Structure of a hemoglobin gene cluster and nucleotide sequence of three hemoglobin genes from the midge Chironomus thummi piger (Diptera, Insecta)

The aquatic larvae of the genus Chironomus (Diptera, Insecta) contain at least 12 different hemoglobin (Hb) variants in their hemolymph. In the present study we have analysed the structure and part of the nucleotide sequence of a Hb gene cluster cloned from the genomic DNA of Chironomus thummi piger. The cluster contains probably 6 different genes, separated by intergenic regions of various lengths. The nucleotide sequence of three putative Hb genes including the intergenic regions is presented. The inferred amino-acid sequences show clearly that two of these putative genes code for subvariants of the Hb variant VIIB. The third gene codes for a so far unknown Hb protein. As known already for other chironomid Hb genes, there are no intron sequences present in the coding regions.     

Apokrine Sekretion der peritrophischen Membran von Chironomus thummi piger Str. (Diptera)

Zusammenfassung Am Beispiel der Larven von Chironomus thummi piger werden die Zellen, die die peritrophische Membran abscheiden, licht- und elektronenmikroskopisch untersucht. Es liegen zwei Zelltypen vor, die einerseits durch ihren Reichtum an granulären E.R.-Schläuchen (ER-Zellen), andererseits durch ihren hohen Gehalt an Mitochondrien (M-Zellen) charakterisiert sind. Die ER-Zellen zeigen Veränderungen, die in vielerlei Hinsicht der klassischen Auffassung der apokrinen Sekretion entsprechen und sich nicht in ein modernes Schema der Sekretionsmorphologie einordnen lassen. Die Zellen gehen im Verlauf der Sekretabgabe weder völlig zugrunde, noch bleiben sie vollständig erhalten. Die M-Zellen, die im Gegensatz zu den ER-Zellen keine Sekretionsgranula besitzen, weisen ähnliche Umwandlungen auf. Das fertige Sekretionsprodukt — die peritrophische Membran — entstammt einmal vorgeformten Sekretionsgranula, zum anderen der umgewandelten, abgeschnürten oberen Zellhälfte. Die peritrophische Membran besteht aus zwei Schichten, wovon die lumenseitige, auf Längsschnitten quergestreifte Lage (Wabentextur) vermutlich aus den Sekretionsgranula hervorgeht und die andere längsgefaserte Lage auf die Umwandlung von Zytoplasma zurückzuführen sein dürfte. Die Mikrovilli der Sekretionszellen sind in keiner Weise für die Strukturierung der Wabentextur verantwortlich.

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On the apocrine secretion in the formation of the peritrophic membrane of chironomus thummi piger Str

Summary Taking the larvae of Chironomus thummi piger as an example, the cells secreting the peritrophic membrane have been investigated with the light- and electron-microscope. Two cell-types can be distinguished which in one case are characterized by abundant rough E.R.-tubules (ER-cells) and in the other case by large quantities of mitochondria (M-cells). The ER-cells undergo changes which in many respects correspond to the individual stages of the classic apocrine secretion, and thus do not fit into a modern scheme of the morphology of secretion. In the course of the discharge of the secretory product the cell neither becomes completely necrotic nor remains totally intact. The M-cells, which do not contain secretion granules like the ER-cells, show similar changes. The final product — the peritrophic membrane — is formed on the one hand by membrane bound secretion granules and on the other hand by the transformed and pinched off upper half of the cell. The peritrophic membrane consists of two layers, the one of which, facing the lumen of the midgut and exhibiting a honey-comb-texture, presumably is formed by the secretion granules, whereas the other layer arises from transformed cytoplasm. The microvilli of the secretory cells are not responsible for the formation of the honey-comb-pattern.

Die Untersuchungen wurden mit Unterstützung durch die Max-Planck-Gesellschaft und die Deutsche Forschungsgemeinschaft durchgeführt.     

Hemoglobins. XXVI. Analysis of the primary structure of the dimeric insect haemoglobin CTT VIIB (Erythrocruorin) from Chironomus thummi thummi, Diptera]

The dimeric haemoglobin CTT VIIB (Erythrocruorin) of Chironomus thummi thummi, Diptera, has been sequenced. Either globin or globin with maleic acid blocked lysines were cleaved with trypsin. The separation of peptides and the sequence analysis are given in detail.     

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.     

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.     

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.     

Cell-free synthesis of Chironomus thummi (Diptera) globins

RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi.     

