No abstract

No abstract

S1 nuclease mapping of the Psammechinus miliaris embryonic histone mRNAs locates the 5' termini in or adjacent to a short sequence homology (5'pyCATTCpu3') downstream from the putative RNA polymerase II regulatory sequence (5'TATAAATA3') or related sequences. The 3' termini map just after a sequence containing GC-rich hyphenated dyad symmetry, a feature of most known terminator sequences.

Previous work has indicated that at least some of the genetic information required for the expression of sea urchin histone genes is recognized following injection of the gene repeat (h22) into Xenopus oocyte nuclei. The ability to elicit the expression of cloned genes and their sequence-manipulated counterparts is proving invaluable in analysing the molecular details of gene expression. Direct injection of such genes into Xenopus oocyte nuclei remains one of the simplest methods for obtaining such expression and a remarkable degree of transcriptional fidelity has been demonstrated using this system with RNA polymerase III genes, and to a lesser extent with rDNAs transcribed by RNA polymerase I. In the case of polymerase II genes there is ample evidence for coupled transcription-translation, but the degree of transcriptional fidelity involved may, as has recently been shown for the ovalbumin gene, be minimal. However, clearly if the oocyte is to be used to investigate transcriptional regulation of such genes, transcriptional fidelity defined as the production of correct RNA termii, rather than the production of 'functional mRNAs' (ref. 15), must pertain. Here we demonstrate such fidelity in the expression of all five Psammechinus miliaris histone genes comprising a repeat unit. However, we find large quantitative variations in the levels of synthesis of the individual correct termini and hence of the mRNAs. In addition to the mRNAs, species with no detectable counterparts in the sea urchin are generated off the coding strand, as are heterogeneous noncoding species.

A method of isolating circular plasmid DNA from cleared lysates of E. coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics or supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dye-centrifugation technique and plasmid DNA of comparable purity and yield is obtained.