Fig 1.
Telomerase activity in Daphnia.
A) Telomeric repeat amplification protocol (TRAP) assay of D. pulex extracts. The amount of cell extract used is indicated above each lane. HI: Heat Inactivated, CE: Cell Extract. TSR8 CT: positive control for PCR step. B) Quantification of the TRAP Assay in 1A. Student T-test was performed, p values are as follows: * = 6.15x10-6, ** = 6.63x10-6 (n = 3).
Fig 2.
Telomeric repeat sequence from Daphnia and sequence alignment of Daphnia telomerase (dTERT) with telomerase proteins from other species.
A) Telomeric repeat sequence of Daphnia and other species. H. americanus: Homarus americanus—Lobster, G. pulex: Gammarus pulex—Freshwater amphipod, B. mori: Bombyx mori—Silkworm/moth, A. viridus: Amaranthus viridus—Beetle, M. musculus: Mus musculus—Mouse, H. sapiens: Homo sapiens—Human. B) Identity and similarity percentages of dTERT with TERT from other species. C) D. pulex telomerase reverse transcriptase (DTERT) sequence alignment for the RNA-binding and reverse transcriptase domains. Green Boxes: conserved residues within the RNA binding domain. Black Boxes: conserved residues within the reverse transcriptase domain. Blue boxes: residues within the reverse transcriptase domain that are essential for nucleic acid binding.
Fig 3.
Telomerase from Daphnia embryos shows high processivity.
A) TRAP assay from egg stage 1 embryos and 1 week old adults of D. pulex and D. pulicaria. 250 ng of Daphnia extract was used in each lane. B) Quantification of the TRAP assay displayed in 2 A. C) Quantification of the processivity of telomerase from each displayed sample. The p values calculated from student T-tests are as follows: * = 6.15x10-6, ** = 5.56x10-6, *** = 0.0005,**** = 1.5x10-5; # = 0.0002, ## = 0.0002 (n = 4).
Fig 4.
Comparison of telomerase activity at different ages in D. pulex and D. pulicaria.
A) TRAP assay was performed using Daphnia extracts prepared at indicated ages. Lanes 1–3: D. pulex, lanes 4–6: D. pulicaria. B) Quantification of the TRAP assay displayed in 4 A. C) Quantification of the processivity of telomerase from each displayed sample. Student T-tests were performed, and p values are as follows * = 0.0005, ** = 0.0006, *** = 0.002, # = 0.001, ## = 0.034, ### = 0.0132, #### = 0.029 (n = 4).
Fig 5.
Life spans and telomere lengths in D. pulex (RW20) and D. pulicaria (LakeXVI-clone11).
A) Survivorship curves of clones RW20 (open squares) and XVI-11 (black circles). Error bars show age-specific standard errors from Kaplan-Meier survival probability estimates. B) Terminal Restriction Fragment (TRF) assay was performed to estimate the average telomere length of the Daphnia at various ages. The age in weeks is indicated above each lane. Lanes 1–3: D. pulex, lanes 4–6: D. pulicaria. M1 and M2: molecular weight markers; lambda HindIII digest and lambda BstEII digest respectively. C) Quantification of 5 A, error bars indicate standard deviation. Student T-tests were performed and p values are as follows: * = 1.03x10-5, ** = 0.007, *** = 2.48x10-5 (n = 3).