Studies on Cloned Human DNA

Munro, June (1987) Studies on Cloned Human DNA. PhD thesis, University of Glasgow.

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This thesis describes recombinant DNA studies on certain families of repeated DNA in the human genome. The initial objective was to isolate and characterise the gene(s) for human heat-shock protein 70 (hsp 70) using a complementary DNA (cDNA) clone (pHS2) prepared previously by others and reported to hybridise to mRNA for hsp70. The insert from this clone was used to screen two recombinant bacteriophage libraries containing human genomic DNA. After certain technical problems had been overcome, a number of corresponding genomic clones were isolated. Restriction maps were constructed for the four genomic clones which possessed the greatest homology to pHS2, as indicated by Southern blotting at high stringency. These maps suggested that three of the clones (lambdaHr02, lambdaHr03 and lambdaHr06) were overlapping clones derived from the same region of the genome, whereas the remaining clone (lambdaHr05) was derived from a completely distinct region. The nucleotide sequence of the insert of pHS2 was determined as a preliminary to sequencing the genomic clones. Analysis of the sequence indicated that it was a GC-tailed cDNA copy of a fragment of ribosomal RNA (rRNA), rather than a heat-shock sequence. The rDNA fragment encompassed a region extending over nucleotide 3600-3923 of human 28S rDNA with only one mismatch. Nevertheless it was clear from genomic blots, comparisons of restriction maps and the frequency of genomic clones isolated that the genomic clones did not contain members of the rDNA tandem repeat. They were therefore analysed further. A portion of lambdaHr02 containing the region hybridising to pHS2 was subcloned and the nucleotide sequence of a 823 base-pair region determined. This showed that the region in lambdaHr02 recognised by pHS2 was a rDNA pseudogene. It was related to a 451 base-pair segment of human 28S rDNA, extending over nucleotide 3627-4105. This rDNA pseudogene has suffered a number of mutations and is not immediately flanked by other rDNA sequences. Regions further upstream and downstream of this pseudogene were analysed by Southern blotting, using two fragments of the human ribosomal clone pA4 (which contain areas not recognised by pHS2) as probes. No sequences corresponding to these regions were detected in the genomic clone, lambdaHr02. This indicated that this rDNA pseudogene (designated H28S-01) was a dispersed member of the tandem rDNA repeat, an 'orphon', and is the first ribosomal orphon to be characterised in detail. Additional Southern blotting evidence suggests that there are some repeated DNA sequences surrounding this pseudogene. The sequence immediately 3' to this pseudogene was shown to possess homology to a region extending over nucleotide 146-170 of the genomic 1.9 kb Hind III repeat, a portion of the human long interspersed middle repetitive sequence, L1Hs. Southern blot and restriction map analyses indicate that clone lambdaHr05 also contains a dispersed rDNA pseudogene. Clone lambdaHr04 may contain an even more diverged rDNA pseudogene as it possesses even less homology to pHS2 than the other genomic clones, as determined from Southern blotting at high stringency. Two mechanisms are considered for the generation of H28S-01. One possibility is that it was generated by a DNA-mediated mechanism, as proposed for other orphons. Unequal crossing-over caused by misaligned tandem repeats might have resulted in a looped out segment being excised and reintegrated at a different site in the genome. A second possibility is that this rDNA pseudogene was generated through an RNA-mediated mechanism. An rRNA fragment with suitable secondary structure may have primed its own reverse transcription, the resulting complementary DNA being subsequently integrated non-specifically into the genome.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76646
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 13:58
Last Modified: 19 Nov 2019 13:58

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