**2.2 Why is this important in regulation of gene expression?**

We know that both the rate of a reaction, as well as the equilibrium that a reversible reaction establishes, are related inversely and logarithmically to the activation energy or the free energy difference between the reactants and products, respectively. It implies that small changes in the binding affinities of DNA-binding regulatory proteins, important for regulation of gene expression, can profoundly affect the rates and equilibrium established in the cell. Also, gene regulatory proteins in the cell do not exist as monomeric units, but as components of multiple different protein complexes with different physical dimensions destined for a variety of purposes in the cell. Thus it can be argued that the same protein in a multimeric complex might not be able to play identical gene-regulatory roles if the sequences flanking its binding site are altered compared to when they exist in their chromosomal context. It is conceivable that some of this disparity in DNA-binding affinities resulting from binding to sites with altered flanking sequences could eventually manifest itself in altered tissue specificity of expression of the gene.

There is also the issue of how one goes about selecting potential gene-regulatory elements to stitch together in a small plasmid designed for expressing a gene of interest. While one can make informed guesses one cannot be unbiased, as much of the players involved in the finetuning of gene expression remain largely unknown. Using BACs functionalized with suitable reporter genes, and integrated into the chromosome, to explore regulation of genes by *cis*-acting elements appears to circumvent many of these difficulties.

We have developed one such approach using BACs retrofitted with enhancer-traps (14). The overall effect of using this enhancer-trap technology in BACs is somewhat akin to scanning the BAC DNA from one end to the other with a "mine-sweeper" that is capable of unearthing transcription-enhancing factor binding sites. Functional comparisons between individual enhancer-trap BACs are thus more meaningful as the modifications in each remain constant. It is important to note that this approach to identifying gene-regulatory elements is likely to be less biased than a targeted approach, because there is no prior selection of sequences for testing their gene-regulatory potential (14, 15). In situations where regulatory function is conserved without sequence similarity (43), choosing the correct sequence to test might be a hit-or-miss phenomenon with a targeted approach. Thus using enhancer-traps to scan BACs offers an effective and relatively unbiased alternative to other targeted approaches to functionally map *cis*-acting gene regulatory elements. Important transcription enhancing elements have been discovered using this approach in non-coding DNA from the intron, and 30 kb upstream of the APPb gene transcription start site [reference (14), and Shakes, Du, Sen, Abe, Kawakami, Wolf, Hatcher, Norford and Chatterjee manuscript in preparation].

The BAC enhancer-trap technology described above has three additional features that should prove beneficial: i) allows sampling much larger sequences of DNA, and consequently multiple discontinuous regulatory domains simultaneously compared to small plasmids, ii) the context of regulatory DNA with respect to the gene and chromosome is preserved and, iii) can be used with BACs in established libraries from a wide variety of organisms, and tested in several species. Although the methodology does not allow generation of internal deletions, truncations from the end opposite to the enhancer-trap can be made with a lox511 transposon [as seen when iTol2-end DNA cassettes were introduced at the other end of BAC DNA, (Figures 5 and 6)] to explore functions of candidate regulatory regions in a limited way. Sequences bending DNA (45-47), or phasing nucleosomes and other transcription factors (48-50) are left unaltered using BACs compared to characteristics of the gene region found endogenously. Bringing exogenous pieces of DNA together to create artificial junctions in small plasmids to trans-activate reporter genes does not adequately address the endogenous role of the regulatory sequence; and this is avoided using BACs. It is probably worth noting that DNA structure surrounding regulatory factor binding sites have evolved over long periods, and these are also left unaltered here. We noted earlier that there were 52 sites with six or more consecutive Aresidues, known to cause bends in unpackaged DNA (46), in the ~28 kb upstream regulatory sequence identified as important in regulating APPb (14).
