The genes for mouse globin and the post-transcriptional control of their expression

In this work I have investigated the possible role of gene reiteration, amplification or deletion, as a means of regulating the expression of the globin gene during tissue differentiation in the mouse. This has employed a probe of complementary DNA (CDNA) prepared by copying reticulocyte 9S RNA using avian myeloblastosis virus reverse transcriptase. Since this RNA contains the messenger RNAs for alpha and beta globin, the cDNA produced is complementary to the alpha and beta globin genes. The fidelity of this cDNA said its use as a probe for globin sequences in DNA and RNA are rigorously justified. The rates of hybridisation of this probe to whole mouse embryo DNA, mouse erythroid DNA from 14 day foetal livers, and mouse germ line DNA from sperm are compared. The small corrections required for the size differences of the hybridising molecules, said the slight mismatching in the cDNA are estimated. The study demonstrates that there are 1-2 copies of the globin alpha and beta genes in all tissues studied, and thus mechstnisms other than gene amplification or deletion are required to explain the control of globin gene expression. The existence of post-transcriptional mechanisms by which this regulation may occur was then investigated. The first of these is the translational role of the poly(A) segment found on the 3' of eukaryotic mRNAs. This segment was removed from reticulocyte 9S RNA, using polynucleotide phosphorylase . Several experiments were performed to demonstrate the absence of the poly(A) sequence from the mRNA. The ability of this mRNA to code for alpha and beta globin chain synthesis in several cell-free protein synthesising systems was compared with intact mRNA. The results demonstrate that the poly(A) segment is not required for efficient initiation, elongation, termination or reinitiation of protein synthesis in a cell-free system. The role of the poly (A) sequence in determining the nuclear metabolism of globin RNA was then investigated. Low levels of globin RNA sequences were detected using cDNA in the nucleus of several non-erythroid tissues, adult mouse brain and liver, and a cultured lymphoma cell line, L5178Y. Very much lower levels of globin RNA sequences were detected in the cytoplasm of these cells. In contrast, in the erythroid 14 day foetal mouse liver, the amounts of globin RNA sequences found in the cytoplasm were very much higher than that in the nucleus. This indicates the operation of a post-transcriptional regulatory mechanism, whereby the nuclear said cytoplasmic levels of globin RNA sequences are regulated independently. However, this mechanism appears not to be mediated through association of nuclear globin RNA sequences with poly(A) since in both erythroid and non-erythroid tissues, where the fraction of total cell globin RNA sequences in the cytoplasm varies considerably, the proportion of polyadenylated nuclear globin RNA sequences is the same. At the present time, the possible functions of this low-level transcription of the globin gene in non-erythroid tissues are unclear