Boll weevil (Anthonomus grandis) eggs contain two yolk proteins, YP47 and YP160. Using anti-YP160 antiserum as probe, a partial-length complementary DNA (cDNA) was isolated from a lambda gt11 adult female cDNA library. A second partial-length cDNA was isolated from a lambda gt10 adult female cDNA library by differential screening with male vs. female cDNAs. Northern blot analysis showed that each cloned cDNA hybridized to a 6-kb female-specific transcript. These cDNAs were used to probe a genomic library, and two overlapping genomic clones were obtained that span the boll weevil vitellogenin gene. The entire transcription unit was sequenced, and introns were mapped by a combination of primer extension experiments, S1 nuclease protection experiments, and polymerase chain reaction-mediated synthesis of two additional cDNA clones. Based on these data, the vitellogenin mRNA is 5511 nucleotides [plus a poly(A) tail of undetermined length] and specifies a provitellogenin of 1790 amino acids. The deduced protein has a Glu+Gln content of 16.3%, which is a relatively high value that is typical of most vitellogenins. Protein sequence similarities including Cys clusters conserved between boll weevil vitellogenin and Xenopus laevis A2 or Caenorhabditis elegans vit-5 vitellogenins indicated that the boll weevil protein is a member of the ancient nematode-vertebrate vitellogenin family. Moreover, the six introns in the boll weevil vitellogenin gene interrupt the coding region at positions closely or exactly corresponding to a subset of the positions of the 34 vertebrate vitellogenin introns, further supporting the argument for a common evolutionary relationship. This report represents the first complete nucleotide sequence and structural analysis of a nondipteran insect vitellogenin gene.
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.
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.
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