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Material and Methods

RNAi in C. elegans embryos:

The preparation of double stranded RNA and imaging were performed as in (Kaitna et al., 2002).

Protein sequence family searches and multiple alignment procedures

Prior to database searches, query sequences were routinely screened for compositionally biased, low-complexity regions (Wootton and Federhen, 1996), transmembrane segments (von Heijne, 1992) and coiled coils (Lupas et al., 1991). Protein sequence database searches with remaining query sequence parts were carried out with PSI-BLAST (inclusion E-value threshold 0.001, no filter but compositional based statistic (Schaffer et al., 2001)). Multiple alignments of hits within one family were generated with T-coffee (Notredame et al., 2000) and their superposition was produced with the profile-to-profile alignment option of Clustalx (Jeanmougin et al., 1998). The resulting alignments were used as starting points for the generation of hidden Markov substitution models (HMM) (Eddy, 1998), which have been used for searches in the non-redundant database in the local alignment mode. Similarly, the alignments were transformed into position specific scoring matrices (PSSMs) for usage with RPS-BLAST (Schaffer et al., 2001).

References:

Eddy, S. R. (1998). Profile hidden Markov models, Bioinformatics 14, 755-63.[PUBMED]

Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G., and Gibson, T. J. (1998). Multiple sequence alignment with Clustal X, Trends Biochem Sci 23, 403-5. [PUBMED]

Kaitna, S., Pasierbek, P., Jantsch, M., Loidl, J., and Glotzer, M. (2002). The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis, Curr Biol 12, 798-812.[PUBMED]

Lupas, A., Van Dyke, M., and Stock, J. (1991). Predicting coiled coils from protein sequences, Science 252, 1162-4.[PUBMED]

Notredame, C., Higgins, D. G., and Heringa, J. (2000). T-Coffee: A novel method for fast and accurate multiple sequence alignment, J Mol Biol 302, 205-17.[PUBMED]

Schaffer, A. A., Aravind, L., Madden, T. L., Shavirin, S., Spouge, J. L., Wolf, Y. I., Koonin, E. V., and Altschul, S. F. (2001). Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements, Nucleic Acids Res 29, 2994-3005.[PUBMED]

von Heijne, G. (1992). Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule, J Mol Biol 225, 487-94.[PUBMED]

Wootton, J. C., and Federhen, S. (1996). Analysis of compositionally biased regions in sequence databases, Methods Enzymol 266, 554-71.[PUBMED]