Analysis of rabbit β-like globin gene transcripts during development

Abstract

We have analyzed the differential expression of a family of β-like globin genes during the development of rabbits, from four days post implantation to one week before birth. The family is composed of four genes, arranged 5′-β4-β3-Ψβ2-β1–3′ on the chromosome; Ψβ2 is an inactive pseudogene. Using the technique of hybrid-arrested translation in vitro, we have identified the embryo-specific globin polypeptides encoded by genes β3 and β4. The β3 and β4 globins are replaced by the adult β1 globin halfway through gestation; this corresponds temporally with the switch in site of erythropoiesis from the embryonic yolk sac to the fetal liver. The decline in production of β3 globin polypeptide precedes the decline in β4 globin. Transcripts from genes β1, β3 and β4 were analyzed at progressive stages of gestation by a blot-hybridization assay and by an S 1 nuclease protection assay. Mature messenger RNA and presumptive precursor RNAs from genes β3 and β4 are synthesized abundantly in embryonic erythroid cells but only at very low levels later in fetal development. Conversely, precursor and mature mRNA from gene β1 are found at very low levels in embryos but are abundant in fetal and adult erythroid cells. The co-ordinate appearance of precursor RNA, mRNA and polypeptide from all three active genes indicates that the primary developmental regulation of this gene family is exerted at the level of transcription. RNA species larger than the expected precursors were observed when the RNA was denatured with formaldehyde but not when methylmercury was the denaturant. These large RNAs are a formaldehyde-generated artifact, possibly a result of cross-linking globin transcripts to ribosomal RNA. We observe no extensive stable transcripts from the 5′ or 3′ flanking regions of these genes.