Brent H. Cochran

The regulation of c-myc by growth signals.

c-myc mRNA is a cell-cycle associated transcript whose induction is regulated by agents that initiate the first phase of a proliferative response. Specifically, c-myc mRNA levels are increased between 10 and 40 fold within one to three hours after incubation of lymphocytes with 1ipopolysaccharide or Concanavalin A or fibroblasts with platelet-derived growth factor (PDGF). This induction of c-myc does not require the synthesis of new protein species and therefore is regulated directly by the biochemical events that immediately follow PDGF binding in fibroblasts and mitogen binding in lymphocytes. In fibroblasts that have been synchronized with respect to the cell-cycle by a short incubation in PDGF, c-myc mRNA levels are maximal in early G0/G1 and diminish to a quiescent level by the time the cells enter the S phase. Thus, c-myc RNA is a labile transcript with a maximal half-life of three hours. Finally, our experiments provide data that places the action of two oncogenes in a hierarchy: c-sis, the putative structural gene for PDGF, regulates the expression of c-myc.

[6] Differential colony hybridization: Molecular cloning from a zero data base

Publisher Summary This chapter provides a guide to differential colony hybridization for the cDNA cloning novice. Differential colony hybridization has been used to study developmental programs of adipocyte and slime mold differentiation. The potential applications of this technique are virtually endless and the number of laboratories using it is rapidly expanding. Procedures can become obsolete rapidly. The protocols, detailed in the chapter, are adaptations of procedures developed by many others and are the ones that are currently being used by laboratory. The chapter emphasizes on practical as well as theoretical considerations in the choices of alternative procedures and tries to point out commonly encountered problems with the procedures. The chapter deals with a cloning strategy that requires no preexisting database of knowledge. The strategy of “differential colony hybridization” or “+/– screening” requires nothing more than the existence of two closely matched tissues. Genes are selected on the basis of unequal levels of expression in the two-cell populations. Using this technique, it has been possible to clone genes whose expression is tissue specific, stage specific, cell cycle regulated, or hormone inducible.

Cell Cycle Control of C-Myc Expression

c-myc mRNA is a cell-cycle associated transcript whose induction is regulated by agents that initiate the first phase of a proliferative response. Specifically, c-myc mRNA levels are increased between 10 and 40 fold within one to three hours after incubation of lymphocytes with lipopolysaccharide or Concanavalin A or fibroblasts with plateletderived growth factor (PDGF). This induction of c-myc does not require the synthesis of new protein species and therefore is regulated directly by the biochemical events that immediately follow PDGF binding in fibroblasts and mitogen binding in lymphocytes. Thus, the c-myc oncogene encodes a product which may function as an intracellular mediator of the growth response to mitogens. PDGF is the putative translation product of the sis gene. The product of the v-sis gene, obtained as a supernatant from v-sis transfected NRK cells, induces c-myc expression in quiescent BALB/c 3T3 cells. Also, v-sis transfected BALB/c 3T3 cells display induced levels of c-myc mRNA following prolonged incubation in platelet poor plasma. Thus, oncogenes may be linked in functional hierarchies: the product of one oncogene, sis, regulates the expression of a cellular proto-oncogene, c-myc.