David S. Hogness - US grants

The proposed research is directed toward a definition of the structure of the human visual pigments and toward an understanding of how these structures mediate human color vision. More specifically, we wish to test the model that the visual pigments in the three types of cones resemble the rhodospin of the rods in consisting of an apoprotein (opsin) covalently linked to the common 11-cis retinal chromophore, that differences in the absorption spectra among the visual pigments result from structural differences among the opsins, that those structural differences result from nucleotide sequence differences among the members of a multigene family encoding the opsins, and that the well known genetic variations in color vision result from mutations in those members that encode the cone opsins. To this end, we propose to clone, isolate and sequence the genes that encode the protein moieties of the human visual pigments fom both normal and mutant individuals. From these cloned genes we will produce the encoded proteins in appropriate host-vector systems, attach them to the 11-cis retinal and study the reconstituted visual pigments in vitro. This approach should allow a direct test of the model. Detailed analyses of many mutants should provide insights into the molecular structure and mechanism of action of the visual pigments that are important both to our basic understanding of vision and to its pathologic anomalies. Our strategy is (i) to isolate and characterize at the nucleotide level the gene encoding the best studied visual pigment, bovine rhodospin (ii) to use coding sequences from this gene as hybridization probes to isolate at least one of the human opsin genes ( e.g., that for human rhodopspin) from a library of genomic DNA clones, and (iii) to similarly use such a human gene isolate to identify and isolate other members of the postulated opsin multigene family in humans. The first step in this plan has been acccomplished.

The proposed research is directed toward solution of the problem of how genes dictate development in higher animals. Drosophila melanogaster is the preferred organism for attempting this solution because its genome is among the smallest and best defined, because more genes specifying development have been isolated, and because cloned DNAs can be easily inserted into its germ line. This grant focuses on the genetic regulatory functions of proteins encoded by the homeotic Ubx gene of the bithorax complex (BX-C) and on the regulation of this gene by the products of other genes. This gene provides the major determinants specifying the developmental pathoways for the identities of the metameres comprising the posterior half of the torax and the most anterior part of the abdomen (parasegments (ps) 5 and 6). The Ubx gene consists of a 78 kb transcription unit whose transcripts are differentially spliced to yield at least five mRNAs encoding a family of Ubx proteins characterized by constant N- and C-terminal regions and a variable central region lying just upstream from a homeo domain encoded by the conserved homeo box. One goal of the project is to define the mechanisms controlling his differential splicing. Another is to define the mechanisms by which each of these proteins binds to and regulates the expression of their target genes, two of which are known: the Antp homeotic gene and the Ubx gene itself. A connected goal is to define the other target genes of these regulatory proteins. Cis-acting elements controlling the transcription of Ubx are spread over a 40 kg region (bxd region) lying upstream of the gene. This control region and the Ubx gene comprise one third of the BX-C, whose remainder is occupied by two other complex genes (abdA and AbdB), each containing a homeo box. Another goal of this project is to define the mechanisms by which the bxd control region, and another cis- regulatory region in an intron of Ubx, regulate Ubx transcription, and particularly, how proteins encoded by abdA and abdB, as well as by certain segmentation genes (e.g., ftz) and by ubx itself, modulate this cis-regulation. There is thus an increasing emphasis on regulating proteins in this project. The development of methods for the synthesis and isolation of these proteins from Drosophila host-vector expression systems, and of novel techniques for determining their regulatory functions, is a further aim of this project.

This proposal requests funds to equip an ultracentrifuge facility. Five investigators have proposed projects that require extensive use of ultracentrifugation. These include mapping ofportions of the human genome using very high molecular weight DNA, genetic control of Drosophila development, a study of how cell interactions regulate development and motility in Myxococcus, molecular mechanisms of eukaryotic DNA replication, and the biochemistry of intracellular transport.

The long-term objective of the proposed research is to determine the molecular genetic mechanisms by which steroid hormones control tissue development. This is a general problem in higher animals including humans, and is of particular importance to an understanding of the role steroid hormones play in the generation of cancers. More specifically, this research is directed toward the molecular definition of the genetic regulatory hierarchies that effect the coordination of the developmental pathways of a set of tissues in response to a steroid hormone. Drosophila melanogaster is the preferred organism for these studies because it includes the only example among higher animals of a well defined steroid-activated genetic regulatory hierarchy. This hierarchy is manifested by the program of polytene chromosome puffing induced in the larval salivary glands by a pulse of the steroid hormone ecdysone at the end of larval life - a pulse that initiates metamorphosis to the adult fly by inducing a coordinated change in the developmental pathways of a set of target tissues. The model proposed by Ashburner for the generation of these transcription puffs consists of a genetic hierarchy of at least three ranks. Starting with the gene encoding the ecdysone receptor protein, the transcription of a half-dozen "early" regulatory genes responsible for the first set of puffs is induced by the ecdysone-receptor complex. Proteins encoded by these early genes then induce the transcription of a much larger set of genes responsible for the next set of puffs. The cloning and characterization of the ecdysone receptor gene (EcR) and two of the early genes (E74, E75) in this laboratory have yielded results consistent with the Ashburner model and indicate that genetic regulatory hierarchies akin to that proposed by Ashburner may also govern the ecdysone response of the other target tissues involved in metamorphosis. The resulting "tissue coordination model" proposes that the third rank in these related tissue-specific hierarchies consists of "effector" genes which effect the change in the tissue's morphological and functional properties that constitute the next step in its developmental pathway. The experimental plan is directed toward the testing and further development of this model. In the first phase, the distribution of E74 and E75 expression among the target tissues, and the role played by the EcR gene in generating that distribution, will be determined. Subsequently, other early genes will be cloned and similarly characterized, followed by the cloning of genes regulated by the early gene proteins in particular target tissues and the testing of these genes for effector properties. Mutational studies will also be used to determine the interaction among the genes in these hierarchies.