Heat shock phenomena in Chironomus tentans

Incubation of 4th instar larvae of Chironomus tentans at elevated temperatures leads in salivary and Malpighian chromosomes to the appearance of 4–5 new puffs. Previously present puffs, particularly Balbiani rings in salivary chromosomes, become drastically reduced. The reactions of region IV-5C and Balbiani ring 1 and 2 in salivary glands are quantitatively analyzed. Statistically significant heat shock effects are observed already after 5 min and reach a maximum between 30 and 60 min. The effective temperature range is small (between 33 to 40 ° C) with an optimum at 37 ° C. Above 40 ° C, i.e., at overheat shock temperatures, heat shock reactions are suppressed. Larvae heat or overheat shocked for 1–7 h or 15–30 min, respectively, survive when returned to normal culturing temperatures. The recovery from heat shock of the puffing pattern occurs in two phases: a fast one (10–20 min) and a slow one (up to 5 h) sometimes separated by a period of backlash. Quenching of overheat shocked larvae does not result in a delayed heat shock reaction.     

Chromosomal RNA synthesis in polytene chromosomes of Chironomus tentans

The presence of heterogeneous RNA of high molecular weight has been demonstrated on the giant chromosomes, in the nuclear sap and in the cytoplasm of the salivary glands in Chironomus tentans. The kinetic properties of this heterogeneous RNA have also been outlined in some detail. — Salivary glands were incubated for different time intervals (20, 45 and 180 min) in haemolymph, supplemented with tritiated cytidine and uridine. The different cellular components were isolated by micromanipulation and RNA extracted with an SDS-pronase solution and analysed with electrophoresis in agarose. — Heterogeneous, high molecular weight RNA with a peak around 35 S was saturated with label on chromosome I, II and III in 45 min, although the synthetic capacity was unchanged during at least 180 min incubation. This indicated a complete turnover of heterogeneous RNA on the chromosomes in less than 45 min. The turnover time in the giant puffs (the so called Balbiani rings) on the fourth chromosome, was even shorter and estimated to less than 30 min. No shift in the electrophoretic pattern of this heterogeneous RNA was found to occur on the chromosomes during long incubation times or during actinomycin D experiments. These labelling characteristics of heterogeneous RNA on the chromosomes indicate that all the different molecules in the heterogeneous RNA have a similar and rapid turnover. A conversion to smaller, stable molecules was excluded. — Heterogeneous RNA of a distribution corresponding to that on the chromosomes was found in the nuclear sap and also in the cytoplasm. The activity in both these cellular compartments increased between 45 and 180 min incubation. The distribution pattern for high molecular weight RNA was in all experiments similar on the chromosomes, in the nuclear sap and in the cytoplasm. It appears that at least a considerable part of the high molecular weight RNA leaves the chromosomes to enter the nuclear sap and lateron to some extent the cytoplasm in this high molecular form. Stable molecules of smaller size (6–15 S) did not appear during 180 min incubation. The data indicate, however, also a substantial breakdown of heterogeneous RNA to acid soluble products during this time.     

RNA synthesis in isolated polytene nuclei from Chironomus tentans

An RNA synthesizing system with isolated polytene nuclei from Chironomus tentans is described. This system allows one to monitor the effect of salt concentration on chromosome structure and to assign in vitro RNA synthesis to structural modifications of the chromosome (i.e. nucleoli, Balbiani rings and puffs).-At a salt concentration of 0.15 M monovalent cations (standard salt medium=SSM) chromosomal structure appears to be best preserved during in vitro incubation. At low and high ionic strength the bands decondense and the microscopically visible chromosomal structure is lost completely. These three states of condensation and decondensation are distinguished with respect to RNA synthesis: (1) in low salt overall RNA synthesis is depressed, (2) in SSM ribosomal RNA synthesis predominates and continues for 30 min, (3) in high salt RNA synthesis is stimulated 3–4 fold again. This stimulation is due solely to chromosomal, non-ribosomal RNA synthesis, which proceeds in high salt for more than 10 h, though new initiation of RNA chains is prevented. Molecular weight determinations of the RNA synthesized demonstrate a time dependent increase in size of the newly synthesized molecules under these conditions. — Autoradiographs of nuclei incubated in SSM reveal prominent label in nucleoli, significant label in Balbiani rings and rather reduced activity at other sites. Addition of various exogenous RNA polymerases does not markedly alter this pattern. Autoradiographs of nuclei incubated in high salt exhibit extensive RNA synthesis spread over the chromosomes. Preparations of autoradiographs from isolated chromosomes show that the high salt induced label is localized in single bands. Though the majority of bands is still unlabelled, the actual number of bands exhibiting incorporation in high salt is higher than in any individual functional state in vivo. These results are discussed in terms of activated and preactivated genes.     

Localization of 5 S RNA genes in Chironomus tentans

The genes for 5 S RNA in Chironomus tentans have been located to region 2A of chromosome II by cytological hybridization. RNA from individual chromosomes, nuclear sap and nucleoli of salivary gland cells hybridized with the identified 5 S RNA genes in region 2A of chromosome II. The results suggest a common origin of 5 S RNA in these different nuclear compartments.     

Weak male-determining genes and female heterogamety in Chironomus tentans

Female heterogamety in the midge Chironomus tentans has been previously reported and attributed to a dominant female determiner. Published results are not consistent with the interpretation, and the female heterogamety, if any, can be better explained by a model involving a weakened male determiner. Suggestions are made for crosses between populations with different sex-determining mechanisms that would discriminate between models for the evolution of female heterogamety, and serve to determine whether indeed female development is the norm in the absence of any parental sex chromosomes.     

