Skip to main content
Figure 1 | BMC Biochemistry

Figure 1

From: A sequence-dependent exonuclease activity from Tetrahymena thermophila

Figure 1

Design of oligonucleotide substrates and detection of a telomere-processing nuclease activity. (A) Schematic representation of the substrate structures utilized in detecting the telomere-processing nuclease in Tetrahymena extracts. Solid lines represent non-telomeric sequences, and the numbers indicate lengths of non-telomeric base pairs in the duplex region. Each gray circle represents a TTGGGG repeat unit, and each black circle is a complementary CCCCAA repeat unit. S inside a circle represents the streptavidin protein. The stars indicate the approximate location of radiolabels, and 4-prong symbols are biotinylation sites. The annealing components were: Ia-S1*:S18; Ib-S1:S18*; IIa-S2*:S18; IIb-S2:S18*; IIIa-S1*:S19; IIIb-S1:S19*; IVa-S3*:S20; IVb-S3:S20*; Va-S3*:S21; Vb-S3:S21*; VIa-S4*:S20; VIb-S4:S20*. (B) Tetrahymena crude cell extracts were assayed with Ib and IVb and with/without streptavidin. ~ 1.2 μg total proteins from the extracts were incubated in each reaction for 15 min. (C) Nuclease purification scheme is presented. (D) The nuclease is 100 kDa in size. Chromatographs of gel filtration runs for standards and nuclease samples are shown in a superimposed graph. The peaks of standard proteins are labeled with numbers: peak 1-bovine thyroglobulin, 670,000; peak 2-bovine γ-globulin, 158,000; peak 3-chicken ovalbumin, 44,000; peak 4-horse myoglobin, 17,000; peak 5-vitamin B12, 1,350. The arrow indicates where the nuclease activity was eluted off the column. The apparent molecular weight was calculated as described under Methods. The activity corresponding to the fractions were indicated in the second panel with ~ 300-600 ngs protein per reaction.

Back to article page