Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Factors involved in the expression of gene activity in polytene chromosomes

In order to separate some of the factors involved in the formation of puffs the antibiotic actinomycin D was applied at different stages of puff activity. Puffs were induced by temperature shocks or eodysone.Inhibition of RNA synthesis with actinomycin D before application of a puff inducing stimulus prevents neither the appearance of the stimulus specific puffs nor the accumulation of acidic proteins in the puff regions. The puffs attained under these conditions approximately 1/3 of the size normally produced by the stimulus.Indications were obtained that during puff formation acidic protein accumulation precedes the onset of RNA synthesis.Synthesis and storage of newly synthesized RNA within the puff region was studied on the basis of grain distribution in uridine-H³ autoradiographs after various incubation periods. RNA synthesis appears to be restricted to a particular area of the puff region. After a 3 min temperature shock following injection of uridine-H³ silver grains are located only over a particular area of the newly formed puff. The same area becomes labeled during a 1 min pulse of uridine-H³ applied at a stage of maximum puff development. Longer periods of incubation result in a random distribution of the grains over the whole puff region. Grain counts on different areas of experimentally induced puffs and on the same areas at a stage of puff regression indicate that the newly synthesized RNA becomes transferred from the area where it was synthesized and is stored for a certain period within the puff region. Complete release of newly synthesized RNA from puffs in which RNA synthesis was inhibited by actinomycin D at a stage of maximal activity is accomplished within 30 to 35 min.     

RNS- und Protein-Synthese in Puffs isolierter Speicheldrüsen-Chromosomen von Chironomus

Isolated salivary gland chromosomes of Chironomus tentans incubated in a simple salt or sucrose solution are able to synthesize not only RNA but also protein in their puffs. The migration of radioactively labelled material from isolated nucleoli to isolated chromosomes suggests that not all protein in the puffs is synthesized by the puffs themselves but that part of the puff protein stems from the nucleoli. It is concluded that besides bound RNA polymerase a puff contains ribosomes attached to the growing mRNA molecules.     

Salivary gland function and chromosomal puffing patterns in Drosophila hydei

Summary The salivary glands of D. hydei larvae show differences between the cells in the distal (posterior) part and those of the proximal (anterior) part during the third instar. The first sign of these differences is an increase in cellular and nuclear volume in the distal cells of the gland, beginning at 103 hours after oviposition. After 125 hours the cytoplasm of the extreme distal cells acquires a reticulated structure, and at 130 hours these cells contain large granules or droplets of mucoprotein. From this moment up to puparium formation the number of cells containing these granules increases and the boundary of this type of cells shows a shift in the proximal direction. Just before puparium formation the granules disappear from the cells and a glue substance is secreted by the larvae. At this moment only a few cells in the extreme proximal part still lack granules. Electron-microscopical observations indicate that these cells were active in secretion, whereas all cells containing large granules are inactive in this respect during most of the third instar.During the early third instar a change in cell function occurs, i.e. from synthesis of substances presumed to be digestive enzymes which are secreted, to a synthesis of a glue substance which is stored. This change begins in the extreme distal cells of the gland.Investigation of the chromosomal puffing pattern revealed that a total number of 148 puffs were present during some period of the third instar, prepupal, and early pupal stages. The activity of 110 puffs was evaluated during a series of successive time intervals. Changes in the puffing pattern during puparium formation were compared with those observed during pupation.Proximal and distal nuclei differ in the activity level of a number of puffs, but only puff 47 B is restricted in activity to the distal cells. This puff becomes active at 119 hours and disappears 4 hours before puparium formation (156 hours). Determination of nuclear diameter and DNA in nuclei of both parts of the gland revealed a correlation between a particular DNA content and the function of the cell. Distal cells show higher nuclear diameters than proximal cells after the onset of granule production. The first differences in nuclear diameter can be seen at 103 hours. Cells in the transitional part of the gland, located between distal granulecontaining and proximal granule-negative cells, always show the same DNA content. These cells are found at different locations within the gland during the third instar. This zone of cells shows a shift in proximal direction during the third instar, identical to that of the neighbouring granule-containing cells.The possible interrelation between nuclear DNA content, the activity of puff 47 B, and the production of the glue substance were discussed.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration

The response of the three major classes of puff in salivary gland chromosomes of larval Drosophila melanogaster to varying β-ecdysone concentrations has been studied in in vitro cultured glands. Two (25AC and 68C) of the intermolt puffs regress at a rate dependent upon the hormone concentration. Three rapidly reacting puffs (23E, 74EF and 75B) respond in a graded way to β-ecdysone concentrations over a range of at least 600 ×. In contrast, five late-reacting puffs (62E, 78D, 22C, 63E, and 82F) do not respond below 5 × 10^?8M and at 2.5 × 10^?7M react maximally. The 50% response of the early puff sites 74EF and 75B and of the late puff sites occurs at 1 × 10^?7M. Two points are discussed in detail: whether ecdysone is necessary as a sustained stimulus or only as a trigger for the sequential puffing response and an evaluation of the absolute ecdysone concentration necessary for induction.     

RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS

The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope.     

