Dunn School: Notebook 21, May 2006–Feb 2007
Notebook covers experiments including:
- Experiments 154 and following: Cloning polymerase genes from A/Vietnam/1194/2004, A/Hong Kong/213/03 and A /Vietnam/1203/2004 (RNA from Alan Hay NIMR). Cloning done initially by Brownlee then handed over to post-doctoral student Taka Kashiwagi.
- Experiment 165: Analysis of RNA sent from China (from Hualan Chen, Harbin, China): 2005 and 2006 duck strains of varying pathogenicity in chickens. Brownlee thinks this cloning was handed over to a Biochemistry part II student working with Tao Deng. If Brownlee remembers correctly, this was only partially successful as the student was not overcommitted.
- Brownlee started experiments with chicken and human karyopherin (α importin) clones. The idea was to see if co-transfection experiments with recombinant polymerase might result in enhanced or decreased activity in primer extension experiments. The human karyopherins were available from Ervin Fodor (via Othmar Engelhardt & Peter Palese) but Brownlee had to request (and in one case clone) the chicken ones. Brownlee also corrected the genomic and a deduced cDNA database of the chicken karyopherin α3, which was missing an exon. (Brownlee submitted the correction to GenBank.)
- See experiment 167 for Nomenclature Table of karyopherins and experiment 168 for chicken clones from Buerstedde, Germany. On sequencing chicken karyopherin α2 it had a dinucleotide deletion which altered the reading frame. To resolve this Brownlee used PCR mutagenesis (experiment 182) to correct the cDNA sequence (experiment 188). Brownlee also had to isolate chicken karyopherin α3 cDNA (not available from others) which Brownlee cloned with a TOP-TA cloning kit from chicken mRNA (experiment 170C).
- The co-transfection experiments with human and chicken karyopherins were difficult to interpret (summarized following experiment 192). The problem was that endogenous karyopherins were present, so I could not easily interpret the small increase or decrease in expression levels observed with the added co-transfected karyopherins (either human or chicken) on the level of the vRNA and mRNA of the reporter construct. (These experiments were never published.)
- Brownlee went on (in an attempt to simplify his approach) to study the effect of cotransfecting selected individual tagged karyopherins (detected with a Flag antibody and a secondary fluorphore distinguishable from GFP) with either PB1-GFP or PB2-GFP or PA-GFP expression constructs in VERO cells assaying for colocalization of fluorescence. Again not easily interpretable, probably [as Brownlee states] because of his inexperience in these cell-based assays, and his reluctance to continue this approach. (See also experiment 209 in notebook 22.)
- May 2006–Feb 2007