Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Eukaryotic RNA Polymerases 69

NaC1 appears to release all of the nuclear components required for

specific transcription, including RNA polymerase II, without releasing

tightly bound nuclear proteins that can inhibit specific transcription (40).

5.4. Nuclear Extract (41)

This method uses lysolecithin (a natural membrane lipid--lysophos-

phatidylcholine) to disrupt plasma membranes and requires no deter-

gents or lysis with a Dounce homogenizer. Lysolecithin nuclear extracts

are said to be competent for RNA polymerase II and III transcription,

DNA replication, pre-mRNA splicing, and sequence-specific DNA-

protein binding. Nuclear extracts can be prepared on a small scale ( 107

cells), as well as for preparative purposes by this method.

Lysolecithin is highly toxic and cells lyse within 90 s. An extract

made from HeLa cells at a density of 4-6 x 105/mL provides a suitable

nuclear extract for in vitro transcription analysis. Using HeLa suspen-

sions at 108 cells/mL, 100% lysis can be obtained with a concentration

oflysolecithin at 300-800 gg/mL. In the protocol (41), a concentration

of 400 gg/mL was chosen.

References

1. Slater, R. J. (1987) In vitro transcription, in Techniques in Molecular Biology,
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2. Jacob, S. T. (1973) Mammalian RNA polymerases. Prog. Nucleic Acids Res.
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3. Chambon, P. (1975)Eukaryotic nuclear RNA polymerases. Ann. Rev. Biochem.
   44, 613-638.


4. Roeder, R. G. (1976) Eukaryotic nuclear RNA polymerases, in RNA Polymerase
   (Losick, R. and Chamberlin, M., eds.), Cold Spring Harbor Laboratory, Cold
   Spring Harbor, NY, pp. 285-329.


5. Chamberlin, M. (1982) Bacterial DNA-dependent RNA polymerases, in The
   Enzymes, 3rd ed., vol. 15B (Boyer, P., ed.), Academic, New York, pp. 61-108.


6. Guilfoyle, T. J. (1983) DNA-dependent RNA polymerases of plants and lower
   eukaryotes, in Enzymes of Nucleic Acid Synthesis and Modification, vol. 2 (Jacob,
   S. T., ed.), CRC, Cleveland, OH, pp. 1-41.


7. Beebee, T. J. C. and Butterworth, P. H. W. (1981) Eukaryotic DNA-dependent
   RNA polymerases: An evaluation of their role in the regulation of gene expres-
   sion, in Eukaryotic Gene Regulation, vol. 2 (Kolodny, G. M., ed.), CRC, Cleve-
   land, OH, pp. 1-56.


8. Chandler, D. W. and Gralla, J. (1980) Specific binding and protection of form II
   SV40 deoxyribonucleic acid by ribonucleic acid polymerase II from wheat germ.
   Biochemistry 19, 1604-1612.