Untersuchungen zur Struktur und Funktion der Balbianiringe von Chironomus tentans

Abstract

Untersuchungen zur Struktur und Funktion der Balbianiringe von Chironomus tentans

1. Ein Verfahren wurde entwickelt, polytäne, somatische Interphase-Chromosomen aus larvalen Speicheldrüsen von Chironomus spec. als Ganze nativ zu isolieren. Isolierte und in Speicheldrüsen befindliche Riesenchromosomen wurden strukturell sowie funktionell untersucht und gegenübergestellt.

2. Die charakteristische Musterausbildung in situ und in vitro befindlicher Riesenchromosomen beruht auf der aperiodischen Abfolge 0.1-0.6 µm dicker Banden. 0.2-0.3 µm dicke Banden kommen in Messungen Glutaraldehyd-fixierter Chromosomen am häufigsten vor. Die Hypothese, daß das bandenspezifische Chromomer als kleinste Struktureinheit polytäner Chromosomen durch die differentielle Knäuelung der Chromatide entsteht, wurde elektronenoptisch bestätigt. In den 0.05-0.3 µm dicken Interbanden sind die Chromatiden anscheinend frei von jeglichem Paarungskontakt. Sie zeigen keine erkennbare Knäuelung. Der Chromatidendurchmesser (110-130 A) ändert sich beim Übergang von den Banden in die Interbanden-Regionen nicht. Unterschiede in der Packungsdichte der Chromatiden kommen vor.

3. In Spreitungspräparaten nativ isolierter Chromosomen findet man in den Banden Schleifen unterschiedlichen Entfaltungsgrades. Sie stellen keine Transkripte dar. Diese Konfigurationen sind Übergangsformen zwischen eng gefalteten Chromomeren-Fibrillen und den in Balbianiringen und anderen Puffs beobachteten Chromomeren-Schleifen. Im Chromatidenverlauf kommen diese 0.8-8.4 µm langen Chromomeren-Schleifen in Abständen von 0.6-3 µm vor. Ihr DNA-Gehalt errechnet sich auf 7.2-75 Kilobasen (KB). Im hypotonischen Milieu geht die Stabilität und Faltung der Chromomeren-Schleifen in den Banden polytäner Chromosomen verloren. Vermutlich durch den Verlust von Histon H1. Als ein Nicht-Histon ist RNA-Polymerase B (II) [B (II)-Polymerase] mit der Chromatide assoziiert.

4. In vivo fixierte Chromosomen zeigen in Banden, Interbanden, Puffs und Nukleolen 110-130 A dicke Elementarfibrillen. Demgegenüber erscheinen die gespreiteten Chromosomenfasern bis zu 300 A dick und zeigen eine deutliche Nukleosomenstruktur. In Ultradünnschnitten von in situ fixierten Chromosomen sind unter physiologischen Salzbedingungen keine Nukleosomen sichtbar.

5. Balbianiringe (BR1, BR2, BR6) repräsentieren wahrscheinlich, wie andere Puffs, solitäre Transkriptionseinheiten. Sie unterscheiden sich von ribosomalen Cistrons durch größere Länge (3-6 µm) und die spärlichere Polymerasenbesetzung. BR1, 2, 6 besitzen 300-500 A große RNP-Grana als Transkriptionsprodukte. BR3 unterscheidet sich von allen bekannten Balbianiringen durch kleinere, 130-200 A messende RNP-Grana. BR3 ist ein Sonderfall. Das Molekulargewicht der BR2-RNA liegt schätzungsweise im Größenbereich von 6-10x10 hoch 6 Daltons. Grundlegende Ergebnisse dieser elektronenmikroskopischen Transkriptionsanalyse des BR2-Gens zeigt Abbildung 40.

6. Mit DIMETHYLSULFOXID kann das BR2, 1, 3-Puffing in lebenden Chironomus-Larven spezifisch verändert werden. BR3 entwickelt sich während der 2.-3. Stunde vorübergehend zum größten BR (overshooting). BR2 und BR1 kollabieren währenddessen. Von der 4. Stunde an sind das extreme Puffing (overshooting) von BR1 und BR2 und das „normale“ Puffing von BR3 typisch. Nach dem DMSO-Entzug kollabieren vorübergehend alle BR. BR3 beginnt in der Erholungsphase immer zuerst, BR2 immer zuletzt mit dem Puffing. Zwischen den drei Balbianiringen besteht eine Aktivitätsbeziehung., Mit der DMSO-Stimulation des BR3 nimmt die Anzahl seiner Transkriptionsprodukte zu.

