All previously reported chironomid globin genes are intronless, suggesting that the ancestral chironomid globin gene was also intronless. In this study, the coding regions of the closely linked Chironomus thummi globin (Gbs) II beta and IX genes are shown to be interrupted by noncoding DNA bounded by a 5'-GT and a 3'-AG. Both genes have appropriately placed transcription and translation signals. Polymerase chain reactions on genomic DNA with oligonucleotides flanking and within the putative Gb II beta intron generated products the size predicted for a gene with a 64-nucleotide intron, and sequencing of a cloned PCR fragment also revealed the intron. A partial-length Gb II beta cDNA sequence exactly matches that of the Gb II beta coding regions. We conclude that the intron-containing chironomid globin genes are functional. Regions of the Gb II beta and IX genes spanning the introns are more similar (86%) than the exons themselves (72% similarity), possibly due to partial gene correction. Surprisingly, Gb II beta and IX gene homologues in C. tentans are intronless. If the common ancestor of chironomid globin genes was not intronless, introns were lost in at least three C. thummi globin-gene lineages, and more recently by Gb II beta and Gb IX genes in C. tentans. If, as previously believed, the ancestral chironomid globin gene was intronless, the ancestral chironomid globin gene was intronless, then an intron was recently acquired in only one C. thummi globin sublineage. These alternatives are discussed.

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.

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 subfamily 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.

While globin genes ctt-2beta and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2beta and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species.

Salivary glands in aquatic larvae of Chironomus are responsible for formation of a fiber that larvae use to construct feeding tubes. Major constituents of this fiber include a family (the sp-I family) of high Mr (1 X 10(6) secretory polypeptides. Because of our interest in the polypeptide composition and polymerization of the salivary fiber we conducted a survey of the electrophoretic pattern of sp-I components found in salivary glands obtained from individual larvae. The survey encompassed ten strains of Chironomus tentans, three strains of Chironomus pallidivittatus and four strains of Chironomus thummi. Salivary glands from C. tentans and C. pallidivittatus contained at least four sp-I components (sp-Ia, sp-Ib, sp-Ic and sp-Id) that behave identically with regard to their electrophoretic mobility and detectability when larvae were exposed to galactose or glycerol. Sp-I components in C. thummi were generally fewer and not directly comparable by electrophoretic mobility to sp-I components in the other two species. During this survey two important alterations were observed in the electrophoretic pattern of sp-I components obtained from C. tentans and C. pallidivittatus. First, all four sp-I components exhibited, with a low frequency, double bands that appeared as slow-versus-fast electrophoretic variants of a particular component. Secondly, the relative steady-state level of each sp-I component fluctuated in comparison to other sp-I components in the same extract. This fluctuation varied such that any one sp-I component might appear as a single prominent component.(ABSTRACT TRUNCATED AT 250 WORDS)