Immunological characterization of the haemoglobins of Chironomus thummi (Diptera)

The major fourth instar-specific haemoglobin (Hb) of Chironomus thummi was purified to homogeneity as assessed by five analytical systems. This Hb was further resolved by isoelectric focussing into two variants, differing only slightly in their isoelectric points. These variants proved to be immunologically identical in each of three antigenically distinct components. Cross-reactivity studies indicated the presence of two or more antigenic components in other Hbs of C. thummi and of C. tentans, a related species. The data exclude the possibilities that non-haeme protein contaminants or that Hb (globin) fragments were present in our preparations. Therefore, multiple precipitin lines on double diffusion plates must have arisen from differentially antigenic sub-populations which are not separable by routine purification procedures.     

Regulation of hemoglobin synthesis by ecdysterone and juvenile hormone during development of Chironomus thummi (Diptera)

Chironomus thummi contains nine soluble hemoglobins (Hbs) in the larval hemolymph which can be resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hemoglobins 2 and 3 are stage specific for the 4th instar and are first detected by day 4 of this stage in vivo, being absent in the 3rd instar. Fat-body cultures in the presence of 3H-delta-aminolevulinic acid and 14C-amino acids synthesize and secrete labelled Hbs, as was assayed by acrylamide gel electrophoresis and immunoprecipitation of Hbs recovered from the culture medium. During development from 3rd instar to pupa, Chironomus fat body undergoes functional changes, being actively involved in Hb synthesis in intermolt periods and inactive with respect to Hb production during molting. The repression of Hb synthesis is reversed following the molt from the 3rd instar to the 4th instar. Metamorphosis is related to a gradual and irreversible loss of Hb synthesis and secretion by the fat body. The treatment of fat body in vitro with ecdysterone inhibits Hb synthesis in tissue from intermolt animals, even in the presence of excess methoprene, a potent juvenile hormone analogue. In contrast, immunoprecipitation of the translation products from a wheat-germ cell-free system, using mRNA from ecdysterone-treated 4th-instar fat body as a template, shows significant synthesis of globins, suggesting that ecdysterone does not affect the amount or template activity of globin messages. Methoprene induces the precocious in vitro synthesis of Hbs 2 and 3 in day-2 4th-instar fat body and enhances all Hb synthesis in the absence of ecdysterone. In vitro treatment with methoprene activates newly molted fat body to synthesize Hbs 2 and 3 in vitro. The process of Hb induction by this analogue is completely inhibited by actinomycin D or ecdysterone. Fat body from animals already exposed to high endogeneous ecdysterone titer are insensitive to treatment with this juvenile hormone analogue. Intermolt larvae normally possess stable Hb mRNA molecules, because actinomycin-D administration in vitro does not affect Hb synthesis for as long as 30 h, whereas it effectively inhibits all RNA synthesis in the fat body. Immunoprecipitation of globin translated in vitro from mRNA from 2-day-old 4th-instar larvae treated in vivo with methoprene shows enhanced synthesis of globins 2 and 3, as compared to controls with no treatment. It is suggested that both juvenile hormone and ecdysterone regulate Hb synthesis in Chironomus; juvenile hormone affecting the activity of Hb genes, and ecdysterone modulating the level of Hb gene expression.     

The ontogeny of multiple hemoglobins in Chironomus thummi (Diptera): The effects of a compound with juvenile hormone activity

The multiple hemoglobins (Hbs) of Chironomus thummi show distinct and significant ontogenetic changes during development from the third instar through the fourth instar and metamorphosis into the pupa. A total of nine Hbs are resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hbs 2 and 3, which are stage specific for the fourth instar, are first detected on the fourth day of this stage by electrophoresis and immunoprecipitation. Hb 4 is the predominant Hb species in the early and middle fourth instar, but during the late fourth instar and prepupa, Hb 1 predominates. The concentrations of Hbs 5–9 remain relatively constant in middle instars and decrease during later development. The Hb content of larval hemolymph exhibits changes that coincide with developmental stages; molting is characterized by low Hb content, whereas, the hemolymph of intermolt animals contains relatively high levels of Hbs. Treatment of fourth instars with a juvenile hormone analog, Altosid, prolongs this stage and inhibits the progress of normal development resulting in the formation of larval-pupal intermediates. Altosid also appears specifically to inhibit the accumulation of soluble hemolymph proteins related to pupation and metamorphosis, without affecting the concentration of Hb. Most significantly, it induces the precocious appearance of Hbs 2 and 3, which remain elevated above control levels in the late larval and prepupal stages. The present results strongly suggest that Altosid stimulates the appearance and accumulation of larval-specific proteins in vivo, while it inhibits the appearance of pupation-related proteins.     

