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Lymorphism in the native Bat1 as well as being relevant for the creation of Bat1 derivatives with novel specificity (dBats). We hypothesize that nonRVD polymorphisms may have two functionally relevant, non-mutually-exclusive, effects. (i) The formation of unique but functionally equivalent repeat interfaces that stabilize the superhelical structure formed by tandem-arranged repeats (4,5) (superstr
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Red DNA bases. RVD composition and target sequence are key parameters determining affinity of TALE NA interactions and these were kept constant in our dBat tests as far as possible. For the repeat switch tests, we exchanged repeats with RVDs paired to the same base in BEBat1 allowing the wildtype target construct to be used in each case. For the RVD switch constructs, where possible we exchanged R
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E 6A). Overall, the impact on activity of the RVD switch constructs showed no single trend with some superior and some inferior to the wild type. Of the four repeat switch constructs none reached the activation level of acBat1 (Figure 6B). Notably, dBat repeat switch 3, in which core repeats 11 and 12 were exchanged, was unable to induce the reporter above background levels. Thus the repeat switch
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E 6A). Overall, the impact on activity of the RVD switch constructs showed no single trend with some superior and some inferior to the wild type. Of the four repeat switch constructs none reached the activation level of acBat1 (Figure 6B). Notably, dBat repeat switch 3, in which core repeats 11 and 12 were exchanged, was unable to induce the reporter above background levels. Thus the repeat switch
1
Lymorphism in the native Bat1 as well as being relevant for the creation of Bat1 derivatives with novel specificity (dBats). We hypothesize that nonRVD polymorphisms may have two functionally relevant, non-mutually-exclusive, effects. (i) The formation of unique but functionally equivalent repeat interfaces that stabilize the superhelical structure formed by tandem-arranged repeats (4,5) (superstr
1
Rminal repeats are typically inflexible, though alternative repeat -1 modules have recently been described (24,25). We tested acBat1 deletion derivatives to test if this paradigm applies to Bat1. First, we tested variants of acBat1 lacking 2 ( 18?0), 4 ( 16?0), 6 ( 14?0) or 8 ( 12?0) core repeats (Figure 5A and Supplementary Figure S10). The later half of repeat 20 and repeat +1 were retained in e
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Red DNA bases. RVD composition and target sequence are key parameters determining affinity of TALE NA interactions and these were kept constant in our dBat tests as far as possible. For the repeat switch tests, we exchanged repeats with RVDs paired to the same base in BEBat1 allowing the wildtype target construct to be used in each case. For the RVD switch constructs, where possible we exchanged R
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E the reporter to levels above background. If we assume that each repeat contributes a certain amount of affinity to the Bat1?Nucleic Acids Research, 2014, Vol. 42, No. 11BEBat1 interaction then fewer than 17 repeats may simply be insufficient for an interaction strong enough to lead to reporter activation. This is in accordance with results from TALE repeat arrays showing that a certain number of