to the same extent and the unmethylated sites were readily cleaved. After treatment with the methylase, the supercoiled plasmid was linearized and then digested with Hha I restriction endonuclease. This exposed unmethylated G-C-G-C sites from the insert that had been protected against cleavage in the Z conformation. A chemical reaction was used to study the distribution of the unmethylated cytosine residues. No accumulation of unmethylated cytosine residues was found anywhere along the entire 32-base-pair insert, which is consistent with a cooperative B-Z transition.     

Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA.

DNA can be chemically cleaved at the site of chloroacetaldehyde-modified residues by the chemicals used for Maxam-Gilbert sequencing reactions. Use of this technique facilitates fine structural analysis of unpaired DNA bases in DNA with non-B-DNA structure. This method was used to study the non-B-DNA structure adopted by the poly-(dG).poly(dC) sequence under torsional stress at various ionic conditions. In the presence of 2 mM Mg2+, the 5' half of the deoxycytosine tract is very reactive to chloroacetaldehyde, while the 3' half is virtually unreactive. In the poly(dG) tract, chloroacetaldehyde reaction is restricted to the center guanine residues. In the absence of Mg2+, however, it is the 5' half of the deoxyguanine tract that is reactive to chloroacetaldehyde, while the 3' half is unreactive. And chloroacetaldehyde reaction is restricted to the center cytosine residues in the poly(dC) stretch. These results strongly suggest that the poly(dG).poly(dC) sequence is folded into halves from the center of the sequence to form a tetra-stranded-like structure. Such a structure contains either a triplex consisting of poly(dG).poly(dG).poly(dC) strands in the presence of Mg2+ or a triplex consisting of poly(dC).poly(dG).poly(dC) strands in the absence of Mg2+. The fourth strand, not involved in triplex formation, is closely associated with the triplex and is positioned in such a way that DNA bases are exposed and freely accessible to the chloroacetaldehyde reaction.     

Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA.

Purine residues located within regions of DNA that have the potential to form left-handed Z-helical structures are modified preferentially by diethyl pyrocarbonate; this hyperreactivity is dependent on the degree of negative superhelicity of the circular DNA molecules. As negative superhelical density increases, guanosines in a 32-base-pair alternating G-C sequence and adenosines (but not guanosines) in a 64-base-pair alternating A-C/G-T sequence become 5- to 10-fold more reactive to diethyl pyrocarbonate. The negative superhelical densities at which enhanced reactivity occurs are similar to those reported for the point at which left-handed helices form within plasmids carrying these DNA sequences. Probing of negatively supercoiled pBR322 with diethyl pyrocarbonate reveals a hyperreactive region 31 base pairs in length of which only 9 base pairs are a perfect alternating purine and pyrimidine sequence; the reactivity of purines within this sequence indicates that purines in the anti conformation, or guanosines in the syn conformation with neighboring 3' thymidines, are not hyperreactive in the Z-DNA form.     

In vitro replication of adeno-associated virus DNA.

An in vitro assay for adeno-associated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the plasmid by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I digestion. The following results were obtained. (i) Only extracts of cells coinfected with AAV and adenovirus replicated the excised insert. (ii) Density label experiments showed semiconservative replication. (iii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.     

Ph-negative chronic myeloid leukemia: molecular analysis of ABL insertion into M-BCR on chromosome 22

Leukemic cells from a patient with Ph-negative chronic myeloid leukemia (CML) had a normal karyotype. M-BCR was rearranged and chromosome in situ hybridization showed an ABL insertion between 5' and 3' M-BCR on an apparently normal chromosome 22. The association of 5' BCR and 3' ABL at the 5' junction of the chromosome 9 insert was typical of that found for the BCR-ABL fusion gene in other patients with the standard t(9;22) and CML. With an M-bcr-3' probe, we cloned and characterized a 3' junction fragment. Field inversion gel electrophoresis and chromosome in situ hybridization studies using a probe isolated from genomic DNA 5' of the junction showed that 3' M-BCR was joined to a region of chromosome 9q34 rich in repetitive sequences and lying some distance 3' of ABL. The chromosome 9 insert was at least 329 kilobases long and included 3' ABL and a larger portion of chromosome 9q34. Our results allowed us to exclude transposon- or retroviral-mediated insertion of ABL into chromosome 22. Instead, we favored a two-translocation model in which a second translocation reconstituted a standard t(9;22)(q34;q11) but left the chromosome 9 insert, including 3' ABL, in chromosome 22.     

Isolation of Drosophila proteins that bind selectively to left-handed Z-DNA.