7. Chromosomen IV in DMSO-behandelten Larven zeigen über kollabierten Balbianiringen keine 3H-Uridinmarkierung. Auch in experimentell überkondensierten IS-Chromosomen sind die B (II)-Polymerasen funktionsgehemmt bzw. –inaktiv. Niedriges Salz (0.03 M NaCl) und hohes Salz (0.34-0.4 M NaCl) hingegen führen beide im zellfreien System zur Auflockerung der Chromomeren. Solche künstliche Dekondensation isolierter Chromosomen IV aus DMSO-behandelten Larven mit kollabierten BR1, 2, 3 führt zur Derepression der B (II)-Polymerasen. Auch alle anderen Puffs nativ isolierter Chromosomen I aus behandelten und nichtbehandelten Chironomus-Larven sind nach künstlicher De- und Rekondensation in vitro auf einmal markiert. Riesenchromosomen besitzen also auf dem DNP-Template gebundene B (II)-Polymerasen. Vor allem hohes Salz (0.34-0.4 M NaCl) stimuliert die Polymerasenaktivität. In Gegenwart von alpha-Amanitin bleibt die Bandenmarkierung aus. Die Polymerasenaktivität ist mit Toluidinblau in nativen IS-Chromosomen als metachromatische Färbung in situ nachweisbar. Das Markierungsverhalten isolierter Chromosomen spricht für die Lokalisation des wesentlichen genetischen Materials in Banden. Der in vivo stattfindende Puffing-Prozeß läßt sich hypothetisch in zwei Teilreaktionen untergliedern: Bei der Initialreaktion der Genaktivierung, dem primären Puffing, wird die sterische Hemmwirkung der Chromatiden-Faltung spezifisch aufgehoben, was zu einem örtlich begrenzten Auflockern der Chromatide im jeweils zu aktivierenden Chromomer führt. Das typische Puffing findet in dieser Phase nicht statt. Nach dem primären Puffing falten die B (II)-Polymerasen anscheinend autonom das jeweilige Chromomer weiter auf. Dieses typische, sekundäre Puffing wird als das Resultat des Transkriptionsprozesses selbst aufgefaßt. Daß das sekundäre Puffing im Normalfall ohne gleichzeitige RNA-Synthese stattfindet, ist auszuschließen.

Teile dieser Dissertation wurden veröffentlicht in:

Sass, H. (1980a). Features of in vitro puffing and RNA synthesis in polytene chromosomes of Chironomus. Chromosoma 78: 33-78 http://www.springerlink.com/index/10.1007/BF00291908

Sass, H. (1980b). Puffing und RNA-Synthese in larvalen und imaginalen Polytän-Chromosomen aus verschiedenen Geweben von Chironomus tentans. Biol. Zentralblatt 99: 399-428

Sass, H. (1980c). Hierarchy of fibrillar organization levels in the polytene interphase chromosomes of Chironomus. J. Cell Sci. 45: 269-293 http://jcs.biologists.org/cgi/reprint/45/1/269

Sass, H. (1981). Effects of DMSO on the structure and function of polytene chromosomes of Chironomus. Chromosoma 83: 619-643 http://www.springerlink.com/index/10.1007/BF00328523

Structural and functional studies of Balbiani rings in Chironomus tentans

1) A new procedure is presented for isolating polytene interphase salivary gland chromosomes in its natural state from aquatic larvae of the midge Chironomus tentans. The individual chromosomes I, II, III and IV can be distinguished by their size and shape. By the possession of such ‘pure’ chromosomes new experiments were suddenly made feasible. Great opportunities are now open for the study of chromosome behavior, the chromatid fiber organization, the chromosomal distribution of RNA polymerase B (II) [RNA pol B (II)] and its RNA synthesis in vitro as compared to that in situ, i.e. the physiological environment of intact salivary glands.

