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Insulators are a class of elements that de®ne independent domains of gene function. The Drosophila gypsy insulator is proposed to establish regulatory isolation by forming loop domains that constrain interactions between transcriptional control elements. This supposition is based upon the observation that insertion of a single gypsy insulator between an enhancer and promoter blocks enhancer function, while insertion of two gypsy insulators promotes enhancer bypass and activation of transcription. To investigate this model, we determined whether non-gypsy insulators interacted with each other and with the gypsy insulator. Pairs of scs or scs¢ insulators blocked enhancer function. Further, an intervening scs insulator did not block gypsy insulator interactions. Taken together, these data suggest that not all Drosophila insulators interact, with this property restricted to some insulators, such as gypsy. Three gypsy insulators inserted between an enhancer and promoter blocked enhancer function, indicating that gypsy insulator interactions may be restricted to pairs. Our studies imply that formation of loop domains may represent one of many mechanisms used by insulators to impart regulatory isolation.

Interactions between paired homologous genes can lead to changes in gene expression. Such trans-regulatory effects exemplify transvection and are displayed by many genes in Drosophila, in which homologous chromosomes are paired somatically. Transvection involving the yellow cuticle pigmentation gene can occur by at least two mechanisms, one involving the trans-action of enhancers on a paired promoter and a second involving pairingmediated bypass of a chromatin insulator. A system was developed to evaluate whether the action of the yellow enhancers in trans could be reconstituted outside of the natural near telomeric location of the yellow gene. To this end, transgenic flies were generated that carried a yellow gene modified by the inclusion of strategically placed recognition sites for the Cre and FLP recombinases. Independent action of the recombinases produced a pair of derivative alleles, one enhancerless and the other promoterless, at each transgene location. Transvection between the derivatives was assessed by the degree of interallelic complementation. Complementation was observed at all eight sites tested. These studies demonstrate that yellow transvection can occur at multiple genomic locations and indicate that the Drosophila genome generally is permissive to enhancer action in trans. G ene expression is controlled by regulatory elements that modulate transcription in appropriate temporal and spatial patterns. In eukaryotes, these control elements often reside in large, complex regions, requiring action over long distances to elicit changes in transcription of a target promoter. In some cases, the control elements of paired homologous genes can interact in trans to alter gene expression. Such trans-regulatory effects illustrate processes known as transvection. Transvection was described first in Drosophila (1), which has homologous chromosomes that are paired somatically. Transvection and related processes have been reported in many organisms besides Drosophila (for example, refs. 2-7, and reviewed in refs. 8-11). These observations suggest that transvection may be generally possible for eukaryotic control regions and that these processes may be important for the normal regulation of some genes. For example, chromosomal pairing is proposed to play a role in gene silencing associated with imprinting and X chromosome inactivation (4, 12).In Drosophila, interactions between homologous chromosomes can have either a positive or negative effect on transcription. Examples of negative effects include repression of white gene expression by certain alleles of zeste, trans silencing conferred by the insertion of a block of heterochromatin near the brown gene, and pairing-dependent silencing, as exemplified by the effects of Polycomb response elements (reviewed in refs. 8-11, 13, and 14). Examples of positive effects include events at the Ultrabithorax, Abdominal B, decapentaplegic, yellow, and eyes absent genes (1,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).A useful system for studying the mechanisms involved in posi...

Drosophila melanogaster males perform a courtship ritual consisting of a series of dependent fixed-action patterns. The yellow (y) gene is required for normal male courtship behavior and subsequent mating success. To better characterize the requirement for y in the manifestation of innate male sexual behavior, we measured the male mating success (MMS) of 12 hypomorphic y mutants and matched-outbred-background controls using a y 1 rescue element on a freely segregating minichromosome. We found that 4 hypomorphs significantly reduced MMS to varying degrees. Reduced MMS was largely independent of adult pigmentation patterns. These mutations defined a 300-bp regulatory region upstream of the transcription start, the mating-success regulatory sequence (MRS), whose function is required for normal MMS. Visualization of gene action via GFP and a Yellow antibody suggests that the MRS directs y transcription in a small number of cells in the third instar CNS, the developmental stage previously implicated in the role of y with regard to male courtship behavior. The presence of Yellow protein in these cells positively correlates with MMS in a subset of mutants. The MRS contains a regulatory sequence controlling larval pigmentation and a 35-bp sequence that is highly conserved within the genus Drosophila and is predicted to bind known transcription factors. R EPRODUCTION usually involves heterosexual courtship behavior that is central to the divergence and diversity of animal species and is of obvious adaptive significance. In many species the basic program of courtship behavior is innate. These inborn, instinctual behaviors are likely to be a result of gene action during development that establishes the potential for behavior and motivates the animal to perform the behavior, given the appropriate external stimulation (Baker et al. 2001). It can be hypothesized that there are genes required for building into the central nervous system (CNS) the ability to process information specific to a behavior and the specific neural output pathway for signaling the performance of that behavior (Baker et al. 2001).Individual fruit flies of Drosophila melanogaster routinely perform innate behaviors. One such behavior that is well-characterized is the courtship ritual performed by males for females prior to heterosexual copulation. The male ritual is in essence a series of dependent fixed-action patterns: tapping, following, orienting, horizontal wing extending, wing vibrating (''singing''), genital licking, and attempted copulation (Bastock and Manning 1955;Bastock 1967;Hall et al. 1982;Hall 1994a;Yamamoto et al. 1997;Greenspan and Ferveur 2000). Typically, these behaviors must be performed in the correct sequence with some repetition over a modest period of time (2-10 min) to significantly stimulate a female to be receptive to copulation. Such a stereotypic courtship sequence is common to many animals (e.g., Darwin 1874;Bastock 1956;Morris 1970;Walters 1988;Bradbury and Vehrencamp 1998;Carew 2000;Judson 2002).Many genes whose fu...