Tissue specific gene activities and proteins in the Chironomus salivary gland

Summary The secretory proteins of the larval salivary gland of some Chironomus species were analysed according to a method developed by Grossbach (1969). After reduction of the disulphide bonds and alkylation the secretion of Chironomus thummi was separated by disc electrophoresis at acrylamide concentrations of 4.8 to 9.5% into seven main and some minor fractions. By increasing the acrylamide concentration up to 20% nine main fractions could be resolved. In addition to these high molecular weight structural proteins a number of proteins soluble in simple buffer solutions and separable at pH 8.8 in a 15% acrylamide gel are present in the secretion but only to a very small amount.The electrophoretic pattern of the secretory proteins of Chironomus strenzkei, Ch. luridus and Ch. obtusidens are qualitatively the same as the Ch. thummi pattern. Some quantitative differences may exist. In the secretion of Chironomus plumosus at least eleven main fractions were detected in 20% acrylamide gels. A comparison between the number of Balbiani rings of the salivary gland chromosomes and the number of main protein fractions in the salivary gland secretion yielded no direct correlation.The results are discussed in respect to the question wether the major puffs of the gland, the Balbiani rings, encode the major products of the gland, the secretory proteins.Abbreviations used BR Balbiani ring - bis N,N-Methylenebisacrylamide     

Structure of the smallest salivary-gland secretory protein gene in Chironomus tentans

The salivary gland secretion in the dipteran Chironomus tentans is composed of approximately 15 different secretory proteins. The most well known of the corresponding genes are the four closely related Balbiani ring (BR) genes, in which the main part of each approximately 40-kb gene is composed of tandemly arranged repetitive units. Six of the seven additional secretory protein genes described share structural similarities with the BR genes and are members of the same BR multigene family. Here we report the identification of a new secretory protein gene, the spl2 gene, encoding the smallest component of the C. tentans salivary gland secretion. The gene has a corresponding mRNA length of approximately 0.7 kb and codes for a protein with a calculated molecular weight of 7,619 Da. The sp12 gene was characterized in seven Chironomus species. Based on a comparison of the orthologous gene sequences, we conclude that the sp12 gene has a repetitive structure consisting of diverged 21-by-long repeats. The repeat structure and the codon composition are similar to the so-called SR regions of the BR genes and the sp 12 gene may represent a diverged member of the BR multigene family. Correspondence to: L. Wieslander     

Identification of the spI products of Balbiani ring genes in Chironomus thummi

The spI fraction of high molecular weight secretory proteins was analysed in Chironomus thummi. These proteins are encoded by giant Balbiani ring (BR) genes which develop specifically in salivary gland cells. Each component of the spI fraction was studied electrophoretically from early and middle 4th instar larvae and prepupae, as well from galactose-treated larvae where changes in the relative puffing pattern of BR1 and BR2 are known to occur. The spI fraction consists of at least two bands with electrophoretic mobilities slower than those of the spI components of Camptochironomus. The slow migrating component remains throughout the 4th larval instar, while the amount of the faster component changes, being abundant in early 4th instar and prepupae, but not present (or very weak) in middle 4th instar. The correlated shifts in BR puffing pattern during these developmental stages suggest that the slow and fast components are encoded by BR2 and BR1. The spI fraction is modified by galactose treatment, the fast component being induced in parallel with a decrease in the slow component. These changes are correlated with changes in the steady-state levels of RNA: an increase in BR1 RNA and a decrease in BR2 RNA, and of proteins. These proteins could correspond to the spIb and spIa fractions allocated to BR2 and BR1, respectively, in Camptochironomus. After galactose treatment a new faster band sometimes appears, that could correspond to the spIc fraction of Camptochironomus. A possible spId equivalent was also identified. In conclusion the main features of the spI family in C. thummi are similar to those of spI in Camptochironomus.Abbreviations BR Balbiani ring - spI family of M_r=10⁶ secretory polypeptides     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.     

Nuclear localization of a DNA-binding C-terminal domain from Balbiani ring coded secretory protein.

All known Balbiani ring (BR) genes in Chironomus tentans, coding for giant secretory proteins, the sp-I family, end with a short (110 codons) 3'-end exon which is highly conserved in evolution and is structurally unrelated to the sequences characterizing the core of these proteins. We find that the expressed product, the C-terminal domain, shows sequence-specific DNA binding and that it is likely to be absent in one of the sp-I components, sp-Ib, believed to be coded by the BR2.2 gene. Immunohistochemistry shows that material with reactivity towards antibody against the C-terminal domain is present in the nuclei, and specifically enriched in Balbiani ring 1 and 2. Western blotting of extracts from isolated nuclei demonstrates a component with the same antibody reactivity and of an apparent size somewhat larger than that of the domain. The possibility is discussed that the C-terminal part, which is part of the secretion when derived from some of the BR genes, might be cleaved off and function as a feedback signal to control BR gene activity when derived from the BR2.2 gene.     

