An intensive examination of the role of gene expression in chicken erythropoiesis is proposed. Both primitive (embryonic) and definitive red cell differentiation will be studied. We have isolated and analyzed the structure and organization of the seven chicken globin genes. Further studies on these genes will concentrate on in vitro mutagenesis of these genes, primarily by deletion and linker scanning techniques. Chimeric genes will also be generated by in vitro recombination between globin genes and promoter regions of other genes (histones, c-src, carbonic anhydrase, etc.) and vice-versa. The mutants so generated will be examined for expression in SV40 vectors, and by microinjection and/or cotransformation with selectable genes. The structure and expression of the chicken histone genes including the red cell-specific H5 histone gene will be studied by standard techniques in a variety of chicken tissues. We have demonstrated that most, if not all, of the 50 or so chick histone genes exist within unique environments of surrounding chromosomal DNA. We have also shown that chickens possess a unique H3-type (H3.3) histone gene which contains intervening sequences, and which is differentially regulated with respect to the continuous H3 genes. We intend to examine a variety of histone genes to see if their location and structure can be correlated with a unique expression pattern as is the case for the H5 and H3.3 genes. Other red cell-expressed genes including carbonic anhydrase genes will be studied by cDNA and genomic cloning techniques in order to relate their structure to their expression patterns in both primitive and definitive red cells. Their expression will also be studied in tsAEV-transformed cell lines at various stages of induction and in erythroblast fractions isolated from chickens in the crisis stage of erythroblastosis.