The fourth chromosome of Chironomus tentans malpighian tubules

Morphology and banding pattern of the 4th chromosome in Chironomus tentans Malpighian tubules have been investigated by electron microscopy, using the squash and selection technique. The map we composed from our observations shows a remarkable increase (75%) in band numbers as compared to the map previously presented by Beermann for the 4th chromosome from salivary glands. Extrapolation of this increase to the entire genome would result in a total band number of about 3,500. The mean DNA content of bands can thus be calculated to be about 50 kb. Many bands show a complex structure, including the BR2 band. Some bands seem to result from fusion of smaller components. Minibands have also been observed. Some interbands contain RNP particles. In our material the interbands appeared to be made up of fibrils with a diameter of about 120 Å. On the basis of these results we estimate the DNA in the interbands as amounting to 2% of the entire genome. The results are discussed with respect to the organization of the polytene chromosomes and the functional significance of the banding pattern.     

Physico-chemical properties of chromosomal RNA in Chironomus tentans polytene chromosomes

Labelled chromosomal RNA of the dipteran Chironomus tentans was studied with respect to its migration properties during electrophoresis in agarose. The RNA was isolated from polytene chromosomes which had been microdissected from fixed salivary glands and obtained free from nucleoli and nuclear sap. Labelled material migrates as 4–5 S RNA and as polydisperse material in a range where the lower limit corresponds to 10–15 S, the upper limit to 80–90 S RNA and the maximum in the distribution to 30–40 S RNA. The data indicate that the latter fractions are formed by unbroken, single-stranded RNA molecules, partly of very high molecular weights. It is shown in a number of tests that the distribution is not a consequence of formation of complexes or aggregates between RNA molecules on one hand and DNA, proteins or other RNA molecules on the other.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

Ultrastructural and cytochemical studies of salivary gland regression in Chironomus tentans

Summary The ultrastructural localization of acid phosphatase (AcPase) activity in regressing salivary gland cells of Chironomus tentans was studied with Gomori's lead method. In last instar intermolt larvae AcPase activity is restricted to Golgi vesicles, to small electrondense bodies of about 0.25 diameter, and to larger, more electron-lucid bodies which are considered to be lysosomes. The smaller bodies apparently arise from Golgi vesicles. The average frequency of lysosomes increases as development proceeds. Until the end of the pupal molt, only very few of them contain degenerating fragments of other cellular components.Overt cell regression begins in young pupae. At this stage practically all lysosomes contain degenerating cell components. In addition, cellular breakdown seems to occur outside of these organelles. Regressing cellular areas show in addition free AcPase reaction products (lead deposits), the amount of which closely parallels the degree of regression of the particular area.Possible genetic relationships between the various AcPase-containing cell organelles and the role of lysosomes in the control of gland cell breakdown are discussed.Supported by NSF Grant GB-2639 to U. Clever. The technical assistance of Mr. Hermann Bultmann in part of these studies is gratefully acknowledged.     

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     

Are there female heterogametic strains of Chironomus tentans Fabricius?

Although male heterogamety is the generally accepted method of sex determination in Chironomus, female heterogamety has been reported for some strains of Chironomus tentans. Some new data, combined with a reassessment of the published data, indicate that the proposal of female heterogamety rests on inconclusive data, while male heterogamety provides an adequate explanation of sex determination in C. tentans. A cross which would unambiguously discriminate between male and female heterogamety in these strains is proposed, although it is considered unlikely that female heterogamety exists in this species.     

Biochemische und cytogenetische Untersuchungen zur Natur des Hämoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidivittatus

The haemoglobin of chironomids is dissolved in the body fluid of the larvae and can be separated electrophoretically in Chironomus tentans into 10, and in C. pallidivittatus into 8, different bands. The molecular weight determined under the electrophoretic conditions was 15,000±1,000 for each haemoglobin band. This means that each haemoglobin band represented a single protein chain. In each species 7 haemoglobins could be characterised as species specific according to their different electrophoretic mobilities, developmental characteristics and the fact that one haemoglobin could be correlated genetically with a specific chromosome inversion. The inheritance of all these species specific haemoglobins was found to be co-dominant. With cytogenetic methods it was possible to define the loci of the species specific haemoglobin genes as being restricted to certain parts of chromosome 3. This finding suggests gene duplication as the most likely mecanism of the evolution of haemoglobins in Chironomus.     

RNA synthesis in a Balbiani ring in Chironomus tentans salivary gland cells

Rapidly labelled RNA in Balbiani ring 2 on chromosome IV in the salivary glands of Chironomus tentans was investigated. This RNA is likely to be transcribed from only one chromosomal band, supposed to be a single operational unit in these polytenic cells (Beermann, 1966).Salivary glands were incubated in larval haemolymph, supplemented with tritiated RNA precursors and fixed afterwards. Balbiani rings 2 (in some experiments also Balbiani ring 1 and 3) were isolated with micromanipulation. The labelled RNA was extracted with SDS-pronase and analysed with electrophoresis in agarose.The rapidly labelled RNA in Balbiani ring 2 was as heterogeneous as RNA from the remainder of the chromosome set (10–90 S) but the peak of the distribution of label in BR 2 corresponded to molecules of about 50 S as compared to that of RNA from the rest of the chromosome set which was about 35 S. When the synthetic activity in Balbiani ring 2 was very high, relatively more molecules with very high molecular weights were produced compared with the state when the synthetic activity was moderate or low. The synthetic activity in Balbiani ring 2 compared to that in Balbiani ring 1 was well correlated to the relative sizes of the two Balbiani rings. The results on Balbiani ring 2 are discussed in relation to the size and structure of the chromomere.