We allowed these viruses to evolve within the infected plants for between approximately 60 and 101 days and then conducted an analysis on the mutations generated within that time.
Wild-type p53 construct was made by inserting wild-type p53 cDNA into the pBABE retoviral vector with A161S or C176F mutations generated within wild-type p53 vector using site-directed mutagenesis as described in Neskey et al. The wt, pBABE, A161S and C176F vectors were used to infect PCI-13 cells, which underwent selection with puromycin.
To test the roles of specific amino acids in integrin binding of P66, mutations were generated within the coding sequence of the hypothesized integrin-binding region (Defoe and Coburn, 2001) of P66 using the Stratagene QuikChange II site-directed mutagenesis kit (Agilent Technologies) per manufacturer instructions.
The appropriate control for mutations generated via HR is an allele in which no targeted mutations have been introduced through the engineering process, but which contains a loxP sequence, the only remnant from the HR procedure, within an intron [ 33].
This showed that mutations could be generated within the germline and transmitted to subsequent generations at a very high efficiency.
Eleven of 24 recurrent mutations were silent, whereas 3 of 13 mutations generating amino acid substitutions were observed within the N-terminal domain of the spike glycoprotein gene (positions 21,722, 22,223, and 22,423, determining a G to D, an I to T, and a G to R amino acid change, respectively).
These include 2 frameshift mutations at residues 45 and 200 within LIM domains 1 and 3, respectively, 2 nonsense mutations generating premature termination codons at residues 153 and 198 within LIM domains 2 and 3, respectively, and 2 missense mutations, C209R and C276S, both located within LIM domain 3 [ 177, 191, 192].
Thus, whilst ZA and ZP domain mutations generate two distinct morphological phenotypes, there are subtle differences in severity within each group.
[This expansion follows directly from the definitions of Chi z), cosh z), and Ei z).] We now extend the derivations in Section A.3 to calculate the number of mutations to allele 2 generated within a certain time interval T, conditional on fixation of allele 1.
At low mutation rates, the amount of functionally different mutants generated within the quasipecies is reduced, and separate lineages, for example, parasites, readily evolve.
We identified four frameshifts within homopolymeric repeats, a nonsense mutation generating a truncation, and a missense mutation within a transmembrane region.