2) Here in situ studies in C. tentans by electron microscopy (EM) have revealed that the characteristic pattern of polytene salivary gland chromosomes is caused by the aperiodic appearance of series of bands 0.1-0.6 µm thick. Bands result from the association of identical chromomeres (= compacted fiber segments) at the same level. Bands 0.2-0.3 µm thick are most frequently found. Thickness of interbands varies from 0.05-0.3 µm. The intimate pairing of numerous copies of fibers in compact bands makes it difficult to identify a single chromatid (= haploid chromosome). A breakthrough came when oligotene Balbiani ring (BR)2 chromatid strands were thinly sectioned lengthwise. Pictures are given visualizing that chromomeric DNA has the appearance of irregular folded or coiled chromatid fibers, but the constituent fibers become relative straight and line up in parallel arrays in interchromomeres.

3) Although the multistranded, cable-like and highly polytenized isolated salivary chromosomes of C. tentans seemed virtually hopeless to study by Miller chromatin spreading technique with EM, this present studies demonstrate that one could learn some of the ultrastructural organization. The dispersal of polytene chromosomes in low-ionic-strength buffer results in destabilization of bands. Chromatin extruded from unfolded bands is composed of more or less twisted, individual loops 0.8-8.4 µm long and 0.6-3 µm apart consisting of duplex DNA and proteins. The looped chromatin structures contain 7.2-75 kilobases DNA. Some loops are transcribed and surrounded by portions of ribonucleoprotein (RNP).

4) The basic structural element of interbands and bands including puffs and nucleoli in C. tentans polytene chromosomes is the 110-130 A chromatid fiber, but their width varies from 110 to 300 A. Visually, there is no obvious indication in ultrathin sections that suggests the existing of repeating units, the nucleosomes, which constitute a chromatid. In contrast, the picture suddenly changed, when isolated salivary gland chromosomes were dispersed in low-ionic-strength buffer for EM-inspection. It now appears that the unravelled 110 A fiber shows nucleosomes in a continuous ‘beads-on-a-string’ morphology. Chromatid fibers in spread preparations of polytene chromosomes are at most 200-250 A in diameter. The larger fiber could reflect a higher-order chromatin structure regulated by nucleosomal folding or packaging events.

5) Evidence is presented that the size of a C. tentans Balbiani ring is not necessarily correlated with the length of its transcripts. Ultrathin sections did show, the only apparent difference between more or less puffed (expanded) BR 2s and BR 1s is a greater concentration of large 300-500 A RNP granules in the larger Balbiani rings. Aside from these findings, experimental evidence was provided that BR3 when excessively expanded (overshooting) in response to the drug DMSO (see below) is filled with small RNP granules. Prior to DMSO-stimulation, much less BR3-RNP granules are present at the transcriptionally active BR3 gene. However, BR3 is a special case. The existence of BR3-RNPs only 130-200 A in diameter had hitherto been unknown. Undoubtedly, these results demonstrate convincingly that BR3-RNPs must contain much shorter lengths of RNA than RNP-granules from BR2 and BR1. These results strongly imply a significant difference in gene organization between BR3 and BR2/BR1.

In addition, progress toward understanding how the BR2 gene is transcribed is presented. For the first time, in Miller-spreads of isolated salivary gland chromosome IV, 33/µm BR2 gene transcripts have been identified. This evidence shows, the BR2 gene of C. tentans belongs to the class of intensively transcribed genes. There is one BR2 gene copy present in the haploid genome. Tandem repetition of transcribed BR2 genes was not found. Mature BR2-RNP granules ~ 500 A thick arise from filamentous precursor molecules and are released into the nuclear sap. While it is clear that 33/µm RNA pol B (II) transcribe the C. tentans BR2 gene, the size of BR2-RNA has been estimated to 6-10x10 to the power of 6 daltons. Figure 40 summarizes the fundamental BR2 gene transcription results.

6) A novel regulator mediating alterations of BR3-, BR2- and BR1-formation is dimethyl sulfoxide (DMSO). Exposure of C. tentans 4th instar larvae to 10% DMSO at 18°C first evokes within 2-3 hours of treatment a most extraordinary BR3 expansion. Such BR-overshooting experiments implicate one mode of DMSO-action may be the transcriptional activation of BR3 gene copies on sister chromatids for increased BR3 expression. In addition, this induction of BR3 and repression (or non-induction) of BR2 and BR1 are of only brief duration. After the 3rd hour of exposure to 10% DMSO the over-stimulated BR3 is reduced to normal dimensions, whereas BR1 and BR2 exhibit excessive puffing stimulation. Thus, BR3 and BR2/BR1 transcription are interconnected. After withdrawal of DMSO, a rapid uniform collapse of Balbiani rings and all other puffs occurs. Recovery proceeds as Balbiani rings and other puffs reappear.

