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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Functional multiplicity and structural correlations in the hemoglobin system of larvae of Chironomus thummi thummi (Insecta, Diptera): Hb components CTT I, CTT II beta, CTT III, CTT IV, CTT VI, CTT VIIB, CTT IX and CTT X

Larvae of the dipteran insect Chironomus thummi thummi that burrow in fresh-water muds, contain at least 12 hemoglobin (Hb) components of which the functional properties have not been systematically documented, although their amino acid sequences have been elucidated, showing mutually distinct primary structures. We isolated eight components (the monomeric Hbs CTT I, CTT III and CTT IV and the dimeric Hbs CTT II beta, CTT VI, CTT VIIB, CTT IX and CTT X) and measured in each O2 affinity and cooperativity and their pH dependence, and the effects of temperature, NaCl and ATP. The O2 affinities, Bohr- and temperature effects of the isohemoglobins are discussed in relation to mode of life and the microenvironmental conditions to which the larvae are subjected in nature, and with regard to the molecular mechanisms underlying the Hb-oxygenation reactions.     

Histone H1 heterogeneity in the midge, Chironomus thummi. Structural comparison of the H1 variants in an organism where their intrachromosomal localization is possible

1. Seven subfractions of histone H1 have been isolated and purified from larvae of Chironomus thummi (Diptera). They have been denominated I-1, II-1, II-2, II-3, III-1, III-2, and III-3, according to the order of migration in two steps of preparative electrophoresis. 2. The amino acid compositions are similar to those of other H1 histones. Subfractions I-1 and II-1 were found to contain one methionine and two tyrosine residues, II-2 contained two methionine and three tyrosine residues, and III-1 one methionine and three tyrosine residues. The other subfractions contained one or two methionine and two or three tyrosine residues. For subfractions I-1 and II-1 a chain length of about 252 amino acids was estimated. 3. Peptide pattern analyses after chemical cleavage at the methionine and tyrosine residues, and enzymatic cleavage with thrombin and chymotrypsin, respectively, showed that all subfractions have different individual primary structures. A comparison of peptide sizes and of the positions in the peptide patterns of epitopes recognized by monoclonal antibodies was made to check whether some of the subfractions could arise by proteolytic degradation of others. This possibility can be excluded for five of the subfractions and is very improbable for the two others. Treatment of C. thummi H1 with alkaline phosphatase did not change the pattern of subfractions, while the phosphorylated subfraction of histone H2A disappeared after this treatment. Most and very probably all subfractions are thus H1 sequence variants. 4. Inbred strains and individual larvae of C. thummi were found to comprise all seven variants. The H1 heterogeneity can therefore not be due to allelic polymorphism. Salivary gland nuclei were found to contain variant I-1 and at least some of the other variants. 5. H1 from Drosophila melanogaster and from calf thymus were used as reference molecules in all cleavage experiments and yielded the peptide patterns expected from the sequence. The comparison discriminates the group of C. thummi H1 histones clearly from Drosophila and calf thymus H1. Limited trypsin digestion yielded a protected peptide of uniform size in six of the seven variants which was considerably smaller than the protected central domain of calf thymus H1. 6. Two other species of Chironomidae, C. pallidivittatus and Glyptotendipes barbipes were found to contain five and three H1 subfractions, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Fat body: a site of hemoglobin synthesis in Chironomus thummi (diptera)

Fourth instar larvae of Chironomus thummi were permitted to incorporate labeled amino acids and/or sigma-aminolevulinic acid (sigma-ALA) in vivo and in organ culture. The products secreted into the hemolymph or into the culture medium were examined by acrylamide gel electrophoresis. Nine electrophoretic bands can be resolved as hemoglobins without staining. When gels are sliced for scintillation counting, incorporated amino acids and sigma-ALA are shown to be associated primarily with the same nine hemoglobin bands, suggesting that hemoglobins are assembled and secreted. Staining of gels with Coomassie brilliant blue reveals that there are several bands in addition to the visible hemoglobins. These bands incorporate amino acids, but not sigma-ALA, suggesting that they are non-heme proteins. The results of culturing isolated salivary glands, gut, and fat body demonstrate that the fat body is the major site of hemoglobin synthesis and secretion. Labeled products of the gut represent about 5% of the total hemoglobins produced by the tissues, while no hemoglobins are produced by the salivary glands. Although nine hemoglobins are visibly resolved on gels, labeling techniques reveal as many as 14 hemoglobins. This is the first demonstration of hemoglobin synthesis by specific tissues in culture in an invertebrate.     

