Investigations into Xpat, a novel gene expressed in the germ plasm and primordial germ cells of Xenopus laevis
To determine the expression pattern of XPAT (Xenopus primordial germ cell associated transcript) protein in Xenopus oocytes, XPAT-GFP fusion proteins were generated. When XPAT was amino-terminally tagged with GFP it became localised to the nuclei of stage VI Xenopus oocytes. However, when carboxy-terminally tagged with GFP, XPAT also translocated to the vegetal pole of stage VI oocytes. XPAT-GFP formed particles (1 to 2.5mm in diameter) which aggregated into large (10 to 50mm) granular structures at the vegetal pole. These particles looked exactly like those seen after in situ hybridisation to germ plasm RNAs. The granules of XPAT-GFP were larger than endogenous germ plasm granules seen in stage VI oocytes; they were more consistent with those observed in 2-cell embryos during germ plasm aggregation. Studies involving the use of the anticytoskeletal drugs colcemid, nocodazole and cytochalasin D and the microtubule stabilising agent taxol indicated that microtubular transport was important in the location of XPAT-GFP. Several attempts were made to raise antibodies to XPAT peptides, but at present the endogenous expression pattern of XPAT protein is unresolved. To investigate possible domain structure of XPAT, one carboxy-terminal and three amino-terminal deletion variants of XPAT-GFP were constructed. An N-terminal deletion protein lacking the first 61 amino acids of XPAT was able to form small particles, but none of the deletion proteins exhibited vegetal localisation or formed large aggregates in Xenopus oocytes. The N-terminal deletion proteins all became predominantly localised to the nucleus; protein motif analysis revealed that XPAT contains a putative bipartite NLS in its carboxy-terminal region. The C-terminal deletion protein, which lacked the putative NLS, was evenly distributed throughout the nucleus and cytoplasm of Xenopus oocytes. XPAT was shown to be able to bind to homopolymeric RNAs in vitro. When Xpat mRNA was depleted from stage VI Xenopus oocytes (by injection of an antisense oligo) levels of DEADSouth and XVLG1 mRNAs decreased substantially.