A tissue-specific puff (Balbiani ring a) in Chironomus thummi may contain a gene encoding a 67-kDa protein which exhibits non-tissue-specific expression

A 2.3-kb genomic clone has been isolated from the region where the tissue-specific puff, Balbiani ring a (BRa), is found on chromosome IV of the special lobe of Chironomus thummi salivary gland cells. The clone was characterized by nucleotide sequence analysis. Two clusters of direct tandem repeats were identified, as well as large and small open reading frames (ORFs). The large ORF was fused to an Escherichia coli lacZ gene. Antibodies against the beta-galactosidase/ORF fusion protein reacted selectively on Western blots with a 67-kDa protein. Western-blot analysis and immunoelectron microscopy showed that this protein was distributed in the cells of all larval tissues examined. We concluded that BRa, a tissue-specific puff, whose activity correlates with the synthesis of 160-kDa secretory protein [Kolesnikov et al., Chromosoma 83 (1981) 661-677], may also contain a gene which is not expressed in a tissue-specific manner.     

Secretory protein synthesis in Chironomus salivary gland cells is not coupled with protein translocation across endoplasmic reticulum membranes. Electron microscopic evidence

Fragments of rough endoplasmic reticulum containing polysomes bound to the membranes only at the 5 end were visualized in electron microscopic spreads from Chironomus thummi salivary gland cells. The length of the nascent protein molecules in the polysomes increased from the 5 to the 3 (free) polysome end. The data obtained disagree with the generally accepted model according to which synthesis of secretory proteins is concomitant with the protein transport across the endoplasmic membrane.     

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.     

Insect globin gene polymorphisms: intronic minisatellites and a retroposon interrupting exon 1 of homologous globin genes in Chironomus (Diptera)

Exon 1 of globin gene ct-13RT in clone lambdagb2-1 from Chironomus thummi contains a 444nt SINE (CTRT1). Based on in situ hybridization to polytene salivary gland chromosomes, C. thummi (ct), C. piger (cp) and C. tentans (ctn) contain copies of CTRT1 at multiple chromosomal loci. Genomic PCR amplifications reveal interrupted (ct-13RT) and uninterrupted (ct-13) alleles of the globin gene in the German population of C. thummi maintained in our laboratory, and only uninterrupted alleles or their homologs in different populations of C. thummi, C. piger and C. tentans. PCR amplification did generate different length fragments from cp-13 gene homologs in natural and laboratory C. piger populations that were due to variation in the length of minisatellite expansions of the central introns of the genes rather than a CTRT1-like SINE. Within minisatellite arrays, aligned homologs were more similar than paralogs in a single population, indicating that a tandem cluster of these repeats predated separation of the C. piger populations. The ct-13 genes of several C. thummi populations lack the minisatellites, suggesting their origin in C. piger only after the thummi/piger split. CTRT1 transposition into a ct-13 allele is even more recent, occurring after separation of German and other European C. thummi populations. The nearly intact ct-13RT and comparison with its intact ct-13 allele support a very recent transposition of the CTRT1 SINE into one of at least two already diverged ct-13 globin gene alleles. PCR analysis of DNA from individual adults in C. thummi shows a 1:2:1 distribution of ct-13/ct-13:ct-13/ct-13RT:ct-13RT/ct-13RT genotypes, consistent with a neutral spread of the ct-13RT allele since transposition, and indicating that the hemoglobin encoded by ct-13 is not necessary for survival, at least in a laboratory population of C. thummi.     

MEC: A transposable element from Chironomus thummi (Diptera)

Summary Two genomic clones, pC1.2 and p20D (containing inserts of 2.0 and 1.6 kb, respectively) were isolated from the A2b region to polytene chromosome IV of Chironomus thummi thummi salivary gland cells. Upon in situ hybridization to polytene chromosomes of C. thummi thummi and C. thummi piger, p20D DNA hybridized mainly over the A2b region of chromosome IV, whereas pC1.2 DNA hybridized to at least 90 sites distributed over all the chromosomes. A partial nucleotide sequence analysis showed that these clones were very similar and allowed the detection of a 596 by insert in the pC1.2 clone. This insert possesses all of the essential features of a Class II transposable element and was called MEC. It carries a nearly perfect 107 by terminal inverted repeat containing one mismatch and is flanked by a 5 by direct repeat. The 372 by central region contains a short open reading frame with a coding capacity of 58 amino acids.     

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.     

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.     

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.     

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     

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.     

DNA reduplication cycle during chromosome polytenization in the salivary gland cells of Chironomus thummi larvae. III. The determination of the duration of the DNA synthesis period

The duration of DNA synthesis in the salivary gland cells of Chironomus thummi larvae of the IV instar was determined by means of autoradiography and cytophotometry. Cells of different levels of ploidy differ in the duration of their DNA synthesis period. The tS of 2(10)c and 2(11)c cells was equal to 17 and 22 hours, respectively. The doubling of DNA content of the chironomid salivary gland cells leads to a 1.3 time increase in the duration of S-phase.     

蜂毒溶血肽基因的定点诱变及其在大肠杆菌中的表达

从蜜蜂毒腺中提取总ＲＮＡ,通过ＲＴ－ＰＣＲ方法扩增得到了蜂毒溶血肽前体蛋白的ｃＤＮＡ,再进一步通过定点诱变在蜂毒溶血肽序列前引入了羟胺裂解位点,构建了与β－半乳糖苷酶部分序列相融合的蜂毒溶血肽诱变蛋白表达载体,序列分析结果表明,成功地引入了目的密码子且与β－半乳糖苷酶部分序列构成正确的读码框,并在大肠杆菌中表达了诱变蛋白,为基因工程生产蜂毒溶血肽提供了新途径。     

DNS-Replikationsmuster der Speicheldrüsen-Chromosomen von Chironomiden

The pattern of DNA-synthesis of the salivary gland chromosomes of Chironomus thummi thummi, Ch. th. piger, Ch. annularius, Ch. plumosus and Ch. melanotus was studied using H³-thymidine-autoradiography. Contrary to the previous conception the bands of the salivary gland chromosomes of Chironomus do not begin replication simultaneously. H³-thymidine incorporation in bands of high DNA content begins later than in bands with a lesser amount of DNA. This difference in time is very small in bands outside the kinetochore regions and not comparable to the asynchrony in replication of typical heterochromatin in the salivary gland chromosomes of Chironomus melanotus. Differences in the amount of DNA in homologous bands do not affect the onset of replication. — Bands of high DNA content are replicating during a longer time than those having less DNA. However, certain chromosome regions behave differently. In these regions bands of very low DNA content are synthesizing DNA during the whole replication cycle. Since no excessive increase of DNA could be observed in these regions it is supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which disappears immediately from the chromosome. — At the end of the replication cycle in the salivary gland nuclei of the hybrid Chironomus th. thummi X Ch. th. piger a labeling pattern is found in the chromosomes of Ch. th. thummi which differs from that in the parental subspecies Ch. th. thummi.     

Changes in the cellular structure of the salivary glands in Chironomus larvae resulting from mechanical cell damage

Rapid morphological changes were observed in some cells of hand-isolated salivary glands of Ch. thummi larvae. The nuclear envelope, routinely closely fitting the tightly packaged polytene chromosomes, was seen to lose its contact with the chromosomes and to attain a smooth round shape. Then unfolding of the chromosomes occurred, their banding patterns becoming clearly evident, probably through widening the interband regions; the chromosome length increased by about 20%. We argue that the changes observed were induced during gland isolation by lesions of the cell basal envelope in the sites of the fat body connections to the salivary gland.