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.     

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.     

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.     

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6-bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.     

Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles.

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36- and 45-KDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36- or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles.     

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.     

The fine structure of red and white myotomal muscle fibres of the coalfish (Gadus virens)

Summary The fine structure of the red and white myotomal muscles of a marine teleost, the coalfish Gadus virens, has been examined and ultrastructural measurements and analyses carried out. The sarcomere lengths of the red and white fibres were found to be 1.60 minimum, 1.82 maximum and 1.70 minimum, 1.85 maximum, respectively. No significant difference was found between the red and white fibres in their percentage of sarcoplasmic reticulum and T system. Both were found to have regularly occurring triads at the Z disk level, to have distinctive M lines and to be multiply innervated. Ultrastructurally the two fibres can be distinguished by the thicker Z line and more abundant mitochondria of the red fibre, and by the ribbon-shaped peripheral myofibrils of the white fibres. The structure of the fibres in these two types of muscle is discussed in relation to their possible role in swimming.This work was supported by a research grant from the National Environmental Research Council.     

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.     

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     

The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles.

Specific premessenger ribonucleoprotein (pre-mRNP) particles, the Balbiani ring (BR) granules in the salivary glands of the dipteran Chironomus tentans, can be visualized in the electron microscope when they assemble on the genes, move through nucleoplasm, and bind to and translocate through the nuclear pores. As shown by BrUTP labeling and immunoelectron microscopy, newly synthesized BR RNP particles, released from the BR genes, appear early in all nucleoplasmic regions of the cell nucleus and they saturate the nucleoplasmic pool of BR particles after 2 h of labelling. It is concluded that within the nucleus the BR particles move randomly. Furthermore, estimates of minimum diffusion coefficients for the BR particles are compatible with the view that the particles diffuse freely in the interchromosomal space, although it is not excluded that the random movement could be slightly retarded. Once the particles get bound to the nuclear pore complexes, they seem committed to translocation through the nuclear pores.     

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 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.