A STUDY OF THE NUCLEOLAR MATERIAL IN SCIARA COPROPHILA

In the polytene chromosomes of Sciara coprophila, in addition to a nucleolus, large numbers of nucleolarlike structures or micronucleoli are formed. A detailed mapping localized the nucleolar organizer at one end of the X chromosome and revealed that approximately 18% of the bands of each chromosome are potentially capable of producing micronucleoli. Most of these sites are in regions known from a previous study to show asynchronous DNA replication: DNA puffs and certain heterochromatic regions. Micronucleoli are rarely found in association with bulbs. The RNA metabolism of the polytene chromosomes during late fourth instar was studied using radioautographic techniques. Isolated glands were incubated in tritiated uridine for 10 to 30 min, and radioautographs were made of squash preparations. Despite the wide range of variation found among different larval cultures, the following pattern was observed. Just prior to and at the beginning of DNA puff formation, a period of intense extrachromosomal nucleolar and micronucleolar RNA synthesis occurs. After maximal development of the DNA puffs, the synthesis of extrachromosomal RNA is at a low point, while incorporation into bulbs and DNA puffs remains high. With the onset of the prepupal stage, all nuclear RNA synthesis ceases.     

Hydroxyurea-induced inhibition of DNA puff development in the salivary gland chromosomes of Bradysia hygida

The injection of hydroxyurea at a critical time during the fourth larval instar inhibits the development of all DNA puffs in the salivary gland chromosomes of Bradysia hygida. RNA puff formation is not disturbed and larval development continues. The effect is explained as a result of a selective and general inhibitory action of the drug on DNA synthesis during the time when gene amplification occurs in the salivary glands. The incorporation of uridine into the chromosome regions where DNA puff development has been inhibited is sharply decreased in comparison with the incorporation into non-amplifying parts of the same chromosomes. The interpretation proposed for the cytologic observations seems to offer a better understanding of the nature of the DNA puffs.     

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both RNA and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. — Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puffs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the Drosophila-based model may not be completely applicable to Rhynchosciara.     

Cytologic and Autoradiographic Studies of the Micronucleus at Meiotic Prophase in Tetrahymena pyriformis

Micronuclear changes of variety 1 of Tetrahymena pyriformis during meiotic prophase have been observed by the light microscope. Morphologic changes in the micronucleus are divided into 6 stages. In stage I, chromatin begins to polarize; in stage II, the micronucleus becomes spindle shaped; and in stage III, one end of the micronucleus protrudes to form a “neck.” In stage IV, where the micronucleus elongates to maximal length, the whole micronucleus consists of 2 chromatin threads pairing longitudinally. One thread probably contains one genome. In stage V, the elongated thread becomes shorter and thicker. Finally, in stage VI, separate chromosomes appear and enter into metaphase. To discover the role of the elongation of the micronucleus, called crescent formation, autoradiographic analysis of RNA and DNA synthesis were undertaken using [³H]uridine and [³H]thymidine. Pulse label and chase experiments show that the crescent in stages II and III is actively synthesizing RNA. Though no remarkable DNA synthesis was observed during meiosis, a small amount of DNA synthesis occurred during the 1st and 2nd prezygotic divisions.     

Alcune caratteristiche strutturali dei cromosomi politenici del sospensore embrionale di Phaseolus coccineus in due momenti dell'embriogenesi

Abstract

Some features of polytene chromosomes of Phaseolus coccineus suspensor during two stages of early embryogenesis. – The distribution of DNA and RNA puffs in the whole genome of the giant cells of the Phaseolus coccineus embryo suspensor has been detected in two stages of embryo development. The collected data show that the chromosome regions showing the highest frequency of DNA puffs in both analysed stages are the following: i) band B (the fraction proximal to secondary constriction) of chromosome pair I and band E of chromosome pair V. When the two stages of development are however compared, it is seen that the % of DNA puffs in chromosome pair is at least double in suspensors dissected from the first stage of embryo development (86% in the first stage; 41% in the second stage). As to chromosome pair V band E organizes DNA puffs in 36% and in 50% of observed chromosomes in the first and second stage respectively; ii) band A of chromosome pair II, with a frequency of 52% (first stage) and 27% (second stage); iii) band E of chromosome pair VIII (27% in the first stage and 19% in the second stage). As far as the organization of RNA puffs is concerning it seems possible to outline the following values as the highest percentages:

a) First stage. chromosome b) Second stage. chromosome

I: band B 83% I: band B 91% VI: band E 55%

II: band A 70% II: band A 51% band G 50%

IV: band E 45% band C+D 59% VIII: band E 44%

V: band B+C 57% IV: band E 51% IX: band A 54%

band E 71% band G 51% band E 43%

VI: band E 53% band I 51% band F 53%

VIII: band E 43% V: band E 43%

IX: band E 42%

The differences observed between the two stages are discussed in relation to the function of the suspensor.     

Heat-shock induced puffing changes in Balbiani rings

A 5 minutes exposure of Chironomus larvae to near-lethal temperatures (39–40° C) produces a characteristic sequence of puffing changes in the Balbiani rings of the 4th salivary gland chromosomes: An initial phase of complete puff regression is followed, after a variable time lag, by a phase of rapid recovery to overnormal puff sizes. This is accompanied by RNP droplet formation. RNA synthesis at Balbiani rings during the initial puff regression still occurs. Regression is inhibited by 2,4-dinitrophenol, while recovery can be prevented by both 2.4dinitrophenol and actinomycin D. Regression and recovery are insensitive to cycloheximide. RNP droplets, as observed in Balbiani rings, in the nuclear sap and at the nucleolar rim, are composed of a fine fibrillar matrix which is covered by Balbiani ring granules in various phases of assembly. The results are discussed in terms of a model of puffing based on an equilibrium between RNA synthesis, RNA processing and RNP release from the puff.