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He age-dependent loss of transition zone HaXS8 Purity & Documentation nuclei and earlier look of full-length synapsis in pph4.1 mutants suggests that chromosomes have much less time for you to actively search for partners, and are much less capable to delay synapsis in response to nonhomology, as they age. Even so, younger pph-4.1 mutant animals show no raise in autosomal pairing levels relative to older animals. For that reason we infer that young pph-4.1 mutants retain the capability to delay synapsis in the absence of homologousPhosphatase Handle of Meiotic Chromosome DynamicsPLOS Genetics | plosgenetics.orgPhosphatase Handle of Meiotic Chromosome DynamicsFigure five. DSB initiation is perturbed in an age-dependent manner in pph-4.1 mutants. (A) Wild-type and pph-4.1 nuclei shown with DAPI staining in magenta and a-RAD-51 staining in green. Top, c-irradiation at 10Gy restores RAD-51 staining to pph-4.1 nuclei. Bottom, quantitation of RAD-51 focus formation in wild-type and mutant animals. RAD-51 concentrate numbers are depicted as a box plot, with box indicating imply and quartiles. Significance was assessed by way of the Mann-Whitney test. Three gonads were scored for every situation; the numbers of nuclei scored in zones 1 are as follows: for wild-type, 316, 312, 256, 252, 231, 198, 120; for pph-4.1, 231, 244, 237, 245, 231, 205, 136. (B) Quantitation of RAD-51 foci with increasing maternal age. Numbers of foci in every of 7 zones are depicted with box plots as in (A). Leading, focus numbers compared involving rad-54 and rad-54; pph-4.1 animals at 24 h post-L4. Bottom, comparison at 72 h post-L4. Asterisks indicate important differences as a result of loss of pph-4.1; diamonds involving best and Bottom graphs show significance as a result of age; comparisons had been performed by way of the Mann-Whitney test. Three gonads were scored for each condition; the numbers of nuclei scored in zones 1 are as follows: for rad-54 24 h, 252, 313, 443, 397, 311, 236, 111; for rad-54 72 h, 237, 321, 397, 467, 395, 268, 64; for rad-54; pph-4.1 24 h, 288, 288, 306, 359, 300, 232, 70; for rad-54; pph-4.1 72 h, 255, 230, 262, 251, 229, 218, 118. doi:10.1371/journal.pgen.1004638.gpairing, but this delay will not cause higher pairing levels on account of the absence of PPH-4.1. The SUN-1 protein is ordinarily phosphorylated through the transition zone and early pachytene, and meiotic errors are correlated with persistence of SUN-1 phosphorylation [8]. We therefore tested whether or not the phosphorylation state of SUN-1 in pph-4.1 mutants changed in correlation using the shortened transition zone in older germlines, using an antibody that particularly detects SUN-1 phosphorylated on Ser8 (SUN-1:Ser8p) (Figure 7). We measured the proportion of your gonad occupied by the SUN-1:Ser8p signal at 24 h, 48 h, and 72 h post-L4 as an indication with the persistence of SUN-1 phosphorylation. At all timepoints, we observed a substantial increase in the proportion on the meiotic zone (from TZ entry to cellularization) containing cells positive for SUN-1:Ser8p in pph-4.1 mutants compared to wildtype (Figure 7B). SUN-1:Ser8p is limited to nuclei having a transition zone or early pachytene appearance in all wild-type gonads, and in pph-4.1 mutants at 24 h post-L4. Nevertheless, at 72 h post-L4, pph-4.1 mutant gonads also include late pachytene nuclei with SUN-1:Ser8p staining (Figure S7B). This observation explains the persistence of SUN-1:Ser8p-positive nuclei in aged pph-4.1 mutants with shorter transition zones and suggests that SUN-1 phosphorylation and nuclear morphology are uncouple.