We believe that some fundamental problems of hematopoietic regulation may be answered by combining the concepts and techniques of molecular genetics with those of cell biology. We propose to use this approach to examine the structure, regulation and function of colony stimulating factors (CSFs) that regulate the production of granulocytes and macrophages from precursor cells. Our studies will focus on the mouse as a model organism, but we will also examine human cells. We recently achieved the first translation of mRNA for CSF and we now intend to expand translational studies to other sources and subtypes of CSF. The mRNA will be assayed either by its ability to give rise to biologically active CSF after microinjection into Xenopus laevis oocytes or by immunoprecipitation of CSF after in vitro translation in a rabbit reticulocyte system. The assays will permit us to: (1) examine the levels of CSF in different cellular sources; (2) characterize and compare the mRNA for the two major CSF subtypes; and (3) determine whether induction of CSF production in lymphocytes by lectins occurs at the level of mRNA. We will also undertake the molecular cloning of CSF sequences. The most promising CSF for cloning studies is mouse L cell CSF, which stimulates the production of macrophages. We have access to both purified L cell CSF and a high titer monospecific antibody to the factor. These tools should permit us to utilize the several techniques for isolation of a CSF cDNA, including polysome precipitation, hybrid selection, and protein sequencing combined with construction of synthetic deoxyoligonucleotide probes. The isolation of such a cDNA would likely lead to studies yielding information about (1) the structure and regulation of CSF genes; (2) evolutionary and functional relationships to other growth factors; (3) the structure of CSF at the level of protein; and (4) the expression of CSF genes in bacteria or eukaryotic cells, perhaps resulting in large quantities of the material for biological or clinical studies. Concommitently, we will carry out genetic studies with mice designed to examine the function and expression of CSF in vivo. Preliminary studies have revealed genetic polymorphisms among strains of mice in both the number of precursor cells and their responsiveness to CSF, and these genetic variants may be useful in mapping and characterizing the genes regulating granulopoiesis.