An affinity column for isolating Z-DNA binding proteins was made by attaching brominated poly(dG-dC) to Sephadex. Proteins from Drosophila nuclei were prepared and those that could bind to Escherichia coli B-DNA were removed from the solution. The remaining proteins were passed over the Z-DNA affinity column and then eluted with NaCl. Using both direct and competitive filter binding assays, we found that the eluted proteins bind to brominated poly(dG-dC) (Z-DNA) and poly(dG-m5dC) but not to poly(dG-dC) (B-DNA), native or denatured E. coli or calf thymus DNA, or brominated oligonucleotides. The proteins also bind to negatively supercoiled plasmids carrying Z-DNA sequences but not to relaxed or linearized plasmids in which the Z-DNA conformation is no longer present. Gel analysis reveals a mixture of several large proteins up to approximately 150,000 daltons.     

Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome

The complete 7410 nucleotide sequence of poliovirus type I genome was obtained from cloned cDNA. Double-stranded poliovirus cDNA was synthesized and inserted into the Pst I site of plasmid pBR322, and three clones were derived that together provided DNA copies of the entire poliovirus genome. Two of the clones contained inserts of 2.5 and 6.5 kilobases and represented all but the 5' 115 bases of poliovirus RNA. A third clone was generated from primer-extended DNA and contained sequences from the 5' end of the viral RNA. An open reading frame that was identified in the nucleotide sequence starting 743 bases from the 5' end of the RNA and extending to a termination codon 71 bases from the 3' end contained known poliovirus polypeptide sequence.     

Structure and multiplicity of genes for the human immunoglobulin heavy chain variable region.

Several genes for the variable region of immunoglobulin heavy chains (VH genes) have been isolated from human fetal liver DNA by using a cDNA plasmid probe containing a mouse VH sequence. The detectable VH genes are separated by 12-16 kilobases of DNA, and hybridization experiments show about 23 hybridizing VH genes in DNA of three different individuals. The complete nucleotide sequence of one of these human VH genes shows that it belongs to the human VHIII subgroup. The VH gene appears to contain an intervening sequence (104 bases in length) within a precursor sequence, between residues -4 and -5. The precursor sequence is itself 19 codons in length. The 3' end of the V gene seems to be at codon 93 or 94, and this is followed by the conserved sequences C-A-C-A-G-T-G and G-A-C-A-C-A-A-A-C-C. The presence of these sequences suggests that similar enzymatic mechanisms are involved in the integration of V genes in both heavy and light chains.     

Chicken lysozyme gene contains several intervening sequences.

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.     

Z-DNA binding protein from chicken blood nuclei.

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.     

Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.

The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.     

Site-specific recombination of yeast 2-micron DNA in vitro.

Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two 599-base-pair precise inverted repeats across which a site-specific inversion event occurs in vivo. This inversion is promoted by a plasmid-encoded function called "FLP." We have cloned the FLP gene of 2-micron DNA under control of a strong yeast promoter and transformed yeast cells with a plasmid containing the cloned FLP gene. Cell-free extracts from such a transformant promote highly efficient inversion of 2-micron DNA in vitro. The reaction requires a cation and works efficiently on supercoiled, relaxed circular, or linear DNA. The FLP activity bears certain similarities to the cre protein, a site-specific recombinase encoded by bacteriophage P1.     

Exposure of DNA bases induced by the interaction of DNA and calf thymus DNA helix-destabilizing protein.

The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexes with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone HI at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stopped-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3-fold upon addition of the DNA helix-destabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed.     

Induction of the Z conformation in poly(dG-dC).poly(dG-dC) by binding of N-2-acetylaminofluorene to guanine residues.

Poly(dG-dC).poly(dG-dC) and poly(dG).poly(dC) were modified by treatment with N-acetoxy-N-2-acetylaminofluorene, and their conformations were examined by circular dichroism and susceptibility to nuclease S1 digestion. A sample of poly(dG-dC).poly(dG-dC) modified to an extent of 28% with acetylaminofluorene (AAF) at the C(8) position of the deoxyguanosine residues showed a circular dichroism spectrum that had the characteristics of the Z conformation seen in unmodified poly(dG-dC).poly(dG-dC) at high ethanol or salt concentrations. A sample of poly(dG-dC).poly(dG-dC) modified only 3% by AAF showed a spectrum characteristic of the B form of DNA. However, it was converted to the Z form at ethanol concentrations lower than required to convert unmodified poly(dG-dC).poly(dG-dC) from the B to the Z form. Poly(dG).poly(dC), which does not undergo the B-to-Z transition at high ethanol concentrations, did not show any large conformational changes with high AAF modification. Susceptibility to digestion with nuclease S1 also suggested differences in the conformations of the two modified polynucleotides. Poly(dG-dC).poly(dG-dC) modified by AAF to an extent of 28% was almost completely resistant to nuclease S1 digestion. However, both poly(dG).poly(dC) and DNA modified to similar levels by AAF were highly susceptible to nuclease S1 digestion. Two different conformations for AAF-modified deoxyguanosine are proposed, depending on whether its position is in alternating purine-pyrimidine sequences or in random-sequence DNA.