Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.661833
Title: The role of linker histone globular domains in chromatosome formation
Author: Shen, Chang-Hui
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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Abstract:
Expression of the globular domain of the chicken linker histone H1 (GH1) in E. coli has the potential to allow the study of chromatin structure. In a T7 polymerase expression system only very low levels of the wild type chicken GH1 are produced. However, mutating the third base in each of the first 4 codons of GH1, from G or C to A or T, substantially raised expression levels. To further investigate this observation, 15 mutants, comprising all possible combinations of the altered first 4 codons, were constructed and their expression was compared to that of the wild type GH1. Although each mutant was transcribed with equal efficiency, expression levels varied over a 300 fold range. The results indicate that increased GH1 expression (i) is not a function of mRNA abundance, (ii) does not depend upon codon usage and (iii) is not correlated to changes in intramolecular mRNA secondary structure per se or to its potential influence upon the structure of the Shine-Dalgarno sequence in the translational initiation region of the mRNAs. However, a striking complementarity between the sequence +5 to +19 of wild type GH1 mRNA and a region of the 16S rRNA molecule (1526 to 1510) located only a few nucleotides 5' of the 16S anti-Shine-Dalgarno sequence has been found. This observation could indicate that the potential to form a duplex between the first 4 codons of GH1 mRNA and the 16S rRNA molecule may be the feature responsible for modulating GH1 expression in E. coli. The fact that mutations which increase GH1 expression substantially decrease the likelihood of hybrid formation support this proposal. Furthermore, the expression of Green Fluorescence Protein (GFP) was greatly inhibited when an oligonucleotide which is homologus to the sequence +4 to +19 of wild type GH1 mRNA was inserted immediately after the start codon of GFP.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.661833  DOI: Not available
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