Genomic organization and primary structure of five homologous pairs of intron-less genes encoding secretory globins from the insect Chironomus thummi thummi

From a Chironomus thummi thummi genomic library we have isolated two distinct recombinant phages, CttG-1 and CttG-3, each carrying a cluster of five homologous globin genes. In addition to the previously reported nucleotide sequence of globin gene D (Antoine and Niessing, 1984) we present the chromosomal arrangement, primary structure and predicted amino acid sequence of nine globin genes. The divergently transcribed globin genes all lack introns, they encode secretory preglobins each containing a highly conserved signal peptide. The amino acid sequences deduced from the globin genes correspond to globin III and variants thereof, to globin IV, and to a novel globin, whose direct amino acid sequence has not yet been reported.     

The nucleotide sequence and in situ localization of a gene for a dimeric haemoglobin from the midge Chironomus thummi piger

A cluster containing at least four globin genes was isolated by screening an lambda EMBL3 genomic DNA library of the midge Chironomus thummi piger (Ctp) with a heterologous haemoglobin (Hb) gene IV (HbIV) probe from Chironomus thummi thummi (Ctt). This globin gene cluster was localized by in situ hybridization to chromosome II. One globin gene together with its 5'- and 3'-flanking regions has been sequenced. It can be deduced from the sequence that it is a new member of the dimeric HbVIIB family. The Ctp HbVIIB-5 gene displays 91.8% nucleotide sequence homology to a HbVIIB cDNA sequence, reported previously. There is no evidence for intron/exon structure in the Ctp HbVIIB-5 gene.     

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.     

Molecular evolutionary analysis of theywvz/7B globin gene cluster of the insectChironomus 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 theywvz/7B locus of the dipteranChironomus thummi thummi. One of these (cttv) 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, theC. 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 fromC. 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 thepiger lines, coupled with a gain (globin gene 7139) in thethummi lineage. Comparisons between thethummi andpiger sequences showed thatywvz/713 intergenic regions have maintained a level of 91 % similarity since thethummi/piger divergence: most differences are simply due to single base substitutions or insertion/deletion events in either thethummi or thepiger DNA, but three instances of partially overlapping deletions were also detected. A phylogenetic analysis ofywvz/713 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 theC. th. thummi orC. th. piger genomic clones) are possibly chimeric genes. Given the trend toward expansion of theC. 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. Correspondence to: G. Bergtrom     

Molecular characterization of an insect genome: Chironomus thummi.

DNA extracted from Chironomus thummi larvae was studied by isopycnic centrifugation in CsCl, thermal denaturation and DNA-DNA reassociation techniques. The mean G+C content of the C. thummi DNA is 28-29% as indicated both by centrifugation in CsCl and thermal denaturation. According to optical reassociation analysis of total DNA and of isolated DNA fractions the C. thummi genome is composed of at least four components. About 80% of the DNA is classified as unique with a kinetic complexity of nearly 7 X 10(10) daltons. 6-8% intermediate DNA exhibits a kinetic complexity slightly above 10(8) daltons with a mean repetition frequency of 35. 11-13% fast-reassociating DNA has a kinetic complexity slightly above 10(6) daltons with a mean repetition frequency of 6000. 3-5% of the DNA cannot be properly studied by the optical reassociation technique and probably contains inverted repeats. The thermal denaturation behaviour of isolated DNA fractions indicated that most of the repetitive sequences in the C. thummi genome are tightly interspersed.     

The nucleotide sequence of an unusual non-transcribed spacer and its ancestor in the rDNA in Chironomus thummi.

The nucleotide sequence of the non-transcribed spacer (NTS) in the ribosomal DNA (rDNA) of Chironomus thummi thummi and Chironomus thummi piger, including major parts of the external transcribed spacer, is described. The NTS of the two subspecies are very different in length, (thummi, 7 kb, piger, 2 kb); this is due to the insertion into the NTS of C.th. thummi of a large cluster of highly repetitive DNA sequences which are not present in the NTS of C. th. piger. The repetitive sequences, called Cla elements, are present in high copy number elsewhere in the genome of C. th. thummi and, in lower copy number, in the genome of C. th. piger in which they are mainly in the centromeric regions. Sequencing of the NTS of thummi and piger yielded information on the junctions between the Cla element cluster and the original NTS sequence, as well as on the sequence of the integration site before the transposition has occurred. The integration site is characterized by a dA cluster at the one end and a dT cluster at the other.     

