Fig. 2. Mouse Ciz1 amino-acid sequence features, exon usage and expression constructs. Antibody V was used to clone the gene for p100 from a mouse embryo expression library (see Materials and Methods), which was identified as Ciz1. (A) Alignment of mouse Ciz1 variants. The predicted full-length Ciz1 amino-acid sequence (Full) is identical to a mouse mammary tumour cDNA clone (BC018483), while embryonic Ciz1 (ECiz1, AJ575057), and a melanoma-derived clone (AK089986) lack two discrete internal sequences. In addition, the first available methionine in ECiz1 (Met84) is in the middle of exon 3, which excludes a polyglutamine rich region from the N terminus. Stars indicate threonine residues changed by site-directed mutagenesis in the constructs shown in D. Amino-acids that correspond to codons targeted by siRNAs are underlined. (B) Mouse Ciz1 is encoded by 17 exons. Coding exons are shown in grey, alternatively spliced regions in mouse ECiz1 are black. (C) Sequence features and putative domains in ECiz1. The C terminal `matrin 3 domain' has homology with the nuclear matrix protein matrin 3 (Nakayasu and Berezney, 1991). The positions of sequences absent from ECiz1 are indicated by triangles. (D) ECiz1 and derived truncations and point mutants used in cell-free DNA replication experiments. Numbers in parentheses relate to amino-acid positions in the full-length form of mouse Ciz1, shown in A. Stars indicate putative phosphorylation sites made unphosphorylatable by site-directed mutagenesis.