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.     

Dependence of Balbiani ring induction in Chironomus salivary glands on inorganic phosphate

Galactose or certain other monosaccharides, administered for several days in the culture medium to larvae of Chironomus pallidivittatus, induce a new Balbiani ring, BR6, in their salivary gland chromosomes (W. Beermann, 1973, Chromosoma, 7, 198–259). This also applies to ethanol (Beermann, personal communication) and as found here, to glycerol. Induction of BR6 has previously been found to be paralleled by the appearance of one or two giant proteins (Ic₁ and Ic₂) probably deriving from allelic genes. We record here that the induction also includes the production of a new giant RNA species similar in size to the RNA from the Balbiani rings normally present, BR1 and BR2. Administration of inorganic phosphate together with glycerol prevented the appearance of BR6, as well as of the new RNA and component Ic protein(s); by contrast chloride and sulfate at similar concentrations did not prevent these effects. Administration of inorganic phosphate several days after the inducer and its continued presence reversed the effect of induction. Glycerol caused a marked depression in the level of inorganic phosphate in the hemolymph which persisted throughout its administration; the phosphate level in the glands was, however, unaffected. Inorganic phosphate administered together with the inducer at equimolar concentrations largely prevented the decrease in phosphate levels. It is concluded that a decrease in phosphate level is required for BR6 induction by glycerol. The two other inducers, galactose and ethanol, which were studied in less detail, seem to have a similar action.     

Galactose-induced puffing changes inChironomus thummi Balbiani rings and their dependence on protein synthesis

InChironomus thummi, puffing changes induced by galactose treatment (sugar effect) are restricted to the Br1/BR2 (Balbiani ring) system. No obvious induction of additional BRs such as BR6 inCamptochironomus pallidivittatus occurs. The response to feeding galactose (or other sugars), i.e. BR2 regression and concomitant BR1 activation, usually takes 24–48 h but can be accelerated somewhat by the application of two 6 h galactose treatments separated by an 18 h interval without sugar. In the special cells composing the lateral lobe of the salivary gland galactose causes regression on BR2 without concomitant BR1 activation which, however, appears delayed. The autonomous collapse of BR2 therefore could be considered as the primary effect of galactose at the puffing level. On the other hand, inhibition experiments performed with cycloheximide (CHM) emphasize the relevance of translational events in the control of the sugar effect. At highly inhibitory doses, CHM prevents the induction or causes reactivation of galactose-repressed BR2, suggesting that both induction and maintenance of the galactose effect are dependent on newly synthesized proteins. Present address: Departamento de Biología Cellular y Fisiología, Universidad Autónoma de Barcelona, E-08193, Barcelona, Spain     

Effects of ligation on ethanol-induced Balbiani ring puffing in salivary glands of Chironomus

Treatment of Chironomus larvae with dilute (0.5%–1.0%) ethanol results in puffing changes similar to those obtained with galactose in the Balbiani rings (BRs) of the salivary gland chromosomes. A shift in the relative size of BR1 and BR2 in chromosome 4 of C. pallidivittatus or C. tentans was observed within 1–2 days after ethanol treatment. The exceptional Balbiani ring, BR6 in chromosome 3, began to appear within 1 day after ethanol treatment of C. pallidivittatus and was fully developed after 3–4 days. Prepupae appeared to be refractory to the treatment. To localize possible controls of BR puffing in Chironomus, ligatures were made at various positions along the thorax and the anterior abdominal segments of the ethanoltreated larvae. In surviving larvae, ligated anterior to the brain or posterior to the salivary glands, induction of BR6 could be detected. In contrast, little or no BR6 puff induction was found in animals ligated in the middle of the second segment approximately between the brain and the salivary glands. No shift in the BR1/BR2 relation occurred with any of the ligations combined with ethanol treatment.     

