Linker-Scanning Mutagenesis

Abstract: The B-cell-specific B29 and mb1 genes code for covalently linked proteins (B29 or Ig beta and mb1 or Ig alpha, respectively) associated with membrane immunoglobulins in the antigen receptor complex on B cells. We have functionally analyzed the upstream region of the B29 gene and have identified a 164-bp region which comprises the minimal promoter responsible for B-cell-specific transcription. Linker scanning mutagenesis of this minimal promoter has established that both the previously identified octamer motif and a DNA motif that binds an unknown protein factor are critical for B29 gene expression in a pre-B-cell and B-cell line. Further mutations showed that binding motifs for Ets, microB/LyF1, and Sp1 also significantly contributed to the overall activity of the minimal B29 promoter. However, the relative contribution of certain motifs to promoter activity was different in a pre-B versus a B-cell line. The microB/LyF1 motif was necessary for full promoter activity in the pre-B cells but was not required in the B cells.

Abstract: In this volume experts from industrial and academic laboratories describe easily reproducible and up-to-date procedures for site directed and random mutagenesis. Site-directed protocols include those based on strand selection, PCR (including "splicing by overlap extension" and the "megaprimer" procedure), the ligase chain reaction, positive antibiotic selection, unique restriction site elimination, gapped heteroduplex formation, and solid-phase capture with the biotin/strepavidin system. Many can be used with virtually any double-stranded DNA plasmid. The bookcovers genesis, linker scanning mutagenesis, nested deletion mutagenesis, and a specialized E coli mutator strain.

Abstract: Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal ‘stem’ of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system.