Investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system.

• In the past few years, it has become apparent that Archaea represent a considerable and important proportion of the biomass, with representatives being found in a wide range of environments. The hyperthermophilic Archaea are of particular interest because the inherent thermostability of their proteins has industrial relevance, and analysis of this property is likely to lead to important insights into our understanding of protein structure.

• Due to the fact that the DNA of hyperthermophilic Archaea ranges from felaxed to positively supercoiled under normal growth conditions, more study into this area will help determine if this regulation is an adaptation to living at high temperatures.

• Perform In vitro transcription reactions by using 300fmol of TBP and TFB and 1 pmol of RNAP on 40 fmol of plasmid template, prepare Sulfolobus shibatae extract, and detect transcription products by primer extension using radiolabled T& sequencing primer.

• Incubate negatively supercoiled plasmid at 75 degrees C for 35 min with 1,750, 3,500, or 14,000 units of reverse gyrase purified from S. shibate in a 40-ul reaction mixture, then add preheated NaCl and continue incuabation for 2 min. Extract DNA 3 times with 1 vol of chloroform/isoamyl alcohol and precipitated with ethanol.

• Incubate negatively supercoliled plasmid (10 ug) for 90 min with calf thymus topoisomerase I in 200-ul reaction mixtures to generate pools of incuresing negative superhelicity. Stop reactions by adding lithium dodecyl sulfate and remove ethidium bromide by two extractions with butanol saturated with Tris-EDTA. Recover DNA again

• Resolve positively supercoiled topoisomers by electrophoresis in the first and second dimensions, and resolve negatively supercoiled topoisomers by electrophoresis in TEP buffer. Before staining, treat gels with .25 M HCl for 30 min.

• Assemble reactions in a total volume of 40 ul by suing 20 mg of template with nucleoside triphosphates, incubate for 10 min at 48 degrees C before the addition of DnaseI or KmnO4, add DnaseI and perform footprinting, and then recover DNA by phenol/chloroform extraction followed by salt exchange on a G50 gel filtration column.

• Assemble standard transcription reactions, and after 7 min of incubation, add heparin to 100 ug/ml. After 30 sec, add UPT and elongation was allowed to proceed for and additional 5 min. Recover and analyze RNA.

Primrose, S.B. Principles of Genome Analysis, a guide to mapping and sequencing DNA from different organisms. Blackwell Science, 1995.