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A general tendency of more biased codon usage of genes with longer CDS length to higher expression level was found.
Furthermore, it is also evident from Figure 4B that among Caenorhabditis, NASP2 exhibits slightly more biased codon usage than NASP1.
Highly expressed genes are generally assumed to possess a more biased codon usage, and a higher GC content in comparison to genes with low expression [ 33].
Genes that are highly expressed have been suggested to possess a higher GC content and a more biased codon usage than genes with low expression levels [ 48].
For example, in Drosophila, genes that show more biased codon usage are enriched for GC-ending codons (Akashi 1994; Duret and Mouchiroud 1999) and tend to be expressed at higher levels.
Codon usage bias of the sequences was estimated by ENC (effective number of codons) that varies between 20 and 61, with the lower the value, the more biased codon usage [ 44].
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Our favored method is Figure 2A, which is to ask: As genes become more biased (uneven) in codon usage, which codon(s) for each amino acid increases in frequency?
We assessed the extent of codon bias between the two paralogs and observed that among fish lineages, NASP1 demonstrates more biased trends in codon usage than the NASP2.
In the case of ray-finned fish-specific NASP paralogs, we observed that NASP1 has higher GC content than NASP2 and accordingly shows more biased trends in codon usage.
AT content at 2nd and 3rd codon positions (65%and68%8% respectively) was more biased as compared to 1st codon positions (49%).
By convention, "preferred" codons in Drosophila refer to those identified as being used significantly more often in genes with highly biased codon usage compared with those with low levels of codon bias (Akashi 1995; Bachtrog 2007).
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