Harold Weintraub - US grants

While an outline for the genetic changes responsible for malignancy is rapidly being developed, three major related areas remain relatively obscure. (1)\Does the transformed phenotype result from genetic alterations in controlling sequences that lead to abnormal expression of c-onc genes or do changes in coding regions of c-onc genes result in an altered function of the protein? Or either? (2)\How do genetic alterations in c-onc genes arise? (3)\Once an altered genotype is generated, how does the effected gene product ultimately lead to the transformed phenotype? This research focuses mainly on these three general questions by pursuing in detail the following observations we have made: (1)\RSV transformation leads to the activation of about 1000 new transcription units. (2)\At least one chemical carcinogen (bromoacetaldehyde) reacts preferentially with S1 and DNase I hypersensitive sites in chromatin and supercoiled plasmids. (3)\Tropomyosin synthesis is turned off translationally in RSV-CEF. Additional experiments are designed to identify replication origins in normal and transformed cells; to identify genes and gene products that fully activate the partially activated globin genes in RSV-transformed fibroblasts; and to identify genes and gene products, suppressed after RSV transformation, that are required for myoblast differentiation. (X)

The MyoD cDNA, when expressed under the control of a viral LTR and transfected into a variety of fibroblast and adipoblast cell lines, converts these cells to muscle. While not yet proven MyoD acts as if it were a master regulatory gene for myogenesis. The MyoD protein is nuclear and preliminary work suggests it binds DNA. The present proposal is to: (1) Characterize MyoD using detailed mutagenic analysis. (2) Study its binding to specific DNA sequences; study its interaction with other cellular proteins. (3) Explore preliminary indications that MyoD positively activates its own synthesis but negatively activates its mRNA utilization.

The overall goal of this proposal is to try to identify genes responsible for determining cell type specificity and eventually to try to understand the genetic complexity and logic of how this specificity is generated. We will be focusing on four types of developmental systems: early decision making in specific frog lineages; genes that commit cells to myogenesis; genes that determine whether hematopoietic precursor cells differentiate into macrophages or granulocytes; and genes that seem to link developmental decisions to decisions of growth control. For many of these systems, we begin by using cDNA clones that are cell type or cell lineage specific and we try to elicit a "phenotype" by introducing these sequences back into cells -- in a positive sense, to induce commitment with normal expression of the vector; in a negative sense, to inhibit differentiation with a vector that produces "abnormal" transcripts. We are hoping to create recombinant DNA mutations using 4 approaches. All of these methods must yield dominant mutations to be useful. The first is to use anti-sense RNA; the second is to use standard in vitro mutagenesis of cloned DNA to create a dominant mutation; the third is to overproduce the gene product; the fourth is to express a tissue-specific gene in the wrong cell type. For all of these schemes we will restrict our attention to tissue or lineage specific sequences obtained by "substractive" cloning of the unique RNAs (or cDNAs) peculiar to a given cell type. In most cases, this material is readily available. Estimates from solution hybridization suggest that if the two cell types that are to be compared are closely related, one might expect between 200-1000 sequences in such a difference library. These could be screened either in pools or individually. In the case of red blood cells, the screen is to look for embryos that lack red cells.

The MyoD cDNA, when expressed under the control of a viral LTR and transfected into a variety of fibroblast and adipoblast cell lines, converts these cells to muscle. While not yet proven MyoD acts as if it were a master regulatory gene for myogenesis. The MyoD protein is nuclear and preliminary work suggests it binds DNA. The present proposal is to: (1) Characterize MyoD using detailed mutagenic analysis. (2) Study its binding to specific DNA sequences; study its interaction with other cellular proteins. (3) Explore preliminary indications that MyoD positively activates its own synthesis but negatively activates its mRNA utilization.

MyoD is a bHLH transcription factor which, when expressed in a variety of cell types, converts those cells into muscle. E12 is also a bHLH transcription factor that can bind to similar sequences as MyoD, yet cannot activate muscle specific transcription. Only 3 residues from the MyoD basic region, when placed into E12, converts E12 into a myogenic activator without effecting DNA binding. Similarly, extensive mutagenesis of one of these residues in MyoD (ala-114) leads to a class of "positive control" mutants -- molecules that bind normally, but fail to activate. The primary goal of this proposal is to focus on the nature of this myogenic specificity by determining: (1) whether the positive control mutants bind to target sequences in vivo; (2) what cellular factors "recognize" the basic region "myogenic code"; (3) the 3-d structure of MyoD and MyoD-El2 heterodimers bound to DNA and in solution; (4) the rules for HLH dimerization efficiency; (5) the rules for how the basic region determines a preferred DNA binding site.