Dependence of Balbiani ring puffing on protein synthesis

The possible dependence of puffing of the Balbiani rings (BRs) on the protein synthesis has been investigated by studying the response of these structures to protein synthesis inhibition induced by cycloheximide and anisomycin. When larvae of Chironomus thummi belonging to middle IV instar (BR1 repressed, BR2 expanded) are subjected to short treatment (3–6 h) with these drugs, BR1 and BR2 puffing states remain essentially unaffected. But when the same treatments are applied to galactose-pretreated larvae (BR1 expanded, BR2 repressed), selective reactivation of the collapsed BR2 occurs. These observations suggest that maintenance of a given puffing state can be dependent, to a variable extent, on the supply of newly synthesized proteins. In particular, selective re-expansion of galactose-repressed BR2 induced by the drugs seem to indicate the existence of repressor-like factor whose activity would be triggered by the galactose treatment.     

Cell-specific synthesis and glycosylation of secretory proteins in larval salivary glands of Chironomus thummi

Synthesis and glycosylation of larval salivary gland secretory proteins of Chironomus thummi were analyzed with respect to cell specific differences in the Balbiani ring (BR) pattern and glycoprotein composition of secretion formerly detected by histochemical staining procedures. In the secretion of a special cell type in salivary glands, which is characterized by the appearance of an additional BR, an additional polypeptide with a relative molecular weight (M_r) of 160 kD was found differing in its antigenic properties and tryptic fingerprint pattern from main cell secretion proteins. This so-called ssp-160 component is preferentially synthesized and glycosylated in the special cells. In the same cells, both the synthesis and glycosylation of all other major secretory proteins was found to be diminished or even repressed. In contrast to the conspicuous cell-specific differences at the level of protein synthesis, RNA analyses show the prominent synthesis of 75 S RNA in both main and special cells and gave no clear indication of the synthesis of a smaller RNA fraction as expected from the size of ssp-160 component. — These and further data on synthesis and properties of secretory proteins as well as expression of BR DNA are discussed with regard to the assumption that at least some of the eight major secretory polypeptides are coded for by BR DNA. The BR gene(s) might have originated by manifold duplications and modifications of short repetitive prototype DNA sequences, which are coordinatively expressed.On the occasion of the 60th anniversary of his birth-day we wish to dedicate this paper to Professor Wolfgang Beermann who was the first to detect, by the discovery of cell specific expression of BR 4 of Chironomus pallidivittatus salivary gland chromosomes and the concomitant occurrence of cell specific secretion granules, a causual relationship between the activity of a Balbiani ring and the appearance of a secretion component (Beermann, 1961)addressee for reprint requests     

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.     

Balbiani ring RNA content and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells

The content of RNA with an origin in the Balbiani rings 1 and 2 (BR 1+2) has been determined in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. Together with information on rate and completeness of export this permits an estimation of half-life of this RNA in cytoplasm and its residence time in the nucleus. The quantities in the BR, nuclear sap, and cytoplasm are roughly related as 110200. The 75 S RNA in the nuclear sap with an origin in the BR 1+2 must to a high extent be a precursor to the cytoplasmic 75 S RNA in vivo. The half-life of the cytoplasmic component is about 20 h and the half-life (residence time) for BR 1+2 RNA in the nuclear sap around one hour. The presence of a large pool of BR RNA in the sap explains the previously observed delay in its cytoplasmic appearance in vivo.     

Rapid and concerted evolution of repeat units in a balbiani ring gene

The Balbiani ring (BR) genes in the midge Chironomus, a genus belonging to Diptera, code for large secretory proteins, used to construct the larval tube. The 15-23-kb long core block in each gene consists of an array of tandemly arranged approximately 200-bp long repeat units, where a single repeat unit is composed of a constant and a subrepeat region. In order to investigate the evolutionary fate of highly repetitive coding DNA, the BR1γ core block in Chironomus pallidivittatus was characterized and compared to the orthologous core block in the sibling species Chironomus tentans. We find that the 75-100 repeat units in the BR1γ core block have evolved in an unusual fashion. In all repeat units the constant regions display an expected high degree of homology between the two species, 94% at the nucleotide level. In contrast, the subrepeat regions in all repeat units have diverged concertedly, both as to length, number and sequence of the subrepeats. The observed changes in all repeat units of the core block probably have occurred after speciation of C. pallidivittatus and C. tentans. These findings demonstrate that a tandemly reiterated coding sequence can rapidly and concertedly convert into a related sequence, much in the same way as has been described for satellite DNA.     

