Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus

Linker histone H1 is required for mitotic chromosome architecture in Xenopus laevis egg extracts and, unlike core histones, exhibits rapid turnover on chromatin. Mechanisms regulating the recruitment, deposition, and dynamics of linker histones in mitosis are largely unknown. We found that the cytoplasmic histone chaperone nucleosome assembly protein 1 (Nap1) associates with the embryonic isoform of linker histone H1 (H1M) in egg extracts. Immunodepletion of Nap1 decreased H1M binding to mitotic chromosomes by nearly 50%, reduced H1M dynamics as measured by fluorescence recovery after photobleaching and caused chromosome decondensation similar to the effects of H1M depletion. Defects in H1M dynamics and chromosome condensation were rescued by adding back wild-type Nap1 but not a mutant lacking sites subject to posttranslational modification by glutamylation. Nap1 glutamylation increased the deposition of H1M on sperm nuclei and chromatin-coated beads, indicating that charge-shifting posttranslational modification of Nap1 contributes to H1M dynamics that are essential for higher order chromosome architecture

Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments

Microinjection of oligodeoxynucleotides (ODNs) complementary to cellular mRNAs has been advanced as an experimental approach to degrade target mRNAs in vivo and thereby obtain information as to the function of their cognate proteins. It is shown here that ODNs can induce a variety of aberrations in cell metabolism and structure when injected into Xenopus oocytes. Examination of histological sections of ODN-injected oocytes revealed the frequent abnormal accumulation of heavily staining basophilic material in the area of the germinal vesicle (gv). Ultrastructural analysis detected further abnormalities including blebbing of the plasma membrane, anomalous cytoskeletal structures, hyperorganised annulate lamellae, hyperinvagination of the gv, and formation of irregular nucleoli within the gv. Analysis of newly synthesised proteins by [35S]methionine radiolabelling of oocytes demonstrated that ODN injection can trigger a general decrease in both label uptake and protein synthesis. Qualitative effects on protein synthesis could also be observed, particularly a decrease in synthesis of high molecular weight proteins. The severity of ODN-induced effects is dose-dependent and highly variable from ODN to ODN. The previously reported delay in progesterone-induced maturation observed in oocytes depleted of the maternal mRNA D7 by ODN-directed degradation (Smith R. C., Dworkin M. B. and Dworkin-Rastl E. (1988) Genes and Devpt. 2, 1296–1306) is most likely a result of nonspecific ODN effects in the oocyte. Oocytes injected with effective antisense D7 ODNs that do not display detectable side effects matured with normal kinetics.</jats:p