7) Autoradiographic studies with tritium-labelled RNA precursor 3H-uridine performed on C. tentans salivary gland chromosomes IV revealed that the in vivo labelling corresponds to the altered size relationship. BR3 grows to enormous dimensions and is heavily labelled while BR2 and BR1 are drastically reduced and exhibit only very low 3H-uridine incorporation. After withdrawal of DMSO, all of the Balbiani rings and all of the other puffs recondense temporarily, and hardly incorporate any 3H-uridine in vivo.

In parallel experiments, chromosomes IV with recondensed BR1, BR2 and BR3 were isolated and subsequently labelled with 3H-UTP in high salt (0.34-0.4 M NaCl). A surprising finding in these autoradiograms was that after the chromosomal decondensation in vitro, the BR-regions, in spite of the absence of puffing, do exhibit transcriptional activity of high labelling intensity, although less than in the puffed, ring-shaped in vivo BR-regions. Such rapid increase of radioactive precursor incorporation in vitro aroused much excitement. This key observation led to the conclusion that RNA pol B (II) is bound to the practically inactive BR1, BR2 and BR3 genes. These results prove that the chromosome decondensation under high salt greatly enhances the basic low rate of transcription in the collapsed BR-regions and many sites of chromosome IV and allows RNA pol B (II) to elongate. Under the experimental conditions used here for chromosome incubation in vitro, there is certainly no free RNA pol B (II) available to initiate RNA synthesis.

Further support for the realization that these polytenes are permanently equipped with RNA pol B (II) was obtained by application of the same method to isolated chromosomes I, II and III. The results agree perfectly with those found for chromosome IV. Again, active RNA pol B (II) is present not only at the gene loci which are transcribed at a given stage, but also at all puff sites which are ever found in salivary glands. On the whole, then, there can be no doubt that the evidence from the here presented chromosome transcription autoradiography disproves the common belief that RNA pol B (II) is recruited to the promoters of genes when they become activated. This finding of the widespread chromosomal association of RNA pol B (II) also affords a ready explanation that the transcription elongation of the DNA template is rapidly activated by appropriate stimuli in vivo.

From these advances a concept for the process of RNA synthesis and puffing is deduced. I prefer to postulate that puffing is a two-step mechanism of activation in transcription. In vivo, during primary puffing, which happens on the ultrastructural level and is visible in the electron microscope, there are changes in the arrangement or configuration of the chromatid fiber in bands. The chromomeric DNA is transiently dispersed from its usual tightly packed state. This is a necessary prerequisite to remove the sterical hindrance for RNA pol B (II) and to allow the enzyme the interaction with its chromatin environment to resume transcription elongation. As secondary puffing, different constellations of chromosome puffs, these light-optically visible expanded regions on polytene chromosomes, appear as the consequence of intense RNA synthetic activity at specific gene loci. Secondary puffing is not a necessary prerequisite for RNA synthesis, while, conversely, RNA synthesis is required for puff formation.

Parts of this thesis have been published in:

Sass, H. (1980a). Features of in vitro puffing and RNA synthesis in polytene chromosomes of Chironomus. Chromosoma 78: 33-78 http://www.springerlink.com/index/10.1007/BF00291908

Sass, H. (1980b). Puffing und RNA-Synthese in larvalen und imaginalen Polytän-Chromosomen aus verschiedenen Geweben von Chironomus tentans. Biol. Zentralblatt 99: 399-428

Sass, H. (1980c). Hierarchy of fibrillar organization levels in the polytene interphase chromosomes of Chironomus. J. Cell Sci. 45: 269-293 http://jcs.biologists.org/cgi/reprint/45/1/269

Sass, H. (1981). Effects of DMSO on the structure and function of polytene chromosomes of Chironomus. Chromosoma 83: 619-643 http://www.springerlink.com/index/10.1007/BF00328523