Unlike most organ systems, which have evolved to maintain homeostasis, the brain has been selected to sense and adapt to environmental stimuli by constantly altering interactions in a gene network that functions within a larger neural network. This unique feature of the central nervous system provides a remarkable plasticity of behavior, but also makes experimental investigations challenging. Each experimental intervention ramifies through both gene and neural networks, resulting in unpredicted and sometimes confusing phenotypic adaptations. Experimental dissection of mechanisms underlying behavioral plasticity ultimately must accomplish an integration across many levels of biological organization, including genetic pathways acting within individual neurons, neural network interactions which feed back to gene function, and phenotypic observations at the behavioral level. This dissection will be more easily accomplished for model systems such as Drosophila, which, compared with mammals, have relatively simple and manipulable nervous systems and genomes. The evolutionary conservation of behavioral phenotype and the underlying gene function ensures that much of what we learn in such model systems will be relevant to human cognition. In this essay, we have not attempted to review the entire Drosophila memory field. Instead, we have tried to discuss particular findings that provide some level of intellectual synthesis across three levels of biological organization: behavior, neural circuitry and biochemical pathways. We have attempted to use this integrative approach to evaluate distinct mechanistic hypotheses, and to propose critical experiments that will advance this field.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA proteinbinding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.DnaA protein of Escherichia coli is required for the initiation of chromosomal replication from oriC, the chromosomal origin (1-3). As a DNA-binding protein, it recognizes a 9-base pair sequence, the DnaA box, present 4 times in oriC (3). On binding, localized melting of the AT-rich region by DnaA protein and 1-5 mM ATP (4) is assisted by either HU or IHF that presumably act by DNA bending (5-7). Subsequently, the binding of DnaB helicase from the DnaB-DnaC complex through direct contact with DnaA protein suggests that it orients the binding of the replicative helicase for its subsequent action to promote bidirectional replication fork movement (8). Priming of DNA replication by primase for both leading and lagging strand synthesis is proposed to occur through a physical interaction between primase and DnaB protein (9), thus establishing the elongation phase of DNA replication.The correlation of domains of DnaA protein to its various functions is rudimentary. By comparative analysis of homologs of the dnaA gene from phylogenetically diverse microbes, sequence conservation of a P-loop motif, GX 4 GKT located at residues 172-179 (reviewed in Ref. 10), correlates with the activity of DnaA protein to bind ATP with high affinity (K D ϳ 0.03 M) (11). In p21 ras and RecA protein, corresponding residues interact with the phosphates of the bound nucleotide (12, 13). Missense mutations of the dnaA5 and dnaA46 alleles, as well as others (14), substitute a conserved alanine for valine at amino acid residue 184 near the P-loop. The mutant proteins were defective in ATP binding, suggesting that the substitution was responsible (15, 16).1 Together, these results implicate this region of DnaA protein in binding to ATP.The DNA-binding domain of DnaA protein has been localized to a region from residue 379 to the carboxyl terminus (17). Other domains of DnaA protein involved in purine binding, unwinding, interaction with DnaB protein (8), or interaction with phospholipids that displace the bound nucleotide (18) are unknown. To correlate domains of DnaA protein to its various functions, a collection of monoclonal antibodies were produced and characte...

DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC. By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogaster embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.Transcriptional repression is a critical component of genetic regulation during development, and the Drosophila melanogaster embryo has served as an important model for elucidation of basic repression mechanisms (7,19). Differential gene expression in the early embryo is controlled in large part by the activity of repressor proteins encoded by gap, pair-rule, and other genes (42, 45). Repression of transcription can involve reactions occuring off the DNA, such as the formation of inactive heteromeric complexes. Another mechanism involves competition between activators and repressors for binding sites on DNA. DNA-binding repressors that function by mechanisms other than competitive binding have been termed active repressors (24).An active repressor can repress basal promoters or enhancer elements over a short range (Ͻ100 bp) or, alternatively, over long ranges (Ͼ1,000 bp) (7,17). One model of repression in the embryo suggests that the short-range-long-range distinction results from the recruitment of distinct classes of cofactors (36, 55). Short-range repressors may interact with dCtBP, while long-range repressors interact with Groucho.Long-range repressors are typified by the Hairy protein, a transcription factor that binds the Groucho cofactor (5, 27, 40). Long-range repression complexes regulating the dpp, tld, and zen genes also recruit Groucho (7, 27), as do Engrailed, Runt, and dTCF, Drosophila repressors whose range of action has not yet been determined (3, 9, 50).Short-range repressors present in the early Drosophila em-

Multicellular organisms depend on cell-type-specific division of labor for survival. Specific cell types have their unique developmental program and respond differently to environmental challenges, yet are orchestrated by the same genetic blueprint. A key challenge in biology is thus to understand how genes are expressed in the right place, at the right time, and to the right level. Further, this exquisite control of gene expression is perturbed in many diseases. As a consequence, coordinated physiological responses to the environment are compromised. Recently, innovative tools have been developed that are able to capture genome-wide gene expression using cell-type-specific approaches. These novel techniques allow us to understand gene regulation in vivo with unprecedented resolution and give us mechanistic insights into how multicellular organisms adapt to changing environments. In this article, we discuss the considerations needed when designing your own cell-type-specific experiment from the isolation of your starting material through selecting the appropriate controls and validating the data.