Breakthroughs and Views How reliable re-adjustment is: correspondence regarding A. Fuglsang, ‘‘The Ôeffective number of codonsÕ revisited’’ Sayed-Amir Marashi * , Hamed Shateri Najafabadi Department of Biotechnology, Faculty of Science, University of Tehran, Enghelab Ave., Tehran, Iran Received 22 August 2004 Available online 17 September 2004 Abstract A. Fuglsang [Biochem. Biophys. Res. Commun. 317 (2004) 957–964] suggested that effective number of codons for individual amino acids (Nc-values) should be re-adjusted to the number of synonymous codons of those amino acids, in order to prevent the overestimation of the effective number of codons. Here, it is shown that re-adjustment at the level of individual amino acids results in loss of considerable amounts of information. Furthermore, we have shown that theoretical Nc-values are functions of GC3s (and GC1s); as a result, when an amino acid Nc-value exceeds the related theoretical Nc-value, the implication of re-adjust- ment depends on the GC composition of the gene. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Effective number of codons; Nc; Re-adjustment; Codon usage bias In a paper pertinent to March 2004 by Fuglsang [1], an altered form of Ôeffective number of codonsÕ [3] as a measure for codon bias is established, declared as ^ Nc  ¼ ^ Nc Ala þ ^ Nc Arg þþ ^ Nc Val ; ð1Þ where each of the individual values represents the effec- tive number of codons for the related amino acid, cal- culated according to Wright [3], and where each individual Nc-value is re-adjusted if it exceeds the num- ber of synonymous codons of the related amino acid. ^ Nc  was proposed as an alternative to ^ Nc since it does not overestimate the effective number of codons, as ^ Nc does. We would like to draw attention to the consequence of application of ^ Nc  in the calculation of the effective number of codons for Escherichia coli K12 genes. As shown in Table 1, when individual Nc-values are calcu- lated for 4390 E. coli genes (GenBank Accession No. NC000913), in so many cases re-adjustment should be applied before computing ^ Nc  , showing that this is a more common phenomenon than an exception, like what Fuglsang [1] exemplified with lrp gene. Further- more, if we assume that the data presented in Table 1 can be generalized to other organisms with intermediate GC (which is not an implausible assumption) then, using average relative frequencies of occurrence of amino acids among genomes of different organisms [2], it can be simply calculated that in about 25% of cases, when an individual Nc-value is calculated, re-adjustment is needed. This is more likely to lose a lot of information rather than correcting the previous method of calculation. In addition, this method shows more need to be re- vised when the effect of re-adjustment of individual Nc-values of amino acids on the position of a gene with respect to the plot of ^ Nc vs. GC3s under H 0 of no selec- tion [3] and A = U, G = C is considered. Nc-plots for individual amino acids can be derived using Eqs. (1) and (2) in [1] for large nÕs, as 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.213 * Corresponding author. Fax: +98 21 6491622. E-mail address: marashie@khayam.ut.ac.ir (S.-A. Marashi). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 324 (2004) 1–2 BBRC