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 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.

Intron-containing globin genes in the insect Chironomus thummi.

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.

Nucleotide sequence, structure and developmental regulation of LHP82, a juvenile hormone-suppressible hexamerin gene from the waxmoth, Galleria mellonella.

We cloned and sequenced a composite cDNA corresponding to the 2.6 kb last instar-specific, juvenile hormone-suppressible Lhp82 mRNA from Galleria mellonella. The identity of the cDNA was confirmed by N-terminal amino acid sequencing of the purified Lhp82 subunit. In addition, we sequenced all coding regions and most of the intronic DNA as well as 1300 nucleotides of 5' flanking DNA from the Lhp82 gene. The eight exons of the Lhp82 gene specify a pre-protein of 706 residues, including the signal peptide of 18 amino acids. The deduced amino acid sequence of Lhp82 contains four potential N-glycosylation sites, and the calculated isoelectric point and molecular weight of secreted Lhp82 are pI = 6.43 and 79.9 kDa, respectively. Inspection of the 5' flanking and intronic regions of Lhp82 DNA revealed a 301 nucleotide cassette in intron 6 that appears to be a recently inserted repetitive element. We also performed Northern blot and nuclear run-off transcription experiments in order to determine the basis for Lhp82 gene inactivity after day 2 of the pupal stage. The results of these studies indicate that Lhp82 transcription is permanently shut off by the ecdysteroid pulse which occurs in the absence of juvenile hormone beginning 24 h post-pupation.

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

The boll weevil vitellogenin gene: nucleotide sequence, structure, and evolutionary relationship to nematode and vertebrate vitellogenin genes.

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.

Differential regulation of insect globin and actin mRNAs during larval development in Chironomus thummi.

S1 nuclease protection assays were used to measure changes in the steady-state levels of six different globin (Gb) mRNAs in the midge, Chironomus thummi thummi (C. thummi, Diptera) during larval development. Two distinct patterns of change were observed. GbI, IV, VIIB-4 and VIIB-5 transcripts were present in 3rd instar larvae, rose from low levels immediately post-moult to peak levels by day 2-3 of the 4th instar, and then declined, reaching near-basal levels by day 7-8. In contrast, transcripts of GbIII (known from previous studies to be 4th instar-specific) and VI, which were undetectable in the 3rd instar, rose to high levels by day 2 of the 4th instar, but remained elevated thereafter. Our data further showed that closely linked Gb genes were not necessarily expressed in a coordinate manner. Unlike the Gb mRNAs, actin (Act) mRNA levels (measured by slot-blot hybridization to a heterologous probe) increased progressively as a proportion of total RNA during 4th instar development. Therefore, the regulation of C. thummi Gb transcript levels is specific, differing from that of Act and among the Gb mRNAs themselves. Elevated 20-hydroxyecdysone (HE) titer at the 3rd-4th instar moult correlates with the low steady-state levels of Gb mRNAs immediately post-moult. However, other aspects of Gb mRNA profiles cannot be explained on the basis of a direct repressive effect by HE on Gb gene transcription.

Multiple clustered genes of the haemoglobin VIIB subfamily of Chironomus thummi thummi (Diptera).

The nucleotide and inferred amino acid sequence of three globin VIIB genes from the midge, Chironomus thummi thummi have been determined. These three genes are intronless, like all previously reported Chironomid globin genes. Transcription of globin genes VIIB-4, VIIB-5 (and/or VIIB-6) and VIIB-7 was demonstrated by S1 nuclease protection analysis. Comparison of actual and inferred globin VIIB amino acid sequences reveals that (i) most of the amino acid substitutions are conservative in nature, and (ii) none of the substitutions are expected to interfere with the ability of the globins to bind haem. Nucleotide sequence comparisons suggest that gene duplications and partial gene corrections (conversions) have occurred recently in the globin VIIB subfamily locus.

Deoxynucleotide sequence of an insect cDNA codes for an unreported member of the Chironomus thummi globin family.

Synthetic oligonucleotides served as probes to isolate insect globin clones from a Chironomus thummi cDNA bank. The cDNA insert of one clone (pC-S9) was completely sequenced by the dideoxy termination procedure. Beginning at the 5' end of the coding region, the 584 base pair sequence encodes most of an N-terminal hydrophobic signal sequence and the complete sequence for a mature secreted globin, and contains a polyadenylation recognition site 3' to an appropriate stop codon. The inferred amino acid sequence is that of an unreported variant of hemoglobin VIIB. Based on the number of differences between Hb VIIB chains, the pC-S9 gene has been evolutionarily independent longer than the other (two) members of the globin VIIB subfamily.