Ringbook of Jane Sharps, 1993–1997
Jane Sharps, on Sir Henry Harris' retirement, joined Brownlee as a research assistant working with him, except for a short gap, until shortly before Brownlee's retirement in 2008. This is a summary of her first ringbook working with Brownlee. Her remaining notebooks are still in her keeping and are a useful resource, while she continues as Professor Ervin Fodor's assistant.
- RNA ligation experiments (continuing experiments of post doctoral researcher Ervin Fodor):
- A series of experiments characterizing the length of the polyA tail that was added to a synthetic 49 mer mimic of an influenza RNA genome by an influenza RNP preparation (influenza "cores") in vitro.
- See ABI Prism sequencing charts of 99, 45, 51, 57 and 80 nucleotide long polyA tails in clones (see following p. 46).
- Short length 49-mer mutations (with post-dcotrial researcher David Pritlove):
- See design of promoter (5' strand) mutants of 49-mer constructed in the plasmid pBXPCAT. See design opposite (p.10) experiment 44 (S44) successfully made (see Autoradiograph (AR) of manual sequencing gels, 12.03.97 on p. 11).
- See hand written note by David Pritlove to Jane Sharps, dated 18 Feb 1997.
- Further 49-mer mutations successfully constructed by mutagenesis. (e.g. experiments 53-56, 7', 8', 9' and 10' promoter mutations). See AR labelled 17.3.97. ?All designed 49-mer mutant constructed.
- Experiments with chemically synthesized 25-mer (no 5' strand), 41-mer (shorter loop) and 50-mer:
- Aim was to find our if these RNAs,shorter than the 50 nucleotide long construct, would polyadenylate with influenza "cores" with ApG as a primer; see GGB experiment 509 for 50-mer; see p. 85 and 38 (GGBs notebook) for design.
- RT-PCR experiments using GGB's phased (clamped) primer T15A gave products in correct position on 16% TBE acrylamide gel for 41-mer and 50-mer but not for 25-mer (11 Apr 1997, 14 Apr 1997 and following e.g. 22 Apr 1997 with various RT –PCR controls). Sequencing of 41-mer, after cloning, (19 May 1997) is correct - see ABI Prism traces. [Leo (Leo Poon, DPhil) questions the validity of these experiments wondering if some background bands in the RT-PCR controls derive from endogenous flu RNA in cores. (21 Apr 1997)]
- RT- PCR with non-phased T20 GC primer of 41 flu transcript, followed by gel elution and cloning (Invitrogen, TA cloning kit). Clones with 30, 74, 84, 37, 38 and 50 As found (includes 20 A's in the primer). (See ABI traces Fri Jun 13 1997).
- Conclude: 41-mer segment was successfully polyadenylated.
- Unsuccessful attempt at devising an RT-PCR assay to detect vRNA transcripts from a 49 long cRNA template:
- Experiments started with pBXPCAT (30 Jun 1997) successfully constructing a new clone, experiment 2 (14 Jul 1997).
- T7 transcripts of 49 template (16 Jul 1997).
- RT-PCR was not specific (4 Aug 1997).
- Even with gel purification and DNase treatment of 49-mer, the RT–PCR was not specific even with reduced cycling.
- "Ampliscribe kit" is a useful way of getting higher yields of the 49 nucleotide- long T7 transcript.