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Australian sabbatical visit: Notebook 2, Feb 1987-Jul 1987

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MS. 12364/9

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This was a continuation of Brownlee's sabbatical at the Murdoch Institute, Childrens' Hospital Melbourne, and then from 30 Mar 1987 at the Walter and Eliza Hall, Institute of Medical Research, Melbourne.

Brownlee continued (Topic 1) with Ruth Brown, to try and isolate cosmids that had both human and mouse DNA repetitive sequences, thus potentially marking the translocation between human and mouse sequences in her somatic cell hybrid. He also continued (Topic 2) experiments trying to use RNases to detect base-pair mismatches in DNA-RNA heteroduplexes.

George Brownlee states: "These experiments, using the Indium CTL assay, seemed not well designed. There was no clear hypothesis. Rather I was learning, first hand, how to do various assays which were new to me. No publishable results":
  1. Topic 1 - Isolating cosmids from a translocation site in a somatic cell hybrid:
  2. Experiment 55, Experiment 61 theory: By screening with human and mouse repetitive probes only 4 human positive, mouse negative, clones isolated. Theoretically, about 1% of cosmids would be expected to be human and over a 1000 clones would need to be screened to stand a reasonable probability of isolating a cosmid across the human-mouse translocation site. (Derek Blake, DPhil student & Yvonne Boyd, Genetics, Oxford – mentioned for advice)
  3. Experiments 62, 65: By making a human-enriched mini-library (about 10 fold enriched in human cosmids) from a high density plating of the original library, 22 human clones were isolated in all (p. 54), although it was not determined if all were unique, and stored (left for Ruth Brown). Brownlee did not solve the numbers game in the time available. Results not publishable.
  4. Topic 2 - RNases to detect base-pair mismatches in DNA-RNA heteroduplexes:
  5. Cloning of "approx. 200 nt" RsaI - Dde I fragment of exon f of FIX, its transcription (experiment 66), and annealing and P-RNase (experiment 67). Successful detection of C-T mismatch - a 146 band. No time to follow this up further. Results not published.
  6. Topic 3 - Walter & Eliza Hall (Ken Shortman’s lab):
  7. Ken Shortman worked with γ-labelled Indium oxime and Brownlee first had to learn to make this from 111 Indium chloride (experiment 1) and use the oxime uptake in a CTL quantitative radiochemical assay (working with Eugen Markovski, student).
  8. Testing effect of anti-T cell receptor AB (mimicking antigen) on CTL assay (experiment 6). Positive result (as expected from Ann Kelso's previous results) but not as significant as the positive controls with PMA or ionomycin. Requires IL-2 (experiment 11).
  9. Follow up of Pike & Nossal PNAS (1981) - tolerance experiments in vitro and CTL responders (e.g. experiment 12, where twofold effects observed, but may not be significant (p. 109)). In vivo (experiment 14) tolerization reduced CTL quantitatively very significantly. Also experiment 23.
  10. B and T cells detected by FACS (experiment 15).

Dates

  • Feb 1987-Jul 1987

Extent

1 volume

Shelfmark

MS. 12364/9

Repository Details

Part of the Bodleian Libraries Repository

Contact:
Weston Library
Broad Street
Oxford OX1 3BG United Kingdom