Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, smallsubunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.Recent studies have demonstrated that natural attenuation and bioremediation of metals, radionuclides, and organic contaminants cannot be effectively applied at many sites until we have a better understanding of the physiology, ecology, and phylogeny of microbial communities at contaminated sites (12,38,57). However, the success of many monitored natural attenuation and bioremediation approaches depends largely on our understanding of regulatory mechanisms and cellular responses to different environmental factors affecting the contaminant degradation or metal reduction activity in situ. Microorganisms are often exposed to multiple stress conditions in situ, and the microbial community structure is most likely affected by many different abiotic and biotic variables in a nonlinear fashion (58).

Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system. These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers. In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours. Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration. Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG. In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes. HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor. These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.

Eukaryotic expression vectors designed to produce E. coli Lac repressor protein targeted to the nucleus of mammalian cells were constructed. These constructions carry the lac repressor gene (lacI) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a. When the NLS's were fused to the lacI gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified LacI protein. Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity. When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the LacI protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected. The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacI behind the nine base pair linker, AGC AGC CTG (ser-ser-leu). This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction. The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-terminus of the protein. These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein. This modified lacI gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells.

The recent emergence of inducible expression systems for mammalian cells has greatly facilitated the in vivo analysis of gene function. The ecdysone-inducible expression system is particularly attractive because of (i) extremely low basal expression and high-level induced expression, (ii) the lack of pleiotropic effects caused by the inducer or activator, and (iii) the rapid penetrance and clearance of the inducer. Here, we describe an improved receptor expression vector. The required ecdysone receptor proteins (VgEcR and RXR) are co-expressed from a bicistronic cytomegalovirus (CMV) expression cassette in the vector pERV3. The CMV promoter in this vector can be readily replaced with a cell type-specific promoter of interest. Using the ecdysone analogs, muristerone A or ponasterone A, induction ratios of up to three orders of magnitude were attained in the transient transfection assays and in a cell line stably transformed with both pERV3 and an ecdysone-inducible reporter vector. Fine control of luciferase expression was achieved bv varying both the induction time and inducer concentration. Here, we describe a set of cell lines stably transformed with the vector pERV3, in which the ecdysone receptors are expressed at optimal levels for the high-level induction of gene expression.

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