The origin recognition complex (ORC) binds to origins of replication in budding yeast. We have cloned a Xenopus homolog of the largest ORC polypeptide (XORC1). Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. We have purified Xenopus ORC, consisting of a protein complex similar to yeast ORC. In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases. RLF-M, a component of the replication licensing system, also associates with chromatin early in the cell cycle but dissociates during S phase. We show that the assembly of RLF-M onto chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves b-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in b-casein turnover when the assay temperature is raised from 37 to 45 8C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAGresponsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.Expression of myelin-associated glycoprotein (MAG; siglec 4a), 1 is restricted to myelinating glial cells on myelin membrane adjacent to the axon and is required for maintenance of myelin integrity (1-4). In vitro, MAG inhibits outgrowth of postnatal neurons (5-8), involving activation of Rho GTPase, a key signaling step for the inhibitory effect of myelin on regeneration of neurons in vivo (9). MAG is therefore thought to contribute to the inhibitory properties of myelin, which is in part responsible for the lack of regenerative capacity of the central nervous system after injury or disease (10, 11).Like other siglecs, MAG binds to sialic acid residues at the termini of glycans on opposing cells through a sialic acid binding site located in the N-terminal V-set Ig domain (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). MAG binds specifically to terminal sialic acid residues in ␣2-3 linkage to galactose, which occurs in glycans linked ␤1-3 to GalNAc or GlcNAc or ␤1-4 to GlcNAc (12,[23][24][25]. Use of sialic acid analogues has identified specific groups on sialic acid essential for interaction with MAG (26), consistent with interactions seen between sialic acid and conserved amino acids in the siglec 1 crystal structure (21). It is also thought that the core glycan structure on which the terminal sialic acid is presented plays a role in recognition by MAG (23,26,27). The ability of MAG to bind specific gangliosides bearing terminal ␣2-3-linked sialic acid has been well documented. Gangliosides bind to MAG with the relative potencies GQ1b␣ Ͼ GT1a␣, GD1␣ Ͼ GD1a, GT1b Ͼ Ͼ GM3, GM4, whereas GM1, GD1b, GD3, and GQ1b do not support adhesion (23,27,28).Although the binding of MAG to sialylated glycans and gangliosides is well characterized, the functional importance of these intera...

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