The ability of the polymerase chain reaction (PCR) to quantitate expression of mRNA was examined in the present study. The model chosen was expression of the multidrug resistance gene mdr-1/Pgp in two colon carcinoma cell lines which express mdr-1/Pgp at levels comparable to those found in many clinical samples. PCR was utilized to evaluate differences in mdr-1/Pgp expression in the two cell lines after modulation by sodium butyrate. Thus, comparisons were made across a range of mdr-1/Pgp expression as well as comparisons of small differences. The PCR was found to be both sensitive and quantitative. Accurate quantitation requires demonstration of an exponential range which varies among samples. The exponential range can be determined by carrying out the PCR for a fixed number of cycles on serial dilutions of the RNA reverse transcription product, or by performing the reaction with a varying number of cycles on a fixed quantity of cDNA. By quantitation of the difference in PCR product derived from a given amount of RNA from the sodium butyrate treated and untreated cells, the difference in mRNA expression between samples can be determined. Normalization of the results can be achieved by independent amplification of a control gene, such as beta 2-microglobulin, when the latter is also evaluated in the exponential range. Simultaneous amplification of the control and target genes results in lower levels of PCR products due to competition, which varies from sample to sample. The PCR is thus a labor-intensive but sensitive method of quantitating gene expression in small samples of RNA.
Cellular DNA in bacteria is localized into nucleoids enclosed by cytoplasm. The forces which cause condensation of the DNA into nucleoids are poorly understood. We suggest that direct and indirect macromolecular crowding forces from the surrounding cytoplasm are critical factors for nucleoid condensation, and that within a bacterial cell these crowding forces are always present at such high levels that the DNA is maintained in a condensed state. The DNA affected includes not only the preexisting genomic DNA but also DNA that is newly introduced by viral infection, replication or other means.Key words: Cytoplasmic macromolecule; DNA condensation; Escherichia coli; Macromolecular crowding; Mandatory condensation; Nucleoid DNA condensation in bacteria: general considerationsThe genomic DNA of bacteria is condensed into one or a few nucleoids per cell [1] that are in direct contact with a surrounding cytoplasm containing very high concentrations of macromolecules (e.g. ,~ 340 mg/ml of total RNA and protein [2]). The term 'condensation' is used to indicate adoption of a relatively concentrated, compact state occupying a fraction of the volume available. The DNA of the bacterial nucleoid is estimated to have a local concentration of ,'-50-100 mg DNA/ml (see footnote 6 of [3]) and to occupy 1/8 to 1/5 of the volume within the cell envelope [1].Condensation of DNA within bacterial ceils has been observed by light microscopy of both living and fixed cells, as well as by electron microscopy [4][5][6][7][8] (reviewed in [9,10]). Condensation of DNA in isolated nucleoids is indicated by microscopy as well as by hydrodynamic properties [11][12][13]. Condensation of DNA in model in vitro systems has been assayed by aggregation, as described below. Condensing forcesThe origins and magnitudes of the forces which cause DNA condensation are poorly understood. Supercoiling, macromolecular crowding and the binding of histone-like proteins and of polyamines have all been suggested to contribute to DNA condensation. It is unclear how important each of these fac-*Corresponding author: Fax: (1) (301) 496-0201.0014-5793196/$12.00 © 1996 Federation of European Biochemical Societies. PH SO0 1 4-5793(96)00725-9 tors is in causing DNA condensation in vivo. Model studies of the binding of histone-like proteins under dilute solution conditions indicate that the cellular amounts of DNA-binding proteins in bacteria are 5-10-fold lower than those required for condensation (see [14] for references and discussion). The polyamines putrescine and spermidine can occur in large amounts in E. coli [15]. Spermidine but not putrescine can condense DNA from aqueous solution [16], although the binding reaction is relatively salt-sensitive [17,18] and so may make a restricted contribution over much of the range of intracellular salt concentrations observed in growing cells [19,20]. Enzymatic supercoiling directly yields more compact forms [21].Macromolecular crowding ('crowding', reviewed in [22,23]) can cause DNA condensation by two distinctly dif...
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