Sequence and evolution of the gene for the monomeric globin I and its linkage to genes coding for dimeric globins in the insect Chironomus thummi

We isolated genomic clones containing sequences encoding globins I and IA from a Chironomus thummi thummi genomic library. Three clones contain globin IA (ctt-1A) genes, while one contains a globin I (ctt-1) gene. The coding regions of the four genes are identical except for the single base substitution accounting for the globin I/IA polymorphism. The noncoding DNA flanking the coding region is more than 98% similar, confirming a previous hypothesis that the globin ctt-1 and ctt-1A genes are alleles. Hemoglobins I and IA are monomeric in the insect hemolymph. Earlier in situ hybridization studies suggested that monomeric and dimeric globin genes are clustered at different chromosomal loci. In situ hybridization of ctt-1 DNA to polytene salivary gland chromosomes places the ctt-1 gene on the same band as genes for the dimeric globins II and VIIB, forcing revision of the earlier hypothesis that genes for monomeric and dimeric globin genes are at different loci. The evolution of the ctt-1 and ctt-1A alleles and of the two globin gene loci are discussed. Correspondence to: G. Bergtrom     

Proton nuclear magnetic resonance study of the solution distal histidine orientation in monomeric Chironomus thummi thummi cyanomet hemoglobins. Dynamic stability of the heme pocket as monitored by labile proton exchange.

The 1H nuclear magnetic resonance spectral characteristics of the cyano-Met form of Chironomus thummi thummi monomeric hemoglobins I, III and IV in 1H2O solvent are reported. A set of four exchangeable hyperfine-shifted resonances is found for each of the two heme-insertion isomers in the hyperfine-shifted region downfield of ten parts per million. An analysis of relaxation, exchange rates and nuclear Overhauser effects leads to assignments for all these resonances to histidine F8 and the side-chains of histidine E7 and arginine FG3. It is evident that in aqueous solution, the side-chain from histidine E7 does not occupy two orientations, as found for the solid state, rather the histidine E7 side-chain adopts a conformation similar to that of sperm whale myoglobin or hemoglobin A, oriented into the heme pocket and in contact with the bound ligand. Evidence is presented to show that the allosteric transition in the Chironomus thummi thummi hemoglobins arises from the "trans effect". An analysis of the exchange with bulk solvent of the assigned histidine E7 labile proton confirms that the group is completely buried within the heme pocket in a manner similar to that found for sperm whale cyano-Met myoglobin, and that the transient exposure to solvent is no more likely than in mammalian myoglobins with the "normal" distal histidine orientation. Finally, a comparison of solvent access to the heme pocket of the three monomeric C. thummi thummi hemoglobins, as measured from proton exchange rates of heme pocket protons, is made and correlated to binding studies with the diffusible small molecules such as O2.     

Comparison of insect hemoglobins (Erythrocruorins) from Chironomus thummi thummi and Chironomus thummi piger (Diptera). The primary structure of the monomeric hemoglobin CTP III

The monomeric hemoglobin fractions of Chironomus thummi thummi (CTT) and Chironomus thummi piger (CTP) differ in the ratio of their components. The determination of the primary structure of the component CTP III was achieved by automatic Edman degradation of the native chain, the tryptic peptides and the C-terminal fragment, obtained by cleavage at the single tryptophan residue. It revealed two chains in the ratio 1:1 which share the ambiguity threonine/isoleucine in position 57 with CTT III. Whereas one chain is identical to the CTT III hemoglobin, the other differs in having isoleucine in position 105 and alanine in position 134. The CTP monomeric hemoglobin fraction comprises 8% of a component (CTP IV A) with a more negative charge than CTT IV but with an identical sequence up to position 44. This study reveals a very high polymorphism within Chironomus species and points out the need for more data at the gene level in order to provide better understanding of this striking phenomenon.     

Hemoglobins, XXIX. Sequence analysis of a dimeric hemoglobin (erythrocruorin), CTT-X, of Chironomus thummi thummi (Diptera).

The amino acid sequences of one of the dimeric hemoglobin components, CTT-X, of Chironomus thummi thummi (Diptera) are given. The sequences were determined by automatic Edman degradation of tryptic peptides and peptides obtained by specific chemical cleavages. CTT-X has two different polypeptide chains, each with 151 amino acid residues. The two polypeptide chains differ only in one amino acid. The sequences are discussed in the light of the sequences of other related heme-proteins.     

Nonreciprocal gonadal dysgenesis in hybrids of the chironomid midgeChironomus thummi. IV. Behavior of a female-specific protein,probably a vitellogenin

Using denaturing SDS-polyacrylamide gel electrophoresis, a protein with a subunit MW of about 148,000 daltons could be detected in the fat body of females of the reciprocal hybrids of Chironomus thummi thummi and Chironomus thummi piger, which is not present in males. This protein is presumably a vitellogenin and can be found in both hybrids during the late fourth-instar larval stage until eclosion of the adults, i.e., in early vitellogenesis. After eclosion, the reciprocal hybrids behave differently concerning the 148-kd protein. In females of the piger female x thummi male cross, which are fertile and produce yolky eggs, the 148-kd protein disappears from the fat body immediately after eclosion. In females of the reciprocal cross (thummi female x piger male) which are affected by gonadal dysgenesis and in which the oocytes only rarely contain yolk, the 148-kd protein is still present in the fat body of the adult up to 50 hr after eclosion until the fat body degrades. It is concluded that the inability of the sterile thummi female x piger male females to produce yolky eggs is caused by an impaired uptake of the presumed 148-kd vitellogenin into oocytes and not by a defective vitellogenesis. The impaired vitellogenin deposition into oocytes is taken as another aberrant trait of gonadal dysgenesis of the thummi female x piger male hybrids.     

