Topoisomerase II can unlink replicating DNA by precatenane removal.

We have analysed the role of topoisomerase II (topo II) in plasmid DNA replication in Xenopus egg extracts, using specific inhibitors and two-dimensional gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required for plasmid unlinking. Extensive unlinking can occur in the absence of mitosis.

Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos.

Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.

Low temperature enhancement of reporter genes expression directed by human immunodeficiency virus type 1 long terminal repeat.

Bacteria and eukaryotic cells respond to cold stress by inducing and enhancing the synthesis of specific arrays of proteins. We describe here cold-induced enhancement of expression for two reporter genes; luciferase and beta-galactosidase, both under the control of HIV-1 LTR sequences, observed in mouse fibroblasts and human HeLa cells respectively. Increased expression of luciferase in fibroblasts when shifted to 25 degrees C was detectable at 30 degrees C but was not observed following cold shock at 4 degrees C.

Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency.

The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences. By using chemical and enzymatic methods to probe the RNA secondary structure and site-directed mutagenesis, we established the complete secondary structure of the U3A snoRNA precursor. This is the first determination of the complete secondary structure of an RNA spliced in a spliceosome.

Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation.

In Escherichia coli transcription of individual genes generally requires concomitant translation, and thus the decay of mRNAs cannot be studied without the complication of translation. Here we have used T7 RNA polymerase to transcribe in vivo lacZ genes carrying ribosome binding sites of variable efficiency. We show that neither cell viability nor growth rate is affected by the T7-driven transcription of these genes, provided that they are present as single chromosomal copy.