Nucleotide sequence and expression of the cloned gene of bacteriophage SP6 RNA polymerase.

The coding region of the gene for bacteriophage SP6 RNA polymerase was cloned into pBR322, and its entire nucleotide sequence was deduced. The predicted amino acid sequence for the polymerase consists of 874 amino acid residues with a total molecular weight of 98,561 daltons. Comparison of the amino acid sequence with that of T7 RNA polymerase reveals that regions with partial homology are present along the sequence. The coding region of SP6 RNA polymerase was inserted into an E. coli expression vector. The polymerase gene was efficiently expressed in E. coli cells, and the enzymatic properties of the expressed polymerase were very similar to those of the enzyme synthesized in SP6 phage-infected Salmonella typhimurium cells.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

Characterization of a human ''midisatellite'' sequence.

We have examined the structure and DNA sequence of a human genomic locus that consists of a large hypervariable region made up of repeats of a simple sequence. With several restriction enzymes, the locus shows many restriction fragments that vary quantitatively as well as qualitatively. Other restriction enzymes produce only a single, high-molecular-weight fragment at this locus. Almost all of the fragments are revealed with a simple sequence probe. Southern transfers of the high-molecular-weight restriction fragments produced by the restriction enzymes NotI and SfiI, resolved by pulsed-field gel electrophoresis, gave at most two fragments, demonstrated to be allelic, showing that the majority of the restriction fragments seen in the complex patterns are at a single locus. The estimated size of the region homologous to the probe varied from 250 to 500 kilobases. DNA sequencing indicated that the region consists of tandem repeats of a 40-base-pair sequence. Some homology was detected to the tandem repeating units of the insulin gene and the zetaglobin pseudogene hypervariable regions, and to the "minisatellite" DNA at the myoglobin locus.