A sensitive denaturing gradient-Gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region.

A population study of heteroplasmy in the hypervariable region 1 (HV1) portion of the human mtDNA control region was performed. Blood samples from 253 randomly chosen individuals were examined using a sensitive denaturing gradient-gel electrophoresis (DGGE) system. This method is capable of detecting heteroplasmic proportions as low as 1% and virtually all heteroplasmy where the minor component is > or = 5%. Heteroplasmy was observed in 35 individuals (13.8%; 95% confidence interval [CI] 9.6-18.0). Of these individuals, 33 were heteroplasmic at one nucleotide position, whereas 2 were heteroplasmic at two different positions (a condition known as "triplasmy"). Although heteroplasmy occurred at a total of 16 different positions throughout HV1, it was most frequently observed at positions 16093 (n=13) and 16129 (n=6). In addition, the majority of heteroplasmic variants occurred at low proportions and could not be detected by direct sequencing of PCR products. This study indicates that low-level heteroplasmy in HV1 is relatively common and that it occurs at a broad spectrum of sites. Our results corroborate those of other recent reports indicating that heteroplasmy in the control region is more common than was previously believed-a finding that is of potential importance to evolutionary studies and forensic applications that are based on mtDNA variation.

Comparative identity and homogeneity testing of the mtDNA HV1 region using denaturing gradient gel electrophoresis.

A denaturing gradient gel electrophoresis (DGGE) assay has been developed for comparative identity and homogeneity testing of the mtDNA HV1 region. A total of 49 pairs of sequences, each pair differing by a single unique polymorphism, were tested to verify the reliability of the assay. Discrimination between all pairings was achieved as judged by the resolution of the mismatch-containing heteroduplexes from the fully base-paired homoduplexes. In all but two pairings, resolution of the fully base-paired homoduplexes was also obtained. Sequence pairs differing by multiple polymorphisms were also tested and resulted in a greater separation between the homo- and heteroduplexes. Additional information derived from the technique includes the identification of co-amplifying contaminating or heteroplasmic samples in the independent samples lanes. Thirteen heteroplasmic samples, six at positions distinct from those analyzed in the pairwise comparison study, were analyzed and the heteroplasmic positions identified unambiguously by sequencing the excised bands. The technique constitutes a conceptually simple, accurate, and inexpensive test for determining whether two sequences match within the mtDNA HV1 region, while providing a more definitive control for the identification of co-amplifying contaminating or heteroplasmic sequences than is presently available.

Bleomycin-induced DNA lesions at mutational hot spots: implications for the mechanism of double-strand cleavage.

Using various end-labeled, defined-sequence DNA substrates, we examined bleomycin-induced damage at several G.C base pairs which correspond to mutational hot spots. The most frequent lesions detected were single-strand breaks and single apurinic/apyrimidinic (AP) sites at the C residue, suggesting that this was the primary site of damage. Strand breaks and AP sites also occurred, but less frequently, at a secondary damage site--i.e., the directly opposed G residue in the complementary strand. However, damage at the secondary site occurred only when a strand break was present at the primary site, and AP sites at the primary site were never accompanied by closely opposed damage in the complementary strand. Thus, formation of a strand break at the primary damage site was a necessary though not sufficient condition for attack at the secondary site. Similar patterns were seen at other sequences attacked by bleomycin, although primary and secondary sites were sometimes staggered by one nucleotide position rather than directly opposed. These and other results suggest a mechanism of double-strand cleavage in which bleomycin is reactivated during formation of the first strand break, and the reactivated drug subsequently attacks the complementary strand at a specific position which is not normally a site of bleomycin-induced cleavage. Regeneration of activated bleomycin could result from a reaction between Fe(III).bleomycin and a 4'-peroxyl derivative of deoxyribose, both produced during formation of the strand break.

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage.

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.

Oxidized apurinic/apyrimidinic sites formed in DNA by oxidative mutagens.

Treatment of DNA with any of several agents, including ionizing radiation, hydrogen peroxide, bleomycin, neocarzinostatin and the copper (I) chelate complex of 1,10-phenanthroline, produces apurinic/apyrimidinic (AP) sites containing oxidized deoxyribose moieties. These AP sites, which are formed by specific or nonspecific free-radical attack on deoxyribose, have been shown to involve oxidation of deoxyribose at the C-1', C-2' or C-4' position. Oxidized AP sites are generally more susceptible to chemical cleavage than normal AP sites, but are in some cases resistant to cleavage by repair AP endonucleases. Nearly all of the AP sites produced by neocarzinostatin, and a fraction of those produced by bleomycin, are accompanied by closely opposed breaks in the complementary strand. Sequence specificity data strongly implicate oxidized AP sites in neocarzinostatin-induced mutagenesis. The role of AP sites in mutagenesis by the other oxidative mutagens is less clear, although there is in some cases suggestive evidence for such a role.

Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions.

In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments resulting from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand was seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5' extensions. Double-strand breaks with 3' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)