Individual dorsal morphogen binding sites mediate activation and repression in the Drosophila embryo.

The dorsal (dl) morphogen gradient is responsible for initiating the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the Drosophila embryo. dl encodes a sequence-specific DNA binding protein that belongs to the Rel family of transcription factors. Previous studies have shown that dl activates the mesoderm determinant twist (twi); here we use a combination of site-directed mutagenesis and P-transformation assays to demonstrate that it also functions as a direct transcriptional repressor of a second target gene, zerknüllt (zen). By exchanging dl binding sites between the promoters we show that activator sites from twi can mediate repression when placed in the context of the zen promoter, and that repressor sites from zen can mediate activation in the context of the twi promoter. This represents the first demonstration that common binding sites for any DNA binding protein can mediate both activation and repression in a developing embryo. Evidence is also presented that the affinities of dl binding sites are important for the efficiency of repression, but are not the sole determinants of the threshold response to the dl gradient.

The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in Drosophila.

A gradient of the maternal morphogen dorsal (dl) establishes dorsal-ventral (D-V) polarity in the early Drosophila embryo. The dl concentration gradient is initiated by regulated nuclear transport, and only protein that enters nuclei is active in the D-V patterning process. Here we show that dl is a DNA-binding protein that specifically interacts with distal sequences of the zerknüllt (zen) promoter, one of the genetic targets of the morphogen. These zen sequences have the properties of a silencer element and can act over long distances to repress the expression of a heterologous promoter. The dl protein recognizes a sequence motif similar to that of the mammalian transcriptional activator NF-kappa B, which was shown to contain extensive homology with dl and the oncoprotein rel. We present evidence that the DNA-binding activity of the dl protein is mediated by the region of homology (the rel domain) conserved in the rel and NF-kappa B proteins.

The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila.

The maternal morphogen dorsal (dl) plays a key role in the establishment of dorsal-ventral polarity in Drosophila. We present evidence that the graded distribution of dl protein is initiated by selective nuclear transport. The dl protein is uniformly distributed throughout the cytoplasm of early embryos, but approximately 90 min after fertilization, dl protein present in ventral but not dorsal regions is selectively transported to the nucleus. Mutations in maternally active genes that regulate dl disrupt this transport process, resulting in an inactive, cytoplasmically localized form of the dl protein. Selective nuclear transport of dl protein was reproduced in tissue culture cells. The wild-type dl protein is largely restricted to the cytoplasm, while truncated proteins are predominantly localized within the nucleus. Transient cotransfection assays suggest that dl activates expression from several promoters in an apparently sequence-independent manner. We discuss the role of nuclear transport as a regulated process in gene expression and development.

The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc.

The Drosophila segmentation gene, hairy (h), acts to regulate embryonic segmentation and bristle pattern. We present the DNA sequence of the h gene and of h cDNAs, thereby deducing the organization of the h transcripts. The h gene encodes a 337 amino acid protein that acts in both embryonic segmentation and adult bristle patterning. The h protein includes a domain that shows extensive similarity to a domain of the proto-oncogene N-myc that may be involved in DNA binding and/or protein dimerization. We discuss mechanisms of h action as a transcriptional regulator.

Tissue-specific and pretranslational character of variants of the rosy locus control element in Drosophila melanogaster.

Prior reports from this laboratory have described the experimental basis for our understanding of the genetic organization of the rosy locus (ry:3-52.0) of Drosophila melanogaster, as a bipartite genetic entity consisting of a structural element that codes for the xanthine dehydrogenase (XDH) peptide and a contiguous, cis-acting control element. The present report describes our progress in the analysis of the control element and its variants. Characterization of the control element variants reveals that, with respect to late third instar larval tissue distribution of XDH activity and cross-reacting material, i409H is associated with a large, tissue-specific increase in fat body which is not observed in malpighian tubules. Further data are presented in support of the inference that this differential expression must reflect differential production of XDH-specific RNA transcripts.--Gel blot analyses are described which demonstrate that the phenotypic effects associated with variation in the rosy locus control element relate to differences in accumulation of XDH-specific poly-A+ RNA and do not relate to differences in rosy DNA template numbers.--Experiments are described that provide for unambiguous mapping of control element sites through the use of half-tetrad recombination experiments and the recovery and phenotypic characterization of the reciprocal products of exchange between control element site variants. Thus, we are able to order the sites as follows: kar-i1005 i409-ry.

Studies on the mechanism of heterochromatic position effect at the rosy locus of Drosophila melanogaster.

Experiments are described that extend the characterization of position effect variants of the rosy locus and test possible mechanisms of heterochromatic position effect. Rosy position effect variants exhibit a variegated phenotype with respect to xanthine dehydrogenase activity in malpighian tubules. The breakpoints of the position effect mutations are located on the DNA map of the rosy region outside of the rosy locus DNA; ryps11136 is located in the DNA of the l(3)S12 gene immediately proximal to rosy, whereas ryps1149 is located some 15 kb distal to rosy in the pic locus. Southern blot experiments are described that test and reject the notion that heterochromatic position effect results from underreplication of the position-affected gene. Rather, the results of Northern blots serve to direct attention to position effect as a defect in transcription. Histone region deletion heterozygosity and butyrate-feeding experiments failed to exhibit specific suppression of position effect at the rosy locus.

Heterochromatic position effect at the rosy locus of Drosophila melanogaster: cytological, genetic and biochemical characterization.

This report describes cytological, genetic and biochemical studies designed to characterize two gamma-radiation induced, apparent "underproducer" variants of the rosy locus (ry:3-52.0), ryps1149 and ryps11136. The following observations provide a compelling basis for their diagnosis as heterochromatic position effect variants. They are associated with rearrangements that place heterochromatin adjacent to the rosy region of chromosome 3 (87D). The effect of these mutations on rosy locus expression is subject to modification by abnormal Y chromosome content. The rearrangement alters only the expression of the rosy allele on the same chromosome (cis-acting). The Y chromosome modification is only on the position-affected allele's expression. The recessive lethality associated with the rearrangements relate to specific rosy region vital loci, and for ryps11136, the lethality is not Y chromosome modified. The peptide product of the position-affected allele is qualitatively normal by several criteria. Heterozygous deletion of 87E2-F2 is a suppressor of the rosy position effect. The rosy position effect on XDH production may be assayed in whole larvae and larval fat body tissue as well as in adults.