Protein synthesis in the Chironomus thummi salivary gland

Summary Secretory proteins isolated from the lumen of the Chironomus thummi salivary gland were labelled with radioactive amino acids in vivo and in vitro. Under both conditions all but one of the electrophoretically separated fractions became labelled, the 6 prominent polypeptides already after 10–15 min of incubation. Differences in the labelling pattern during development from early 4th instar larvae to late prepupae were not detected.After synthesis the secretory proteins are stored in the cytoplasm for different times until they are exported into the gland lumen.None of the prominent protein fractions extracted only from the cells of the gland were found to be labelled even after labelling times up to 10 hrs. Therefore, it may be concluded that the Chironomus salivary gland synthesizes predominatly secretory proteins at least after the last larval moult.Long-time treatment of whole larvae with actinomycin D has no striking effect on the protein synthesis of the gland.Some of the results together with data from the literature led us to the speculation that changes of puff patterns (Balbiani rings excluded) do not reflect subsequent changes at the translational level.     

Homology of Balbiani Ring DNA in two closely related Chironomus species

Cytogenetic analysis indicates that Balbiani Ring 2 (BR 2) in the two sibling species Chironomus tentans and Chironomus pallidivittatus arises from identifically banded segments in the salivary gland polytene chromosomes, although chromosomal rearrangements have occurred. In situ hybridization of BR 2 RNA to the polytene chromosomes of each individual species, as well as their F1 hybrids, reveals that the repetitious BR 2 DNA in the two species has, within the limits of the technique, retained identity of nucleotide sequences and degree of repetition. The DNA of the naturally expressed BR 1 and BR 3 in both species and that ot the galactose induced BR 6 in C. pallidivittatus did not hybridize with BR 2 RNA, indicating that these BR's are different from BR 2 with regard to sequence content.     

The repetition frequency of DNA in Balbiani ring 2 of Chironomus thummi

The RNA of Balbiani ring BR2 of polytene chromosomes from Chironomus thummi salivary glands was microisolated and reassociated in the presence of an excess of total larval DNA. BR2 RNA reacts as a single component with a C₀t_1/2 of 8.6. Ribosomal precursor RNA from microisolated nucleoli reassociates under identical conditions with a C₀t_1/2 of 12.3. These C₀t_1/2-values suggest repetition frequencies in the range of 35 and 50 for ribosomal DNA and Balbiani ring 2 DNA, respectively. The data presented here favour the view that the gene for BR2 RNA of C. thummi is internally repeated and contains only one type of DNA sequence.     

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.     

A new member of a secretory protein gene family in the dipteranChironomus tentans has a variant repeat structure

Summary We describe the structure of a gene expressed in the salivary gland cells of the dipteranChironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.     

IN SITU DEMONSTRATION OF DNA HYBRIDIZING WITH CHROMOSOMAL AND NUCLEAR SAP RNA IN CHIRONOMUS TENTANS

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chromosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2). Salivary glands were incubated with tritiated nucleosides. The labeled RNA was extracted from microdissected nuclei and hybridized to denatured squash preparations of salivary gland cells under conditions which primarily allow repeated sequences to interact. The bound RNA, resistant to ribonuclease treatment, was detected radioautographically. It was found that BR 2 RNA hybridizes specifically with the BR 2 region of chromosome IV. Nuclear sap RNA was fractionated into high and low molecular-weight RNA; the former hybridizes with the BR 2 region of chromosome IV, the latter in a diffuse distribution over the whole chromosome set. RNA from chromosome I hybridizes diffusely with all chromosomes. Nucleolar RNA hybridizes specifically with the nucleolar organizers, contained in chromosomes II and III. It is concluded that the BR 2 region of chromosome IV contains repeated DNA sequences and that nuclear sap contains BR 2 RNA.     

A novel giant secretion polypeptide in Chironomus salivary glands: implications for another Balbiani ring gene

Chironomus salivary glands contain a family of high Mr (approximately 1,000 X 10(3)) secretion polypeptides thought to consist of three components: sp-Ia, sp-Ib, and sp-Ic. The use of a new extraction protocol revealed a novel high Mr component, sp-Id. Results of a survey of individual salivary glands indicated that sp-Id was widespread in more than a dozen strains of C. tentans and C. pallidivittatus. Sp-Id was phosphorylated at Ser residues, and a comparison of cyanogen bromide and tryptic peptide maps of 32P-labeled polypeptides suggested that sp-Ia, sp-Ib, and sp-Id are comprised of similar but nonidentical tandemly repeated amino acid sequences. We concluded that sp-Id is encoded by an mRNA whose size and nucleotide sequence organization are similar to Balbiani ring (BR) mRNAs that code for the other sp-I components. Furthermore, parallel repression of sp-Ib and sp-Id synthesis by galactose led us to hypothesize that both of their genes exist within Balbiani ring 2.     

Effect of galactose treatment in the puffing pattern of Chironomus thummi Balbiani rings

Galactose feeding of Chironomus thummi larvae induces the regression of Balbiani ring c (BRc) and the full expansion of BRb, both localized in the IV salivary gland chromosome. This effect coincides with that described on BR2 and BR1 of Ch. pallidivittatus and Ch. tentans. The puffing changes of BRb and BRc throughout development have been studied and also show identical variations as in BR1 and BR2 of Ch. pallidivittatus and Ch. tentans. The similar behaviour of BRb and BR1, and of BRc and BR2 respectively after galactose treatment and throughout development strongly suggests that these BRs play the same physiological role in the three Chironomus species, with BRb = BR1 and BRc=BR2.