It has been believed that XIST RNA requires a discrete window in early development to initiate the series of chromatin-remodeling events that form the heterochromatic inactive X chromosome. Here we investigate four adult male HT-1080 fibrosarcoma cell lines expressing ectopic human XIST and demonstrate that these postdifferentiation cells can undergo chromosomal inactivation outside of any normal developmental context. All four clonal lines inactivated the transgene-containing autosome to varying degrees and with variable stability. One clone in particular consistently localized the ectopic XIST RNA to a discrete chromosome territory that exhibited striking hallmarks of inactivation, including long-range transcriptional inactivation. Results suggest that some postdifferentiation cell lines are capable of de novo chromosomal inactivation; however, long-term retention of autosomal inactivation was less common, which suggests that autosomal inactivation may confer a selective disadvantage. These results have fundamental significance for understanding genomic programming in early development.

cDNAs encoding two cytosolic and two chloroplastic ascorbate peroxidase (AsAP) isozymes from spinach have been cloned recently [Ishikawa et al. (1995) FEBS Lett. 367: 28, (1996) FEBS Lett. 384: 289]. We herein report the cloning of the fifth cDNA of an AsAP isozyme which localizes in spinach glyoxysomes (gAsAP). The open reading frame of the 858-base pair cDNA encoded 286 amino acid residues with a calculated molecular mass of 31,507 Da. By determination of the latency of AsAP activity in intact glyoxysomes, the enzyme, as well as monodehydroascorbate (MDAsA) reductase, was found to be located on the external side of the organelles. The cDNA was overexpressed in Escherichia coli (E. coli). The enzymatic properties of the partially purified recombinant gAsAP were consistent with those of the native enzyme from intact glyoxysomes. The recombinant enzyme utilized ascorbate (AsA) as its most effective natural electron donor; glutathione (GSH) and NAD(P)H could not substitute for AsA. The substrate-velocity curves with the recombinant enzyme showed Michaelis-Menten type kinetics with AsA and hydrogen peroxide (H2O2); the apparent Km values for AsA and H2O2 were 1.89 +/- 0.05 mM and 74 +/- 4.0 microM, respectively. When the recombinant enzyme was diluted with AsA-depleted medium, the activity was stable over 180 min. We discuss the H2O2-scavenging system maintained by AsAP and the regeneration system of AsA in spinach glyoxysome.

Two cDNA clones encoding stromal (SAP28) and thylakoid-bound (SAP22) ascorbate peroxidase were isolated from a spinach cDNA library constructed by greening cotyledons. The SAP22 and SAP28 contained an open reading frame encoding mature protein of 295 and 345 amino acids with calculated molecular mass of 32239 Da and 37710 Da, respectively, preceded by the common transit peptides of 70 amino acid residues. Interestingly, the N-terminal 364 amino acids of SAP22 were 100% identical with SAP28 except for one C-terminal amino acid residue (Asp-365), and the C-terminal of SAP22, which is the putative transmembrane segment, was 50 amino acids longer than that of SAP28.

No abstract

No abstract