Rapid and efficient site-specific mutagenesis without phenotypic selection.
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Coliphages↗
Biomedical subjects
R A Zakour
Publications and source records attributed to R A Zakour.
Introduction, rescue and expression of plasmid genes in mammalian cells and Escherichia coli.
A shuttle-vector system is described for the study of mutational specificity in mammalian cells. Using a plasmid (pGKTK) carrying the E. coli galactokinase gene (gk) and the herpes simplex virus thymidine kinase gene (tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK- mouse fibroblasts) and its efficient rescue back into E. coli. This system makes use of two genes, each of which can expressed in both E. coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host. In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing. In the case of a putative single-copy transformant (T8), about half of the rescued plasmids are identical in size and restriction pattern to the original plasmid. Each of these expressed the tk gene, indicating the fidelity of the rescue system.
Animals↗
Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization.
We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.
Base Sequence↗
Metal-induced mutagenesis in the lacI gene of Escherichia coli.
Mutagenesis in the lacI gene of Escherichia coli has been examined in cells grown in the presence of beryllium, manganese or chromium compounds, metals with suspected mutagenic or carcinogenic potential. 2--3-fold increases in mutation frequency were produced by BeCl2, MnCl2 and K2Cr2O7. Among the cells grown in the presence of Be2+, the frequency of amber and ochre mutants was 3-fold higher than the spontaneous background, suggesting that at least part of the increased mutagenicity was due to base-substitution mutations. The specificity of base-substitution mutations induced by Be2+ and Mn2+ in the lacI gene was analyzed. Among the amber mutations induced in cells grown in the presence of Be2+, an increase in G:C----A:T transitions was detected. In contrast, following growth in Mn2+, no increase in amber and ochre mutation frequencies was observed, and the mutational spectrum resembled that obtained spontaneously indicating that mutations induced by Mn2+ in the lacI gene involve changes that do not yield nonsense mutations. These results suggest that metals may exert a number of different mutagenic effects and that these effects vary for each metal.
Beryllium↗
Site-specific mutagenesis by error-directed DNA synthesis.
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Avian Myeloblastosis Virus↗
Metal-induced infidelity of DNA synthesis.
A number of metals have been demonstrated to be mutagens in procaryotic and eucaryotic organisms as well as carcinogens in experimental animals. Epidemiologic studies have indicated that Ni, Cr, and As are involved in human carcinogenesis. We have hypothesized that the active molecular species is the cation and that metal induced mutations result from incorrect base-substitutions during DNA replication. This is supported by the observations that metal ions diminish the fidelity of DNA synthesis in vitro using a variety of DNA polymerases. There is a significant correlation between the metals that decrease fidelity and those that have been reported to be mutagenic and carcinogenic. Thus, metal carcinogens are no exception to the general postulate that carcinogens can be identified by their effects on DNA.
Animals↗
Metal-induced infidelity of DNA synthesis.
In this paper, we consider the effects of metal ions on the accuracy of catalysis by DNA polymerases. Certain activating and nonactivating metal ions have been shown to diminish the fidelity of DNA synthesis in vitro with a variety of DNA polymerases. There is a significant correlation between the metals that decrease fidelity and those that have been reported to be mutagenic and carcinogenic. Thus, metal carcinogens are no exception to the general postulate that carcinogens can be identified by their interactions with DNA.
Animals↗
Effects of arsenic, selenium, and chromium on the fidelity of DNA synthesis.
The effect of three environmentally important metals, arsenic, selenium, and chromium, on the accuracy of DNA synthesis in vitro has been analyzed. The addition of arsenic to fidelity assays did not significantly alter accuracy. Selenium did not alter fidelity under normal conditions of magnesium activation, nor did it affect the mutagenicity of manganese. Chromium in the form of Cr(III) as well as Cr(VI) diminished the fidelity by which Escherichia coli DNA polymerase I copies polynucleotide templates. Nearest-neighbor analysis of the product synthesized in the presence of chromium indicates that the misincorporated in the presence of chromium indicates that the misincorporated bases are present as single-base substitutions. Chromium was also mutagenic using the recently developed phi chi 174 assay, which measures the fidelity of DNA synthesis with a natural DNA template.
Arsenic↗
Evoluation of Drosophila mitochondrial DNAs. Analysis of heteroduplex molecules.
We have mapped the single block of non-homologous sequences and measured the extent and distribution of base-pair substitutions within the homologous sequences in Drosophila melanogaster: Drosophila virilis heteroduplex mitochondrial DNAs (mtDNAs). Of the 4.8 kilobases long, unusually (A + T)-rich region in D. melanogaster mtDNA, only 0.5 kilobases can react with related, but not identical sequences in D. virilis mtDNA, while the rest (4.3 kilobases in the long arm of a heteroduplex loop) is replaced by a shorter, non-homologous region (1.0 kilobases in the short arm of the loop). No additional heterologous regions are evident. Homologous sequences have accumulated on the average 15.5% base-pair changes. Regionally, these substitutions are relatively uniformly distributed (14.5--16.5%) except for a single, more conserved region (10--13%), which presumably represents the ribosomal cistrons. The lack of general sequence stability suggests that the invariant topographic organization of the nucleotide sequence, previously recognized among Drosophila mtDNAs, is under more stringent selection than the sequence per se.
Animals↗
Evolution of Drosophila mitochondrial DNAs. Comparison of denaturation maps.
In an approach to the functional anatomy of the mitochondrial genome and its evolution, we have compared buoyant densities, contour lengths, and denaturation maps in circular mitochondrial DNAs of the genus Drosophila. Mitochondrial DNAs from three representatives of the subgenus Drosophila (D. virilis, D. hydei, D. funebris) are similar in size (approx. 5 mum or 1 - 10(7) daltons) and buoyant density (approx. 1.685 g/ml), while in two members of the subgenus Sophophora (D. melanogaster, D. simulans), mitochondrial DNAs are longer (approx. 6 mum or 12.4 - 10(6) daltons) and have a lower buoyant density (approx. 1.681 g/ml). The latter mitochondrial DNAs also share one distinctly large early melting region, which in D. melanogaster is equivalent to 1.54 mum of native DNA. The corresponding (A + T)-rich region in D. virilis or D. hydei mitochondrial DNA is 1 mum shorter. Except for this region, denaturation maps of D. melanogaster and D. virilis mitochondrial DNAs are indistinguishable. The addition or deletion of a single block of (A + T)-rich sequences can fully account for the differences in buoyant density and size between the mitochondrial DNAs we have examined. In an appendix, we show that there is an equivalent discrepancy between the extent of strand separation determined by electron by electron microscopy and the actual extent of DNA denaturation, whether this is determined from absorbance changes or inferred from the reduction in contour lengths of individual circular molecules. The reduction in contour length appears to result exclusively from the uniform foreshortening of single-stranded DNA, not only in regions of visible strand separation but also in denatured regions hidden within putatively native segments of molecules. For molecules showing 15--45% strand separation, we estimate that putatively native segments are approximately 50% denatured.
Animals↗