Correlated changes in steady-state levels of Balbiani ring mRNAs and secretory polypeptides in salivary glands of Chironomus tentans

Balbiani rings (BRs) on polytenized chromosomes in Chironomid salivary glands contain members of a homologous multigene family that encodes a family (the sp-I family) of high M _r secretory polypeptides. Each of these BR genes is comprised largely of tandemly duplicated core repeat sequences consisting of related constant (C) regions and intergenically divergent subrepeat (SR) regions. A set of oligodeoxyribonucleotide probes were synthesized that correspond to the transcribed strand of the SR region of BR1, BR2, BR2/, and BR6 core repeats. Under a defined set of conditions, it was possible to show that each oligonucleotide probe hybridized exclusively to its cognate repeat type without hybridization to other repeat types in cloned DNA templates. These BR probes were then used in dotblot hybridization experiments to simultaneously follow alterations in the steady-state level of BR mRNAs in response to prolonged exposure of larvae to galactose. The results indicated that the relative amounts of these four BR mRNAs may change in a noncoordinate manner. These BR probes were also used in experiments to compare simultaneously the salivary gland content of sp-I components and specific BR mRNAs in larvae that exhibited naturally occurring or induced alterations in BR gene expression. A correlation was found which suggested that sp-Ia is encoded in a gene comprised of BR1 repeats, sp-Ib is encoded by BR2 repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2 repeats.     

The 3′ ends of two genes in the Balbiani ring c locus ofChironomus thummi

Summary The 3-end sequences of two nonallelic genes derived from the Balbiani ring c (BRc) locus ofChironomus thummi are described. Only one of the genes appears to be transcribed abundantly in normal late larval salivary glands. The two sequences are highly similar, even in the 3 untranslated regions, but sharply diverge beyond the polyadenylation site. Together with evidence from the 3 ends of BR1 and BR2 genes ofC. pallidivittatus andC. tentans, independently characterized by others, this result suggests the existence of a sequence-homogenization mechanism that operates across the 3 ends of all BR genes characterized to date. The 3-terminal coding region of each BRc gene is divided into two portions by a short intron. The upstream portion is homologous to and continuous with the tandem repeats that make up the internal core of each BR gene; however, that portion is variant in sequence relative to the core, and apparently is not subject to the homogenization process that operates on the core repeats. The portion downstream of the intron encodes a unique, 111-residue polypeptide highly different from the rest of the BRc product. The evolution of the various segments of the BRc genes is discussed.     

Immune response of mice to Thy-1.1 antigen: Studies on congenic lines

The primary response to Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes (PFC). TheH-2 congenic mice (B10.K and B10.BR) carryingH-2 complexes of high responders (CBA and C57BR) on the low-responder background (B10) were found to produce significantly fewer PFC than the corresponding donor of theH-2 complex. On the other hand, C3H.B10 mice carrying theH-2 complex of a low responder on the high-responder background produced significantly more PFC than the donor of theH-2 complex. These findings were interpreted as evidence that alleles at previously described loci believed to be components of theI region of theH-2 complex and controlling immune response to Thy-1.1 are influenced by alleles at another locus. Studies of segregating populations of theH-2 congenic lines supplied evidence that this locus, tentatively calledIr-5, is in chromosome 17 (linkage group IX).     

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes

An antibody was raised against high mobility group nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. — Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. — It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday.