Chironomidae: Ecology, Systematics Cytology and Physiology

SECTION I

CYTOLOGY AND PHYSIOLOGY

Outline

Chapter 1: In Vitro Translation of Balbiani Ring RNA from Chironomus tentans

Chapter 2: Comparative External Morphological and Karyological Characteristics of European Species of Genus Clunio Haliday, 1855 (Diptera, Chironomidae)

Chapter 3: Untersuchungen ueber die Verbreitung der Zuckmuecken Gattung Chironomus in der Schweiz, mit Besonderer Beruecksichtigung von Drei Cytologisch nicht Beschreibenen Arten

Chapter 4: Die Evolution der Gattung Chironomus aus Biochemisch-Genetischer Sicht

Chapter 5: Esterase Evidentiation and Characterization in the Course of Onthogenesis in Chironomus Thummi Kieff.

Chapter 6: The Haemoglobin Synthesising Tissue of Chironomus

Chapter 7: Basic Patterns in Chromosome Evolution of the Genus Chironomus (Diptera)

Chapter 8: Zur Atmungsintensitaet, Bewegungsaktivitaet und Herztaetigkeit nicht Dormanter und Dormanter Larven von Chironomus Plumosus

Chapter 9: Do the Malpighian Tubules Persist Unchanged Throughout the Metamorphosis?

Chapter 10: The Activities of Glycerinaldehyde 3 Phosphate Dehydrogenase, Lactate Dehydrogenase and Phosphoenol Pyruvate-Carboxykinase in Chironomid Larvae and Chaoborus Cristallinus Larvae

Chapter 11: Circadian Eclosion Rhythm in Chironomus Thummi: Ecological Adjustment to Different Temperature Levels and the Role of Temperature Cycles

Chapter 12: The Relation of Dry Weight and Temperature to Respiration in Some Benthic Chironomid Species in Lough Neagh

In Vitro Translation of Balbiani Ring RNA from Chironomus tentans

P.A. Hardy and C. Pelling,     Max-Planck Institut für Biologie, Abteilung Beermann, Spemannstrasse 34, 7400 Tübingen 1, Federal Republic of Germany

ABSTRACT

Balbiani rings are sites of particularly active RNA synthesis on the polytene chromosomes of Chironomids. Three such sites occur on the fourth chromosome in the salivary glands of Chironomus tentans. The RNA products of two of them (BR1 and BR2) have molecular weights of over 10 × 10° Daltons and age thought to code salivary proteins, which include fractions of more than 5 × 10⁵ Daltons. In order to obtain insight into the nature of their primary translation products, RNA was isolated from salivary glands and translated in a cell-free system from rabbit reticulocytes. Salivary gland RNA induces the synthesis of a set of polypeptides, having molecular weights of up to 5 × 10⁵. Immunoprecipitation studies, using an antiserum directed against total saliva, indicate that the very large species synthesized in vitro are immunologically related to constituents of saliva. These data provide evidence to support the contention that the unusually large proteins present in saliva are primary products of translation and are most probably coded by Balbiani ring RNA.

KEYWORDS

Balbiani ring RNA

in vitro translation

reticulocyte lysate

immunoprecipitation

salivary proteins

INTRODUCTION

The fourth polytene chromosome in the salivary glands of Chironomus tentans is characterized by the presence of three Balbiani rings. These chromosomal sites synthesize large amounts of RNA (Pelling, 1964), The RNA products of two of these giant puffs, BR1 and BR2, have been isolated and characterized biochemically and by in situ hybridization. The kinetics of in situ hybridization indicate that both Balbiani rings contain repeated sequences. The RNA is polyadenylated and has a sedimentation constant of 75S (For review, see Case and Daneholt, 1977). Its size has been determined by electron microscopy to be about 12 × 10⁶ Daltons(Case and Daneholt, 1978). 75S RNA emerges from the nucleus without detectable size change and is incorporated into polysomes. It thus functions as messenger RNA. The main protein products of the salivary gland are constituents of the saliva, which is secreted into the lumen of the gland and eventually the medium. They have been investigated by Grossbach (1969, 1977) and include species whose molecular weights exceed 500,000 Daltons. The observed correspondence between the high rate of RNA synthesis at BR1 and BR2 and the production of salivary proteins has been taken to indicate that Balbiani ring RNA codes for fractions of saliva. It has been suggested that BR RNA is translated into small units of 40 – 60K. which are later polymerized into the larger forms found in saliva in the lumen (Rydlander and Edström, 1975), but Grossbach (1977) could find no evidence to support this hypothesis.

