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B Daneholt

Publications and source records attributed to B Daneholt.

At least 73 records · Page 4Linked to original sources
PubMed1982

Visualization of active 75 S RNA genes in the Balbiani rings of Chironomus tentans.

The active 75 S RNA genes in the Balbiani rings of Chironomus tentans were investigated by two complementing electron microscopy procedures: spreading of isolated chromosomes according to Miller and serial sectioning of Balbiani rings. The Miller spreads show that the transcription products, the ribonucleoprotein (RNP) fibers, increase gradually in length along the 75 S RNA genes. The morphology of the active genes in situ suggests the following sequence of events: during transcription a 20 nm RNP fiber is formed which is oriented roughly perpendicular to the chromosomal axis. When an RNP fiber corresponding to one quarter of the gene has been generated, packaging of the fiber into a dense globular structure begins at the free end of the RNP fiber. The processes of transcription and packaging go on in parallel in such a way that the length of the 20 nm fiber is kept constant, while the globular part is steadily increasing in size. When transcription is completed, a globular product is released, 50 nm in diameter. A thin (5 nm) chromosomal axis as well as a low DNA compaction (3.6) imply that the DNA in the chromosome fiber of an active 75 S RNA gene is more extended than the DNA in a nucleofilament. On the basis of our electron microscopy data and other information available on the Balbiani ring genes, we discuss the activation of the 75 S RNA genes in terms of a two-step process.

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PubMed1981

Modulation of 75S RNA synthesis in the Balbiani rings of Chironomus tentans with galactose treatment.

Galactose has been used as a tool to modify gene activity in the giant puffs Balbiani ring 2 (BR2) and Balbiani ring 1 (BR1) on chromosome IV in the salivary glands of Chironomus tentans. BR2 decreased gradually and was absent or almost absent after a four day galactose treatment. Concomitant with this morphological change, the labelling of the population of growing 75S RNA molecules in BR2 decreased, and was essentially abolished after four days in galactose. Since the elongation rate at the 75S RNA genes proved to be the same in the galactose treated glands as in the control glands, the decreased labelling in BR2 was likely to correspond to a decreased production of 75S RNA. No changes in the size distribution of the growing 75S RNA molecules were noted during the galactose treatment, suggesting that the modulation of the activity was most likely accomplished at the initiation level, but regulation of a very early premature termination could not be excluded. When galactose was removed from the medium, BR2 attained its normal size and its ordinary RNA labelling. BR1 was studied in parallel with BR2 and it behaved strikingly different: BR1 expanded during the galactose treatment and the amount of growing 75S RNA increased, indicating an enhanced production of this 75S RNA species. Also the modulation of BR1 RNA synthesis was reversible. During the galactose treatment no changes in the labelling of chromosome I-III and of nucleolar RNA were observed suggesting that during the four day treatment, galactose exerted its effect mainly on the synthesis of BR2 and BR1 transcription products. The significance of these observations are considered in relation to the information available on the synthesis of the corresponding secretory polypeptides and the formation of the tube-like burrows. We also discuss the implications of the results for models of the regulation of gene activity and of the puffing process.

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PubMed1980

Identification of the Balbiani ring 2 chromomere and determination of the content and compaction of its DNA.

The giänt puff Balbiani ring 2 (BR 2) in the salivary glands of Chironomus tentants is known to contain transcriptionally active 75S RNA genes. The corresponding chromosomal superstructure, the BR 2 chromomere, has been identified and characterized in the present study. It was demonstrated by cytological methods that BR 2 originates from the broad band IV-3B10, since all the bands except the 3B10 band could be detected as intact bands in the vicinity of BR 2. In situ hybridization of BR 2 RNA to squashed Malpighian tubule chromosomes lacking BR 2, showed that all or almost all the DNA sequences complementary to BR 2 RNA are located in the 3B10 band. The DNA amount in the 3B10 band, designated the Br 2 band, was measured in relation to the DNA content of the whole chromosome set by both direct and indirect microspectrophotometry of Feulgen-stained Malpighian tubule chromosomes. From a determination of the haploid DNA content in sperm cells (0.25 pg), the amount of DNA in at BR 2 chromomere could then be calculated to be 5.1 x 10-4 pg DNA or 470 kb DNA. A minimum value of the DNA compaction within the BR 2 chromomere was estimated to 380 from the B-form length of BR 2 DNA (160 mum) and the thickness of the BR 2 band (0.42 mum). Since there are only between 1 and 4 75S RNA genes, 37 kb in size, in a BR 2 chromomere, most of the BR 2 DNA must consist of DNA not coding for 75S RNA. The nature of this DNA is discussed in relation to the high average AT-content of Chironomus DNA. The orgnization of the chromosome fiber in the BR 2 chromomere is considered in relation to the gene activation process in the salivary glands, i.e. the formation of a 75S RNA transcription loop from a protion of the tightly packed chromosome fiber in the BR 2 chromomere.

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PubMed1979

Characterization of active transcription units in Balbiani rings of Chironomus tentans.

