Rapid and efficient site-specific mutagenesis without phenotypic selection.
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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↗
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↗