Beta -Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

Previous work showed that human beta-globin mRNAs harboring a premature termination codon are degraded in the erythroid tissues of mice to products that lack sequences from the mRNA 5' end but contain a 5' cap-like structure. Whether these decay products are the consequence of endonucleolytic or 5'-to-3' exonucleolytic activity is unclear. We report that this beta-globin mRNA decay pathway is recapitulated in cultured mouse erythroleukemia (MEL) cells and targets nonsense-free mRNA to a lesser extent than nonsense-containing mRNA.

Amplified primer extension assay for psoralen photoproducts provides a sensitive assay for a (CG)6TA(CG)2(TG)8 Z-DNA torsionally tuned probe: preferential psoralen photobinding to one strand of a B-Z junction.

An amplified primer extension assay has been developed for quantitatively mapping the sites of psoralen photoaddition to DNA. This assay was applied to a torsionally tuned Z-DNA-probe that was specifically designed for the primer extension assay. The torsionally tuned Z-DNA forming sequence, (CG)6TA(CG)2(TG)8, forms Z-DNA in vitro at negative superhelical density: sigma = -0.

Torsionally tuned cruciform and Z-DNA probes for measuring unrestrained supercoiling at specific sites in DNA of living cells.

We describe the development and application of "torsionally tuned" Z-DNA and cruciform probes for analyzing the level of unrestrained supercoiling at specific sites in the DNA of living cells. This approach is applicable for the analysis of dynamic differences in supercoiled DNA in different parts of plasmid, bacterial, or eukaryotic chromosomes. Using a psoralen-based assay, we have shown that the Z-DNA forming sequence (CG)6TA(CG)6, cloned into plasmid pUC8, exists as Z-DNA in 30 to 40% of plasmid molecules in wild-type Escherichia coli.

Topoisomerase I is the predominant nuclear protein from avian erythrocytes that can be covalently linked to DNA.

Nuclear extracts of erythrocytes contain proteins which stably or possibly covalently bind to DNA. These proteins can be detected by an assay which was originally developed to quantify stable binding of topoisomerases to DNA [Trask, DiDonato & Muller (1984) EMBO J. 3, 671-676].