['Although the sequence requirements for translation initiation regions have been frequently analysed, usually the highly expressed genes are not treated as a separate dataset.', 'To investigate this, we analysed the mRNA regions downstream of initiation codons in nine bacteria, three archaea and three unicellular eukaryotes, comparing the dataset of highly expressed genes to the dataset of all genes. In addition to the detailed analysis of the nucleotide and codon frequencies we compared the N-termini of highly expressed proteins to the N-termini of all proteins coded in the genome.'] Do highly expressed proteins have an increased frequency of alanine in the second amino acid position?