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Figure 4.
Copper-resistance and U4-cs1 suppression analyses of various
β-finger mutations. (A) All β-finger mutations grow similarly
to the WT at 30°C, 18°C, and 37°C. Only one
concentration point in the serial dilution is shown. (B)
Copper-resistance assay indicates that V1860D, T1865K, A1871E,
and T1872E grow worse than the WT in both the BSG and UuG
reporters at 0.05 mM Cu^++ concentration, characteristic of
first-step alleles. Mutant H1863E grows better than the WT in
both the BSG and UuG reporters at 0.2 mM Cu^++ concentration,
characteristic of second-step alleles. V1862D behaves similarly
to the WT and does not demonstrate a clear first- or second-step
allele phenotype. Known first-step allele R1753K and second-step
allele prp8–162 (V1870N) are used as positive controls and
labeled with +. (C) Primer extension experiment indicates that
V1860D, T1865K, A1871E, and T1872E are first-step alleles, which
demonstrate increased lariat intermediate, reduced mRNA product,
and reduced second-step efficiency compared with the WT. H1863E
is a second-step allele, which demonstrates decreased lariat
intermediate, increased mRNA product, and increased second-step
efficiency compared to the WT. V1862D is neither a clear first-
nor second-step allele. R1753K and prp8–162 (V1870N) are used
as positive controls for first and second-step alleles, and the
corresponding lanes are labeled with +. pBR322 DNA digested with
MspI is used as a molecular weight marker. (D) V1860D (positive
control, designated with +) but no other β-finger mutants
tested suppress the U4-cs1 phenotype at 18°C. Only one
concentration point in the serial dilution is shown.
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