In order to gain some insight into the nature of the primary translation products of Balbiani ring RNA, we have isolated RNA from salivary glands and translated it in vitro in the reticulocyte lysate system.

MATERIALS AND METHODS

Isolation of Salivary Gland RNA

Total salivary gland RNA was isolated by a modification of the method of Strohmann et al. (1977). Glands were homogenized in 50mM Tris pH8.0, 5mM EDTA; 2mM aurin tricarboxylic acid; 70mM mercaptoethanol; 8M guanidinium chloride. The homogenate was extracted three times with an equal volume of chloroform: isobutanol (4:1). RNA was precipitated by the addition of a half-volume of ethanol at −20°C and extracted twice more with 8M guanidinium chloride. The final pellet was washed with 3M acetate pH5.2 and with 80% ethanol and used for translation.

Preparation of Antiserum to Salivary Proteins

Salivary proteins were isolated by brief fixation of the glands with 35% ethanol and subsequent mechanical removal of gland cells. This material was injected into rabbits using the double emulsion technique (Weir, 1978). Antiserum was characterized using a protein A binding test (Kronvall and coworkers, 1970) and by rocket Immunoelectrophoresis (Axelsen, Kroll, and Weeke, 1973).

In Vitro Translation of Salivary Gland RNA

Aliquots of RNA were translated in the reticulocyte lysate kit from New England Nuclear (Dreieich, FRG) using the following salt conditions: 160mM K+, 1mM Mg, and including 5uM S-adenosylmethionine. 35S methionine was the labelled amino acid. Incubation was for 2 hrs. at 30°C.

Immunoprecipitates were isolated with fixed Staphylococcus aureus cells after the method of Kessler (1975).

The products of translation were examined by electrophoresis on SDS Polyacrylamide gradient gels (3 – 20%) with the buffer system of Laemmli (1970). Labelled bands were detected by fluorography (Bonner and Laskey, 1974).

RESULTS AND DISCUSSION

Figure 1 shows the pattern obtained on electrophoresis of cellular and salivary proteins in SDS Polyacrylamide gradient gels (3 – 20%). The saliva contains two prominent proteins (referred to as A and B) with mol.wts. of 55oK and 18oK respectively (these are probably equivalent to fractions 1 – 3 of Grossbach (1964)) as well as fractions of 15 – 20K. In addition, several minor bands are present.

Figure 1 shows gel separations of cellular (left) and salivary proteins (right). Positions of markers are also shown.

Figure 2 shows the results of a pulse-chase experiment in which larvae were injected with luCi of ³⁵S-methionine (Pelling, 1964). Cellular and salivary proteins were isolated after the times indicated and separated on gels. Labelled bands were detected by fluorography. It can be seen that 55oK material becomes labelled within 5 mins. Several discrete bands can be distinguished in this region of the gel. After some time, smaller polypeptides appear, including two bands of about 18oK, one of which is actually a doublet. The first protein to emerge into the lumen is Table 1: A constant amount of ¹²⁵I-labelled Protein A was incubated with aliquots of antiserum and/or isolated saliva in phosphate-buffered saline containing 1% BSA at 37°C for 2 hrs. Bound Protein A was recovered by centrifugation and counted in a gamma-ray counter. Results are expressed as a percentage of the input counts. Binding is observed only in the presence of both antiserum and antigen, indicating that Protein A is specifically recognizing immune complexes.a single species of 550K. A group of bands of 20 – 25K are also detectable after 30 min. Of the two heavily labelled proteins of 180K and 150K, which are present in cells, only one (B) appears in saliva.

TABLE 1

Protein A Binding Test

Figure 2 shows a fluorograph of cellular and salivary proteins separated after injection of larvae with 1μCi of ³⁵S-methionine. Left: Cell proteins 5 mins. after injection. Centre: Cell proteins 30 mins. after injection. Right: Salivary proteins 30 mins. after injection.

The observation that several species are found in the region of fraction A in cellular material but only the largest is apparently secreted into the lumen, possibly indicates that some post-translatory modifications occur. Indeed it is known that the salivary proteins are glycosylated (Grossbach, 1969) and the extra bands may represent intermediates in this