Bhuvanagiri et al (2014) have identified a means to allow mutant genes containing aberrant stop codons to become useful.
Moreover, approximately one-third of inherited genetic disorders and many forms of cancers are associated with mutant genes containing PTCs [ 23- 26].
The human γD-crystallin (CRYGD) and mutant genes containing N-terminal hexa-His tags were inserted into the pQE.1 vector (Qiagen), and protein was produced in transformed Escherichia coli M15 cells, essentially as described previously (26).
The PCR mixture (50 μL) for amplifying the full-length mutant gene contained 40 ng of each megaprimer, 10 μL of 5× GC buffer, 3% DMSO, 0.4 mM dNTPs, and 1 unit of Phusion DNA polymerase.
Soybean codon optimized mutant SEB genes containing sequences encoding the native SEB N-terminal signal peptide sequence and the native soybean glycinin N-terminal signal peptide sequence were synthesized from GeneArt (Life Technologies Carlsbad, CA) and DNA 2.0 (Menlo Park, CA), respectively.
Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.
To gain further insight into the relationship between active histone demethylation and DNA methylation at silent loci, we compiled a collection of homozygous transfer DNA insertion mutants in genes containing JmjC domains in Arabidopsis.
To determine whether predicted catalytic triad of AHO-3, Ser 191, Asp 256 and His 285, is required for thermotactic plasticity, we constructed mutant aho-3 genes containing mutations that disrupt the probable Ser His Asp catalytic triad and expressed it pan-neuronally in aho-3(nj15) mutants (Fig. S15A E in Supporting Information).
Plasmids expressing wild-type or mutant human MECP2 3′UTR reporter genes containing a 415-bp fragment including the most conserved miR-130a binding site (shown as the "240 site" in Fig. 4A) were constructed using psiCHECK2 vector (Promega).
Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer.
To meet these requirements, we have generated transgenic mice harboring a single copy of the full-length human mutant DRPLA gene containing 76 CAG repeats (13).
