Temperature-induced Balbiani rings in Chironomus thummi

The formation of a new telomeric Balbiani ring in the right arm of chromosome III (T-BR III) has been induced in Chironomus thummi larvae by applying a wide range of temperature treatments (33 °–39 ° C). In this paper we present some kinetic and functional characteristics of this structure. T-BR III incorporates tritiated uridine, and during its formation accumulation of acidic proteins takes place. However, induction and maintenance of this puff structure appear to be insensitive to Actinomycin treatment. An additional T-BR can be induced in chromosome I by employing the most drastic temperature treatments (37 °–39 ° C). We also report the existence of a group of puffs active after heat treatments in Chironomus polytene chromosomes which could be homologous with the T-puffs of Drosophila.     

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.     

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.     

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.     

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.     

An AT-rich DNA component in the genomes of Chironomus thummi thummi and Chironomus thummi piger

A DNA fraction has been isolated from total Chironomus thummi thummi DNA which is discernible from the bulk Ch. th. thummi DNA by a lower thermal stability. In situ hybridizations with polytene salivary gland chromosomes of Ch. th. thummi and Ch. th. piger made localization of this DNA fraction possible. Hybridizations with bands which contain different amounts of DNA in the two subspecies indicate that the isolated DNA fraction mostly consists of those sequences which represent the genetical difference between thummi and piger.This paper is dedicated to Professor Dr. H. Bauer on the occasion of his 75th birthday     

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