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The best-characterized insulator in Drosophila melanogaster is the Su (Hw)-binding region contained within the gypsy retrotransposon. In the [y.sup.2] mutant, Su (Hw) protein partially inhibits yellow transcription by blocking the function of transcriptional enhancers located distally from the yellow promoter with respect to gypsy. Previously we have shown that yellow enhancers can overcome inhibition by a downstream insulator in the [y.sup.rh1] allele, when a second gypsy element is located upstream of the enhancers. To understand how two insulators neutralize each other, we isolated various deletions that terminate in the regulatory region of the [y.sup.rh1] allele. To generate these alleles we used DNA elongation by gene conversion of the truncated chromosomes at the end of the yellow regulatory region. We found that gypsy insulator can function at the end of the truncated chromosome. Addition of the gypsy insulator upstream of the yellow enhancers overcomes the enhancer-blocking activity of the gypsy insulator inserted between the yellow enhancers and promoter. These results suggest that the gypsy insulators do not form separate transcriptional domains that delimit the interactions between enhancers and promoters.

The B-Peru allele of the maize b regulatory gene is unusual relative to most b alleles in that it is expressed in the aleurone layer of the seed. It is also expressed in a subset of plant vegetative tissues. Transgenic maize plants containing the B-Peru gene with the first 710 bases of upstream sequence conferred the same levels of aleurone expression as nontransgenic B-Peru plants, but no pigment was made in vegetative tissues. Transient transformation assays in aleurone tissue localized the aleurone-specific promoter to the first 176 bases of the B-Peru upstream region and identified two critically important regions within this fragment. Mutation of either region alone reduced expression greater than fivefold. Surprisingly, the double mutation actually increased expression to twice the native promoter level. Our results suggest that these two critical sequences, which lie close together in the promoter, may form a negative regulatory element. Several lines of evidence suggest that the B-Peru promoter arose through the translocation of an existing aleurone-specific promoter to the b locus. Immediately upstream of the aleurone-specific promoter elements and in the opposite orientation to the b coding sequence is a pseudogene sequence with strong similarity to a known class of proteins. Our findings that novel aleurone-specific promoter sequences of the B-Peru transcription factor are found adjacent to part of another gene in a small insertion are quite unexpected and have interesting evolutionary implications.

A hybrid transcription factor comprising a fusion of the DNA-binding domain of Saccharomyces cerevisiae GAL4 and the transcription activation domain of maize C1 was expressed in stably transformed Arabidopsis. Additional transgenic lines were created containing test genes controlled by a synthetic promoter consisting of concatemeric copies of the cis-acting site recognized by GAL4 ([UAS.sub.G]) fused to a minimal promoter. The GAL4/C1 effector line was crossed to two lines containing a synthetic promoter/GUS fusion. Both histochemical staining and GUS activity assays indicate strong activation of GUS expression was achieved only after crossing. The GAL4/C1 effector line was also crossed to 15 lines containing a synthetic promoter/antisense adenylosuccinate synthetase gene. Severely retarded growth, and in some cases lethality, was observed in 40% of the [F.sub.1] lines. This system of activation by crossing is generally useful for activating expression of test transgenes.

Bone morphogenetic proteins (BMPs) are secreted signaling molecules that control important developmental events in many different organisms. Previous studies have shown that BMPs are expressed at the earliest stages of skeletal development, and are required for formation of specific skeletal features, strongly suggesting that they are endogenous signals used to control formation of skeletal tissue. Despite the importance of BMP signaling in normal development, very, little is known about the mechanisms that control the synthesis and distribution of BMP signals in vertebrates. Here, we identify a large array of cis-acting control sequences that lay nut expression of the mouse Bmp5 gene in specific skeletal structures and soft tissues. Some of these elements show striking specificity for particular anatomical features within the skeleton, rather than for cartilage and bone in general. These data suggest that the vertebrate skeleton is built from the sum of many independent domains of BMP expression, each of which may be controlled by separate regulatory elements driving expression at specific anatomical locations. Surprisingly, some of the regulatory. sequences in the Bmp5 gene map over 270 kb from the Bmp5 promoter, making them among the most distant elements yet identified in studies of eukaryotic gene expression.

The suppressor of Hairy-wing [SU (HW)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the SU(HW) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. In these studies, we address whether repression resulting from the Polycomb group genes can be blocked by the SU(HW) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group Response Element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. We demonstrate that the SU(HW) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome.

Analysis of Drosophila promoter database of 252 independent promoter entries reveals the presence of significant nucleotide sequence irregularities in the proximal promoter region. This region has a tripartite structure, with initiator at plus or minus 5 bp, TATA at -25/-30 bp and a new group of downstream elements at +20/+30 bp from the RNA start site. The strand-specific downstream elements vary from the initiator and TATA in their length, position, representation and multiple sequences. An extended region with increased GC- and decreased A-content with several binding sites for Drosophila transcription factors constitute the overall sequence organization of the promoter region.

Six different transcripts of length 6.4, 5.6, 5.4, 2.5, 1.9 and 1.1 kb are all encoded by the forked gene of Drosophila melanogaster which affects the development of bristles and causes irregularities in large bristles in the head, scutellum and thorax of adults. One polyclonal antibody enables the identification of forked proteins on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The results show that changes in forked protein ratio give rise to irregular arrangements of fibrillar cytoplasmic configuration essential for bristle morphogenesis.

We report here the isolation of a new P-element-induced allele of the vestigial locus [vg.sup.2a33], the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was [vg.sup.21], which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing [vg.sup.2a33] in a dysgenic cross, we isolated the [vg.sup.2a33] allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3'-proximal copy of the 11-bp internal repeat from the P element of [vg.sup.21]. To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5'-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from [vg.sup.21]. Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter.