Cloning of Non-Globin Messenger RNAs From Mouse Reticulocytes

Casimir, Colin Maurice (1981) Cloning of Non-Globin Messenger RNAs From Mouse Reticulocytes. PhD thesis, University of Glasgow.

Abstract

The development of red blood cells in the mouse is a useful model system for studying gene expression during differentiation. Erythropoiesis entails the sequential expression of a coordinated set of gene products, culminating in the formation of a highly specialised cell, the erythrocyte. Many of these gene products can be used as recognisable markers of differentiation, for example, spectrin, glycophorin and the globins themselves. To date, however, it has really been possible to analyse only globin gene expression in terms of its molecular mechanisms. This has been due to the fact that reticulocytes synthesise predominantly globins and therefore the messenger RNA population is similarly highly enriched in globin-specific sequences. In order to acquire a greater understanding of the genetic control mechanisms at work during erythroid differentiation, it would be highly advantageous therefore to be able to study concomitantly the regulation of other erythroid-specific gene sequences. To this end genetic engineering techniques have been employed to construct a complementary DNA (cDNA) "library" of recombinant plasmids, using the total reticulocyte mRNA population as template. It was hoped therefore to select out clones from the library which contained "non-globin" sequences, with the aim of using these as molecular hybridisation probes to further investigate the process of differentiation. As a preliminary study, the feasibility of using density-gradient centrifugation to provide a convenient method of enriching the mRNA preparations for "non-globin" sequences was investigated. By use of appropriate size markers, suitable centrifugation conditions were obtained; however, when messenger RNA processed in this fashion was analysed by molecular hybridisation techniques the "non-globin" sequence content remained only some 10% of the total messenger population. A closer analysis, however, indicated that mRNA prepared by standard methodologies was significantly lower than expected in "non-globin" sequences. Such a conclusion was confirmed in a subsequent study of "non-globin" protein synthesis in reticulocytes during induced anaemia. Proteins were isolated from cells lab elled in vitro, fractionated on Sephadex G100, and the "non-globin" fractions examined by electrophoresis on SDS/polyacrylamide gels. In addition, this analysis demonstrated that in immature reticulocytes obtained earlier in the phase of anaemia, "non-globins" represented a significantly greater proportion of total protein synthesis than in their more mature counterparts (from which messenger RNA was usually prepared). In order to utilise immature reticulocytes it was then necessary to boost yields of these cells from the low level (17%) obtained at this stage in the anaemia. By manipulation of the dosage of phenyl hydrazine administered to induce anaemia it was found possible to obtain blood containing a very high (>98%) proportion of circulating reticulocytes. Messenger RNA prepared from blood of this type was shown, by in vitro translation, to direct greater synthesis of "non-globin" proteins. This mRNA was used therefore as a template for synthesis of cDNA and the single-stranded product converted to a double-stranded form by incubation with DNA polymerase. The double-stranded cDNA was then treated with S-1 nuclease to destroy the hairpin loop which remains joining the two strands together. Attempts were made to insert this double-stranded cDNA into linearised pAT153 plasmid DNA by the complementary homopolymer extension technique. Unfortunately, this approach was not successful in producing recombinant bacteria and was thus rejected in favour of the technique of "blunt-end ligation". This methodology involves the covalent linking of flush-ended plasmid DNA and cDNA prior to bacterial transformation. Recombinants resulting from the use of this procedure were screened by filter-bound colony hybridisation (Grunstein/Hogness technique), to select out clones containing reticulocyte cDNA sequences. From 100 clones so identified, ten putative "non-globin" recombinants were selected by a competition hybridisation assay. For this procedure duplicate nitrocellulose filters bearing DNA from colonies lysed in situ were challenged with either an unfractionated reticulocyte cDNA probe, or with a similar probe which had previously been hybridised, in solution, to a large excess of plasmid DNA containing and globin cDNA sequences. Colonies which showed evidence for competition, by reduction of hybridisation signal, were considered globin recombinants and were eliminated. Colonies unaffected by competition were considered putative "non-globin" recombinants and subjected to further study. These colonies were rescreened by the competition assay and additionally by a filter hybridisation using alpha and beta globin cDNA sequences purified from a total reticulocyte cDNA. The putative "non-globin" clones were then characterised by restriction analysis to ascertain the length of the inserted cDNA sequence and confirm its insertion into the Barn HI restriction site of the plasmid vector. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Paul Harrison
• Keywords: Biochemistry
• Date of Award: 1981
• Depositing User: Enlighten Team
• Unique ID: glathesis:1981-76128
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Dec 2019 09:15
• Last Modified: 19 Dec 2019 09:15
• URI: https://theses.gla.ac.uk/id/eprint/76128