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Free-floating sections were stained with cresylechtviolett and four series of every tenth section were mounted on slides and coverslipped (see http://mbl.org/procedures/tissue_proc.htmlfor further details).
Sequence searches were performed with the web-based programs Blast and PsiBlast at http://www3.ncbi.nlm.nih.gov/BLAST/for sequences that were deposited in data bases and by tblastn at http://www.ncbi.nlm.nih.gov/MicrobJ3last/unfinishedgenome.htmlfor unpublished microbial sequences.
The genomic sequences of different COP1 orthologs were identified by BLASTn search with HsCOP1 or MmCOP1 cDNA sequence, or by tBLASTn search with HsCOP1 protein sequence in the following genomic blast sites: http://www.ncbi.nlm.nih.gov/genome/seq/HsBlast.htmlfor human genome; http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.htmlfor mouse genome; http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/agambiae.htmlfor mosquito; and http://www.ncbi.nlm.nih.gov/BLAST/fugu.htmlfor fugu genome.
The design of the evolution experiment has been described elsewhere [ 19 ] . In short, 12 lines were derived from a single, strictly asexual clone of E. coli B that has been in the laboratory for several decades (see: http://myxo.css.msu.edu/ecoli/strainsource.htmlfor more information).