MOLECULAR EVOLUTIONARY ANALYSIS OF THE YWVZ/7B GLOBIN GENE-CLUSTER OF THE INSECT CHIRONOMUS-THUMMI

We report the sequence of 8.1 kb of DNA containing the 3' end of one and seven other complete intronless globin genes from the YWVZ/7B locus of the dipteran Chironomus thummi thummi, One of these (ctt-v) appears to be a pseudogene by virtue of a premature termination codon, whereas the others encode apparently functional globin polypeptides. Taken together with previously published data, the C. th, thummi YWVZ/7B locus codes for at least 11 globins, five of which differ from one another by no more than two amino acids, In contrast, only nine globin genes are found in a comparable genomic clone isolated from C. th. piger. As indicated by sequence alignment, this difference in copy number can be attributed to a loss of one gene (fusion of globin genes 7B8 and 7B10) in the piger lines, coupled with a gain (globin gene 7B9) in the thummi lineage. Comparisons between the thummi and piger sequences showed that YWVZ/7B intergenic regions have maintained a level of 91% similarity since the thummi/piger divergence: most differences are simply due to single base substitutions or insertion/deletion events in either the thummi or the piger DNA, but three instances of partially overlapping deletions were also detected. A phylogenetic analysis of YWVZ/7B gene products was conducted, from which a plausible reconstruction of the evolutionary history of the locus was obtained. In addition, alignment of globin 7B amino acid sequences suggested that globin genes 7B2 and 7B3 (reported at the protein and cDNA level, respectively, but not contained on the C. th. thummi or C, th. piger genomic clones) are possibly chimeric genes. Given the trend toward expansion of the C, thummi globin gene family in general and of the globin 7B sub-family in particular, we propose that increased copy number of these genes has been positively selected as a mechanism to achieve a high Hb concentration in the larval hemolymph.