The amino acid sequences of erabutoxins, neurotoxic proteins of sea-snake (Laticauda semifasciata) venom

1. Erabutoxin b was reduced, S-carboxymethylated and hydrolysed with trypsin. Seven tryptic fragments were isolated by column chromatography and paper electrophoresis. Some of the fragments were further hydrolysed with alpha-chymotrypsin, pepsin, Nagarse, Proctase A or Proctase B. The amino acid sequences of the fragment peptides were determined by subtractive Edman degradation. 2. From the tryptic digest of reduced, S-carboxymethylated and trifluoroacetylated erabutoxin b two fragments were isolated. From the amino acid composition of the fragments and from the terminal sequence studies on the reduced and S-carboxymethylated erabutoxin b, the sequence of the above seven tryptic fragments was elucidated. 3. The tryptic digestion of reduced and S-carboxymethylated erabutoxin a gave fragments, only one of which was different from the corresponding fragment from erabutoxin b. The amino acid sequence analysis of the fragment peptide showed that the only difference between erabutoxins a and b was that the former had asparagine and the latter had histidine at position 26.     

Amino acid sequence of normal (microheterogeneous) porcine immunoglobulin lambda chains.

The partial amino acid sequence of pooled, microheterogeneous pig immunoglobulin lambda chains was determined previously (Fran?k, F. (1970), FEBS Lett. 8, 269; Novotny, J., and Fran?k, F. (1975), FEBS Lett. 58, 24). In the present study, citraconylated pig lambda chains were digested by trypsin under conditions in which some of the epsilon-amino groups of lysine residues unmask. The resulting fragments were purified by gel filtration and ion-exchange chromatography at pH 3.0 in buffers containing urea; some of the fragments were found to be of intermediate size (i.e., larger than normal tryptic peptides but smaller than "citraconyl" peptides), thus permitting overlap information and amino acid sequences of all the 14 tryptic peptides to be deduced from amino acid compositions and partial amino acid sequences of selected fragments. In addition to completing the major amino acid sequence of pig immunoglobulin lambda chains, the present study demonstrates that it is possible to sequence microheterogeneous proteins with a suitable fragmentation strategy.     

Selected carboxymethylation of ferri-hemoglobin from insect larvae Chironomus thummi thummi]

Specific modification of the monomeric fraction III of ferri-hemoglobin from insect larvae Chironomus thummi thummi (Hb CTT) was studied on histidyl residues His-G19 (pK 4,8), His-E5 (pK 7,3) and Met-H22 at different pH using iodacetamide and spin label 2,2,6,6-tetramethyl-4-bromacethyl-piperidin-1-oxyl, an analogue of bromacetate. The analysis of the products of carboxymethylation (CM) showed that at pH 5,0 two products of modification CM-(His-G19)-Hb CTT, and CM-(Met-H22)-Hb CTT were obtained. In the case of modification at pH 7,2 with a spin label dicarboxymethylatid product CM-(His-G19)-CM (His-E5)-Hb CTT is obtained. In all products the degree of modification was one spin label per mole protein. Based on the data on the primery and tertiary structures Hb CTT and the results of the investigation, different reactivity of His-G19 and His-E5, as well as the cause of the absence of the product of carboxymethylation on His-G2 have been discussed. By analizing the absorption spectra of carboxymethylated derivatives of hemoglobin in the ultraviolet and visible region, as well as from the pH dependence curves of the absorption at Soret band in the interval pH 5,5-11,5 it has been shown that carboxymethylation of His-G19 and His E5 is not accompanied by any substantial disturbance of the structures of aquous-complexes Hb CTT. Modification of Met-H22 leads to strong changes in the absorption spectrum and to the absence of pH dependence of the absorption at Soret band, which indicates a change in the aquous-complexes Hb CTT structure.     

The amino acid sequence of rape (Brassica Napus L) cytochrome c

The amino acid sequence of rape (Brassica Napus L) cytochrome c was determined using 1 mumole of protein. Analysis of chymotryptic and tryptic peptides by the dansyl-Edman method showed that the molecule consisted of 111 residues and was homologous with other mitochondrial plant cytochromes c. The amino acid sequence was found to be identical with that previously reported for the cytochrome c from cauliflower (Brassica oleracea L).