The sequence of a dimetric hemoglobin (component IIbeta, Chironomus thummi thummi, Diptera) (author's transl)]

The primary structure of the dimeric hemoglobin CTT 11beta from the insect larva Chironomus thummi thummi (Diptera) is given. The sequence of a dimeric hemoglobin is presented for the first time. Some details of this primary structure are discussed and compared with human alpha-chains. The sequence was determined automatically.     

Different repetition frequencies of a 120 base-pair DNA-element and its arrangement in Chironomus thummi thummi and Chironomus thummi piger

The repetition frequency of a highly repetitive DNA sequence has been measured in the genomes of Ch. thummi thummi and Ch. th. piger. This sequence is known to be involved in the evolutionary duplication of defined chromosomal segments leading to a significant increase in the genome size of Ch. th. thummi. Reassociation of this highly repetitive DNA sequence which has a repeat length of 120 base-pairs, with total Ch. th. thummi and Ch. th. piger DNA has shown that the repetition frequency in the Ch. th. thummi DNA is 5.5 fold higher than in Ch. th. piger. In both genomes a 120 base-pair sequence is present as tandemly repeated sequence as shown by Southern analysis.     

New foldback transposable element TFB1 found in histone genes of the midge Chironomus thummi

A new Foldback transposable element (TFB1) has been found in the histone H1-H3 intergenic region in the midge Chironomus thummi thummi. TFB1 has long terminal inverted repeats, composed of short, degenerate subrepeats and is flanked by nine or ten base-pair "target site" duplications. TFB1 is present in at least two adjacent histone gene units in Ch. th. thummi, indicating a homogenization of histone gene repeats. The copy number and chromosomal distribution of TFB1 are different in the closely related subspecies Ch. th. thummi and Ch. th. piger. showing that amplification, elimination and transposition of TFB1 have occurred recently during evolution.     

The primary structure of a dimeric hemoglobin (erythrocruorin); component CTT VI of Chironomus thummi thummi, Diptera (author's transl)]

The complete amino acid sequence of 147 residues was determined automatically for a major dimeric component (CTT VI) of the insect larva Chironomus thummi thummi (Diptera). The molweight was found to be 32411. All tryptic, maleylated tryptic and cyanogen bromide peptides were isolated. The handling of some large fragments was facilitated by maleylation and subsequent ion exchange chromatography. Some details of the primary structure are discussed. The alignment of the amino acid sequence with that of human alpha-chains shows only 29 identical positions.     

Different hybrid effects in reciprocal crosses betweenChironomus thummi thummi andCh. th. piger including spontaneous chromsome aberrations and sterility

Hybrids from reciprocal erosses between twoChironomus thummi thummi andCh. th. piger laboratory stocks show four abnormalities in comparison to the parental stocks. One cross direction (Ch. th. thummi x Ch. th. piger ) is characterized by chromosome aberrations, reduced hatchability and malformations, whereas in reciprocal hybrids both sexes are sterile. Sterility is the consequence of rudimentary or non developed gonads.InCh. th. thummi x Ch. th. piger crosses chromosome aberrations were analysed in salivary gland nuclei. These aberrations are all somatic in origin, and they are induced during the first 40 h of embryonic development, prior to the onset of polytenization. The chromosomes of both subspecies are equally affected. In all four chromosomes breaks occur preferentially at specific regions. Reduced hatchability and malformations are presumably caused by chromosome mutations because within egg-masses a correlation exists between the rate of salivary gland chromosome aberrations and the rates of hatchability and malformations.     

An AT-rich DNA component in the genomes of Chironomus thummi thummi and Chironomus thummi piger

A DNA fraction has been isolated from total Chironomus thummi thummi DNA which is discernible from the bulk Ch. th. thummi DNA by a lower thermal stability. In situ hybridizations with polytene salivary gland chromosomes of Ch. th. thummi and Ch. th. piger made localization of this DNA fraction possible. Hybridizations with bands which contain different amounts of DNA in the two subspecies indicate that the isolated DNA fraction mostly consists of those sequences which represent the genetical difference between thummi and piger.This paper is dedicated to Professor Dr. H. Bauer on the occasion of his 75th birthday