Specific active transcription units on chromosome IV in the salivary glands of Chironomus tentans have been visualized by the Miller spreading technique and in situ by conventional electron microscopy. These units are likely to be located in the two most conspicuous puffs on chromosome IV, Balbiani ring 1 (BR 1) and Balbiani ring 2 (BR 2). The transcription units in these Balbiani rings generate 75S RNA molecules constituting putative messenger RNA species for the predominant cellular product, the salivary polypeptides. Solitary active transcription units with a mean length of 7.7 micron were observed most frequently. The lateral ribonucleoprotein (RNP) fibers of each unit formed a single length gradient. The number of fibers per unit was 123 (+/- 24), or about 16 growing RNP fibers per micron of chromosome fiber. The considerable variation in the number of RNP fibers per unit suggests that transcription can be modulated at the level of the individual gene. The modulation is probably achieved via the initiation event and/or via an early pretermination step, but a change in the elongation rate could not be excluded. The number of polymerases starting to traverse the whole gene was estimated to be six per min and transcription unit, and the rate of RNA chain elongation was calculated to be 31 nucleotides per second at 18 degrees C. The properties of the chromosome fiber within the active 75S RNA units and also within their vicinity were studied in the Miller spreads. The inactive chromosome fiber exhibited a uniform beaded conformation, while the active fiber was sparsely and irregularly beaded. Furthermore, the chromosome fiber was more extended in the active 75S RNA unit than in inactive regions (DNA packing ratios of 1.6 and 1.9, respectively). By comparing the properties of the active 75S RNA gene with those of active genes in other systems, it was inferred that the loss of beads and the extension of the fiber in the active unit is probably directly related to the level of transcriptive activity. Finally, a smooth nonbeaded segment of 0.18 micron in length was found to precede the RNP fiber gradient. This segment may have a role in the process of transcriptional regulation. On the basis of comparison with the active transcription units in spread preparations. It was possible to identify active units in the Balbiani rings in sectioned material using conventional electron microscopy. In both BR 1 and BR 2 an active unit appeared as a loop, consisting of a fiber axis and having RNP granules attached to the loop axis by stalks. The growing RNP fibers therefore seem to be organized into granular structures during the transcription process, and the final products in BR 1 and BR2 are granules, 500 A in diameter, each containing a 75S RNA molecule.

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PubMed1978

The 75 S RNA transcription unit in Balbiani ring 2 and its relation to chromosome structure.

A defined transcription unit in the Balbiani ring 2 (BR 2) region of chromosome IV in the salivary glands of Chironomus tentans has been characterized on the basis of analysis of the corresponding primary transcript, 75S RNA, and its functional significance. The available information on the transcription unit and its relations to chromosome structure can be summarized in the following way: 1. The size of the 75S RNA transcription unit in BR 2 is on the order of 30 000 base pairs. 2. The unit is likely to contain a long coding segment (at least 6000 base pairs), probably corresponding to information for salivary polypeptides. 3. The sequences are distributed in more than one chromomere (probably in 3--5 chromomeres). Further studies are needed before it can be stated whether or not there is a simple one-to-one relation between chromomeres and transcription units in the BR 2 region.

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PubMed1977

Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75S RNA.

Polysomes from the salivary glands of Chironomus tentans were investigated to determine whether Balbiani ring 75S RNA is incorporated into polysomal structures, and thus probably acts as messenger RNA. A new extraction technique for obtaining ribonucleoproteins was applied that gives a high yield of polysomes with only moderate degradation of the cytoplasmic, high molecular weight RNA. The polysomes sedimented in a broad region (200-2,000S) with a peak value of about 700S, which suggested that they were partly of very large sizes. This was confirmed by visualization of the polysomes in the electron microscope: 400S polysomes contained mainly 11-16 ribosomes, and 1,500S polysomes about 60 ribosomes per polysome. However, polysomes containing 100 or more ribosomes were also observed. It was further established that most of the cytoplasmic 75S RNA was located in polysomes, preferentially in the most rapidly sedimenting ones. From the available information on Balbiani ring RNA in cytoplasm and the present demonstration of 75S RNA molecules in polysomes, it was concluded that at least some Balbiani ring RNA, generated as 75S RNA within the Balbiani rings, eventually enters polysomes without being measurably changed in size. The present information on the potential amino acid coding sequences in 75S RNA is discussed in relation to the large size of the polysomes observed.

Animals↗
PubMed1977

Demonstration of Balbiani ring RNA sequences in polysomes.

A polysome extract from salivary glands of C. tentans was sedimented in a 15-60% sucrose gradient. Fractions from the heavy polysome region (1,000-2,000S) and fractions from the light polysome region (200-1,000S) were pooled separately, and the long-term labeled RNA was released by Sarkosyl/pronase and analysed by in situ hybridization. The results showed that BR 1 and BR 2 sequences were present in the heavy and the light polysome regions of the sucrose gradient. From control experiments with EDTA-treated extracts, it was concluded that most of the recorded BR 1 and BR 2 sequences were in fact located in polysomes. The finding that BR products enter polysomes suggests that they act as messenger RNA molecules. This study therefore strongly supports the concept that chromosome puffs represent active genes.

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PubMed1975

The relationship between molecular size and electrphoretic mobility in agarose gels as determined from a single population of growing RNA molecules.

Growing RNA molecules obtained from a defined transcription unit, the 75 S RNA transcription unit in Balbiani ring 2 of Chironomus tentans, have been analysed by electrophoresis in agarose. This set of RNA molecules forms a broad distribution of molecules with sizes ranging from very small up to the final product of giant size corresponding to 75 S RNA. The relative molecular weights along the migration path were calculated from a simple activity relationship derived from the expected distribution properties of a single population of growing RNA molecules. It was concluded that there is a linear relationship between the molecular weight and the logarithm for the mobility.

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