Cell-free processing of the secreted globins of Chironomus thummi (Diptera)

Poly(A)⁺ RNA from fourth instar larvae of the insect Chironomus thummi was efficiently translated in vitro. Up to 10% of the total cell-free translation products was immunoprecipitable with antiserum specific for chironomid haemoglobins. Six immunoreactive preglobin products were resolved on SDS-17.5% polyacrylamide gels, and were cotranslationally processed by dog pancreas microsomes to 2 major bands that comigrated with authentic secreted globins.     

Development and localization of microsatellite markers for the sibling species Chironomus riparius and Chironomus piger (Diptera: Chironomidae)

Five variable microsatellite loci are reported for the nonbiting midge species Chironomus riparius and Chironomus piger. All loci show considerable intraspecific variation and species‐specific alleles, which allow to discriminate among the two closely related species and their interspecific hybrids, and to estimate genetic diversity within and between populations. Additionally, the loci were localized on C. riparius polytene chromosomes to verify their single copy status and investigate possible chromosomal linkage. The described markers are used in different studies with regard to population and ecological genetics and evolutionary ecotoxicology of Chironomus.     

Tissue-specificity of hemoglobin synthesis: Localization of heme synthesis in the subepidermal fat body of Chironomus thummi (Diptera)

Possible sites of heme synthesis in the fourth instar of Chironomus thummi were investigated by means of autoradiography of specific isotope incorporation. “Body wall” preparations, which include subepidermal and visceral fat body, oenocytes, muscle, epidermis, and cuticle, were cultured for 1 h in a medium containing tritiated-δ-aminolevulinic acid, a specific precursor to heme biosynthesis. Light-microscopic examination of autoradiographs of sections of the body walls indicates that the subepidermal fat body is the major site of incorporation of the precursor into heme. The visceral fat body shows few silver grains. Oenocytes, as well as muscle and epidermis, are characterized by absence of silver deposits. These findings indicate that the subepidermal fat body of Chironomus is the primary site of heme synthesis, and are discussed in relation to specific hemoglobin synthesis.     

Structure-related changes of the electron spin resonance spectra of the monomeric nitrosyl haemoglobin IV from Chironomus thummi thummi

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) is an allosteric protein characterized by pH-dependent ligand affinities (Bohr-effect). The ligand-linked proton dissociation gives rise to a t r conformational transition. Furthermore, the Bohr-effect is ligand-dependent and decreases in magnitude following the order of ligands, O₂ > CO > NO. Although the Bohr-effect for NO is smallest, the electron spin resonance (ESR) spectra of frozen solutions of ¹⁵NO-ligated CTT IV measured as higher derivatives at 77 K reflect this pH-dependent conformation change. g Tensor and hyperfine constants coinciding with the principal directions of the g tensor have been evaluated for ⁵⁷Fe, ¹⁵NO, ¹⁴N_E-imidazole, and ¹⁴N-pyrroles.Hyperfine parameters and g values of both conformation states of this haemoglobin, i.e., of the t state at low pH with low ligand affinity and of the r state at high pH with high ligand affinity, are characteristic for a hexacoordinated nitrosyl haem complex. The change in pH leads to a variation of the Fe-N-O bond angle which is larger at high pH (r conformation) than at low pH (t conformation). Furthermore, the spin transfer from NO into iron orbitals is larger at high pH than at low pH. These results are consistent with the assumption that the interaction of proximal imidazole and iron is smaller in the r conformation than in the t conformation.Binding of anionic detergents to nitrosyl CTT IV causes a conversion of the native (t, r) into a denatured (super-r) structure. The latter, on the basis of hyperfine and g values, apparently contains a pentacoordinated nitrosyl haem complex. Because of the extreme displacement of the proximal imidazole in the super-r structure, the Fe-N-O gouping is nearly linear and a large spin transfer from NO into iron orbitals occurs. Removal of anionic detergents from the protein leads to a full reconversion of the super-r into the native conformations.These structure-related changes of hyperfine constants and g tensor further support the assumption that the trans-effect of the proximal imidazole is an important link of allosteric interactions in haemoglobins.