Figure 2.

Replacing endogenous Rpb1 with one that has 29 consensus heptads produces healthy homozygous flies

(A) Schematic for CRISPR-mediated replacement of the endogenous Drosophila CTD. Rpb1_HA and Rpb1_FLAG have the wild-type Drosophila CTD sequence with a HA or FLAG-tag attached to the end of the C-terminus. Rpb1^29con has 29 YSPTSPS motifs substituted for the wild-type CTD and a FLAG-tag attached to the end of the C-terminus. The sequence of the CTD (orange) in each fly line was verified by sequencing the PCR product amplified from genomic DNA with primers flanking the CTD (green arrows).

(B) Hatch rates of embryos at 24°C, measured 36h after egg deposition, n>300 for each genotype.

(C) Percentages of hatched embryos that developed into adults when raised at 24°C, n>200 for each genotype.

(D) Hatch rates of embryos at 30°C, measured 36h after egg deposition, n>300 for each genotype.

(E) Percentages of hatched embryos that developed into pupae when raised at 30°C, n>200 for each genotype.

(F) Quantification of recovery time after 14h of cold shock on ice, n=70 for Rpb1_HA, n=64 for Rpb1_FLAG; n=64 for Rpb1^29con (equal numbers of male and female adults were assayed). Dots represent individual adults. Red bars show the medians and interquartile ranges.

(G) Immunofluorescence with anti-HA and anti-FLAG antibodies on polytene chromosomes derived from salivary glands of third-instar larvae (FLAG-Rpb1^29con/Rpb1_HA) under non heat shock and heat shock conditions. Chromosomes derived from salivary glands lacking either tagged form of Rpb1 (FLAG-Rpb1^29con/+ or Rpb1_HA/⁺) served as negative controls. The heat shock puffs are readily visible as pointed by yellow arrows.

Two-sided chi-squared tests were used for (B) to (E). Mann-Whitney U tests were used for (F). ns: not significant (p>0.05), *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001 values were considered statistically significant from Rpb1_FLAG.