S1 Supplemental Data Wee1B Is an Oocyte-Specific Kinase Involved in the Control of Meiotic Arrest in the Mouse Seung Jin Han, Ruby Chen, Maria Paola Paronetto, and Marco Conti Supplemental Results and Discussion Wee1A, and homologous sequences are found in human and Japa- nese silver crucian carp (ginbuna) [S1–S4]. Mouse Wee1B shares 36% identity in the N-terminal region, 79% homology in the central Properties of the Mouse Wee1B Gene The mouse Wee1B clone of 2430 bp nucleotides corresponds to a kinase region, and 55% homology in the short C-terminal region with Xenopus Wee1A. However, it is clearly different from the previously gene in chromosome 6, and its translation predicts a protein of 555 amino acids (Figure S1A). Mouse Wee1B is orthologous to Xenopus described mammalian Wee1 because mouse Wee1 and Wee1B se- Figure S1. Sequence and Domain Organiza- tion of Wee1B Protein (A) Alignment of amino acid sequence of Wee1 family members. Xenopus Wee1A (XeWee1A, GenBank accession number P47817), human (AAD04726), and deduced amino acid sequences of the cloned mouse Wee1B were used for alignment of amino acid sequences. Mouse Wee1B sequence was de- posited with accession number DQ011691. Sequence alignment was performed with ClustalW 1.8 routine. Residues identical among two or more sequences are boxed in black. Numbers at left refer to adjacent amino acids, and the initiating methionine (M) is des- ignated as +1. Asterisks indicate the amino acids that are conserved among all known members of the Wee1 kinase family, but not in other eukaryotic protein kinases [S5]. A downward arrow indicates a potential 14-3-3 protein binding site (RSXS, [S13]), and up- ward-pointing arrows indicate the putative 14-3-3 binding site in the mouse Wee1B pro- tein. A solid bar indicates the conserved non- catalytic sequences required for optimal Wee1 kinase activity [S1]. The amino acid (K237) marked with a dot was changed to methionine to generate the kinase-dead mu- tant, mWee1B. The dashed line indicates the amino acid sequence of the peptide used to generate mWee1B-specific antibodies in rats and rabbits. (B) Comparison of domain organization in mouse Wee1 and mWee1B proteins. The two proteins share a highly conserved kinase do- main (73%) and have weak homology at the N- (26%) and C-terminal (38%) regions. Puta- tive PKA, Plk, and Cdc2 phosphorylation sites are indicated by the different shapes reported in the figure, and putative nuclear localization signals (NLSs) are indicated by boxes. The human Wee1 protein’s region shown to bind SCF -TrCP after plk and Cdc2 phosphorylation is reported as a line. (C) Phylogenic tree for Wee1 proteins from various organisms. The compared proteins are Xenopus laevis Wee1A (XeWee1; acces- sion number U13962), human Wee1 (huWee1; U10564), mouse Wee1 (mWee1; D30743), Drosophila melanogaster Wee1 (DmWee1; AAC46913), S. pombe Wee1 (M16508), Saccharomyces cerevisiae Swe1 (ScSwe1; X73966), human Wee1B (AAD04726), Xeno- pus Wee1B (AF358869), and mouse Wee1B (DQ011691).