J. L. Díez

Thermotolerance and heat shock proteins in Chironomus

Abstract The acquisition of thermotolerance in Chironomus thummi has been analysed. When larvae were subjected to 15 min exposure at 40°C, over 90% of larvae died a few hours later. Under these conditions, no heat shock protein synthesis could be detected in cells from larvae tissues. If a conditioning treatment (35°C 1 h) was given prior to the test treatment, the larvae were thermotolerant between 3–72 h after the conditioning treatment. The optimal post-test treatment level of survival was reached at 8–24 h after the conditioning treatment. When thermotolerance was induced, the cells acquired the ability to synthesize heat shock proteins after the second heat exposure. This second synthesis took place during the recovery period and also occurred if a carbon dioxide treatment was applied as the conditioning treatment. If heat shock protein synthesis was inhibited by blocking transcription with Actinomycin- d (AMD) it was also possible to induce thermotolerance, but with some differences: the posterior survival was restricted to 24–48 h after test treatment. Actinomycin- d itself could induce the same state of thermotolerance. Our data indicate that it is possible to induce thermotolerance in C. thummi which can then be expressed as two different types of survival: a short-term survival achieved independently of heat shock protein synthesis, and a long-term survival which seems to be connected with the synthesis of heat shock proteins after the second treatment.

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.

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.

Histochemical localization of reverse transcriptase in polytene chromosomes of chironomids

Abstract.The localization of a reverse transcriptase-related protein in salivary gland polytene chromosomes was investigated by immunohistochemistry in two species of Chironomus. The antibodies used were raised against a recombinant protein containing phylogenetically conserved motifs of reverse transcriptases and derived from an abundant non-LTR element previously identified in Chironomus. Immunoreactive protein was found in some telomeres, in a centromeric region, in a few interstitial bands and in Balbiani ring 3. The telomeric signal was probably dependent on transcription and increased dramatically when telomeric heat shock puffs were induced. A correlation with transcription was also seen in Balbiani ring 3, the immunobinding of which disappeared after inhibition of transcription with actinomycin D.

Telomeric DNA sequences differentially activated by heat shock in two Chironomus subspecies

The patterns of puffing, transcription and protein synthesis under heat shock were analysed in polytene nuclei of Chironomus thummi piger, in comparison with those obtained in the closely related subspecies C. th. thummi. Most chromosomal heat shock puffs, as well as heat shock induced polypeptides, in C. th. piger paralleled those previously reported for C. th. thummi. Nevertheless, we found a striking difference in behaviour in the induction of telomeric Balbiani rings by heat shock in the two subspecies. Although homologous sequences were present at all the telomeres in both subspecies, they were not always transcriptionally activated by heat shock. The most frequently puffed telomeres were that of chromosome III R in C. th. thummi and that of chromosome IV R in piger. Transcription of the same sequences from both telomeric Balbiani rings (T-BR-III and T-BR-IV) occurred under heat shock. The enigmatic behaviour of telomeres and the functional significance of T-BRs are discussed in relation to possible equivalents in other Diptera.

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.

Induction of the heat-shock response by carbon dioxide in Chironomus thummi

The effects of a set of stress treatments on gene expression of Chironomus thummi salivary gland cells have been analyzed. Among the treatments assayed, only during recovery from carbon dioxide have we observed a response similar to that previously described after heat-shock treatment: induction of the heat-shock puffs and synthesis of the heat-shock polypeptides. In these conditions, puffing and transcription of telomeric regions were observed, which led to the appearance of the temperature-inducible telomeric Balbiani ring T-BR-III. Other treatments failed to induce the heat-shock response, despite promoting real stress conditions to C. thummi larvae or salivary gland cells.

DNA-RNA hybrids and transcriptional activity in Chironomus polytene chromosomes

The distribution of DNA-RNA hybrids was studied in fixed Chironomus polytene chromosomes by means of specific antibodies directed against DNA-RNA hybrids. Attention was mainly focused on the relationship between detection of hybrids and local transcriptional activity. As a model to test such a relationship Balbiani rings, whose transcriptional activity was experimentally modified, and a new set of puffs induced by heat shock (t-puffs), were chosen. With a few exceptions DNA-RNA hybrids appeared, and when air-drying of the slides was avoided, in actively transcribing loci. When the slides were air dried, the antibody detected no DNA-RNA hybrids unless the preparations were submitted to conditions promoting denaturation and subsequent reannealing, or to a mild pronase digestion. Denaturation/renaturation led to the detection of DNA-RNA hybrids in heat-shock-induced as well as in heat-shock-inactivated loci. On the other hand, a mild pronase digestion led to the detection of hybrids only in the heat-shock-induced puffs. These results strongly suggest that in air-dried preparations the hybrids are masked by protein and that they are unmasked by a mild pronase digestion.

Effect of cycloheximide on RNA synthesis in Chironomus polytene chromosomes

Modifications in the synthesis of salivary gland RNA were induced by treatments with 10 μ/ml cycloheximide (CHM) on 4th instar larvae of Chironomus pallidivitattus. After 3, 6 and 24 h CHM treatment, RNA was labeled “in vitro”, by incubating the salivary glands in a medium containing H3-uridine. The electrophoretical analyses corresponding to the 3 and 6 h treatment showed a stimulation of the non-ribosomal components of the newly synthesized RNA, while preribosomal RNA synthesis appeared depressed. This fact was also confirmed at cytological level, since autoradiograms made after 3 h of CHM treatment showed a reduced H3-uridine label over the nucleolus and an increase of diffuse labeling over the chromosomes. Longer treatments (24 h) causes a considerable inhibition of the synthesis of all RNA species. The role played by protein synthesis inhibition in the aforementioned effects is discussed. — Some of the morphological implications of CHM treatment, such as modifications of the nucleolar structure (nucleolar segregation) are also reported. The use of a squash technique based on glutaraldehyde fixation of the salivary glands, considerably facilitates such studies.

Induction of heat-shock Balbiani Rings after RNA synthesis inhibition in polytene chromosomes of Chironomus thummi

We have studied the effect of RNA synthesis inhibitors, such as Actinomycin D, α-Amanitin, or DRB, on the heat-inducible puffs of Chironomus thummi. While the appearance of most of the T-puffs is blocked by the inhibitory treatments, the induction of a heat-shock Balbiani Ring (T-BR-III) takes place in the presence of these drugs. No transcriptional activity could be detected in this structure. However, its frequency and approximate size values are similar to those obtained for T-BR-IIIs induced under normal conditions. Accumulation of acidic proteins also takes place in T-BR-IIIs in the presence of these inhibitors. On the other hand, puff III-A3b, a major T-puff of Ch. thummi, seems to be resistent to DRB treatment at both the puffing and the transcriptional levels. These results are discussed in the context of puffing-RNA synthesis relationships.