Restriction enzyme, molecular cloning and DNA annealing techniques have been used to study mRNA and DNA coding for the embryonic feather keratins of the chicken and the DNA genomes of coliphages 186 and P2. The coliphage DNAs were used to develop the techniques for application to the keratin system which awaited the availability of appropriate bio - hazard containment facilities before being undertaken. The following results were obtained. 1. Restriction endonuclease cleavage of chick DNA with BamHI, BgïII, EcoRI, or HindIII, fractionation on agarose gels, immobilization on nitrocellulose filters and annealing to DNA complementary to purified 12S mRNA isolated from the developing embryonic feather and coding for embryonic feather keratins, yielded a complex pattern of major and minor bands. These patterns consisted of 4 - 6 major bands and many minor bands. No simple repeat length could be deduced from these patterns, suggesting that keratin - coding DNA is heterogeneous in coding sequences, non - coding sequences or both. 2. Keratin gene expression was shown to be independent of DNA rearrangement, as the complex pattern of restriction fragments was identical in DNA isolated from germ - line tissue ( sperm ) the differentiated feather tissue and somatic tissue not synthesizing keratins ( erythrocytes ). Keratin gene expression must therefore involve the activation of pre - existing control regions in the DNA. 3. The purified 12S mRNA coding for feather keratin was transcribed into double - stranded DNA and individual species isolated by molecular cloning in E. coli. Sequence variation between species was confirmed by restriction enzyme analysis. 4. Preliminary analysis of the cloned species revealed the existence of two distinct groups of species comprising 12S mRNA : Group I ( the more abundant group ) and Group II ( the less abundant ). The fact that filter - bound DNA of individual Group I species bound more 12s cDNA than equal amounts of Group II species DNA and that pure Group I species and total 12S mRNA sequences ( coding for keratins in cell - free translation systems ) annealed to exactly the same complex set of EcoRI, HindIII, or BgïII restricted chick DNA fragments, compels the conclusion that Group I species represent true keratin coding sequences. Group II species annealed to restricted chick DNA fragments which were totally different to those annealing, to either Group I species or total 12S mRNA sequences. Different Group II species appeared to anneal to certain common fragments, suggesting that this less abundant group was comprised of a family of sequence related species and were not simply contaminating mRNA species coding for ' housekeeping ' functions. Their exact nature is at present, however, uncertain. 5. Group I species, the presumptive keratin - coding species, are members of a family of homologous species present in the chick genome. This is demonstrated by the fact that the two Group I species which have been examined so far, shown to be non - identical by restriction analysis, and total 12S mRNA sequences from which they were derived, annealed to the same set of between 20 and 30 BglII, HindIII or EcoRI restricted chick DNA fragments under annealing and washing conditions of low stringency, ( high salt ). Under stringent ( low salt ) washing conditions, however, all except between 1 and 3 of the duplexes formed by these fragments and the Group I species were differentially lost from the filter, indicating that the majority of duplexes were mis - matched and therefore that these multiple copies were homologous and not identical. In addition the two non - identical Group I species annealed to EcoRI generated chick DNA fragments of different sizes under the stringent ( low salt ) washing conditions, demonstrating that differences must exist in the sequence of adjacent non - coding and / or intervening sequences ( should they exist ) for these two species. 6. Although the two Group I species discussed above annealed to different EcoRI generated chick DNA fragments under the stringent ( low salt ) washing conditions, they both annealed under these conditions to a HindIII generated chick DNA fragment of size 3.0 kb. Assuming that this is a single fragment and not two fragments co - electrophoresing by chance, sequences identical to or with very close homology to both of these species lie on the same fragment and are therefore linked in the genome. The exact nature of this linkage and of the extent of gene clustering, should it exist, was not determined. 7. Restriction cleavage maps of coliphages 186 and P2 were determined for the enzymes BamHI, BglII, EcoRI, HindIII, PstI, SaïI, XbaI, and XhoI. These maps were used to analyse four insertion or deletion mutants affecting the major control region of 186. 186ins2 and 186ins3 were shown to be insertions of an IS3 element in the cI. gene and int gene respectively. 186dell and 186del2 were shown to carry the same deletion affecting the cI gene, but 186del2 carried a cryptic insert in the repressor binding site ( operator ). / Thesis (Ph.D.)--Department of Biochemistry, 1979.
| Identifer | oai:union.ndltd.org:ADTP/263879 |
| Date | January 1979 |
| Creators | Saint, Robert Bryce |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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