Vincenzo Pirrotta - US grants

The developmental biology and differentiation of higher eukaryotes require many genes to be subject to multiple control mechanisms which regulate the time of expression, the tissues in which it occurs, and even the physical pattern of the cells in which the gene is active. We will approach the molecular basis of these mechanisms using the white (w) gene of Drosophila and its interactions. This gene, most intensively studied genetically, is a unique system to study specificity of expression, dosage compensation: the sex specific regulation which affects X chromosome genes and transvection. This last phenomenon depends on the zeste (z) locus and affects the white gene and at least two other loci, bithorax (BX-C) and decapentaplegic (dpp), of fundamental developmental importance. We will study these mechanisms by analyzing the effects of an array of mutants on white gene expression, its specificity and distribution, using Northern blots, S1 mapping, assays of w-product, and in situ detection in thin sections from different developmental stages. We will dissect the functions of the regulatory region using P-mediated transformation to reintroduce altered versions of the w gene into flies. We have cloned and will complete the analysis of the zeste gene, including the isolation of its product. The interaction of the z-product with chromosomes and their constituents will be explored in situ. In particular, we will look at its interaction with the DNA of its targets in w, BX-C, and dpp loci. The mode of action of the z-product will be analyzed also using mutations in suppressor of zeste loci. We will then attempt to clone these loci which suggest an involvement of zeste in vital cellular processes. The analysis of insertional mutants in w and z will also be continued to study their effects on transcription, termination, and splicing. The pursuit of one of these, w-a and a second site suppressor mutation promises to lead us to the mechanism of transcription termination.

This project proposes to dissect the molecular mechanisms that determine the pattern of expression of the Drosophila white gene in specific tissues, specific cells and specific developmental stages and modulate it according to the dosage of X chromosome material present in the embryo. The sequence of the different tissue specific determinants in the white regulatory region, their relationship to one another, to the promoter and to the zeste binding sites will be studied using a variety of transposon constructions introduced into the genome by germ line transformation. The transformed lines will be tested for the tissue and cell specificity of expression and for the effects of zeste and zeste mutations both on the functioning of the white promoter and on interchromosomal transvection-like effects. In particular, these experiments will permit the testing of current hypotheses on the role of the zeste product in governing the interaction between regulatory sequences and the promoter. Similar transposon contructions will identify sequences in the white regulatory region that are able to confer X-chromosome dependent dosage compensation on gene expression. This will be done using DNA segments from white placed in front of the xanthine dehydrogenase gene. Affinity chromotography will then be used to isolate a protein factor that binds specifically to the dosage compensation determinant. This work will test basic mechanisms underlying the functional organization of genetic information. These mechanisms could lead to new insights on the control of larger genetic domains such as the globin gene cluster in man.

The overall objective of this project is to isolate overlapping clones representing the euchromatic part of the Drosophila genome. This will be accomplished in three steps. First, the euchromatic portions of the salivary gland chromosomes will be microdissected into regions of approximately 200-300 kb and the genomic DNA from each region cloned into a lambda bacteriophage vector. This will yield approximately 600 minilibraries representing part of the sequence complexity of each interval. Second, the unique sequence clones in each minilibrary will be used to screen an ordered and complete library, constructed in a cosmid vector designed for efficient use of the clones for subsequent chromosomal walking and/or P-factor mediated transformation experiments. The overlaps between cosmids from each microdissected region will be established by restriction mapping. For the majority of regions, it is likely that the overlapping cosmids will define a single continuous stretch of genomic DNA (a contig). In addition, many of the interval contigs representing adjacent microslices will be connected simply from the restriction maps. Third, given sufficient time, the remaining gaps will be closed by limited chromosomal walking between unconnected contigs. This project will advance substantially an understanding of the structural organization of the genome of this important model organism and contribute to the development of techniques important for characterizing more complex genomes. Furthermore, the clones obtained will be invaluable to many Drosophilists for studying specific genes and their environs.