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B Daneholt

Publications and source records attributed to B Daneholt.

At least 55 records · Page 3Linked to original sources
PubMed1991

Transport of Balbiani ring granules through nuclear pores in Chironomus tentans.

Balbiani ring granules are premessenger RNP particles synthesized in the larval salivary glands of the dipteran Chironomus tentans and containing the genetic information for large-sized secretory proteins. The granules in the nucleoplasm consist of a thin elementary RNP fiber tightly packed into an RNP ribbon, which is bent into a ring-like shape. When the 50-nm granule is translocated through the nuclear pore, it attains an elongated conformation. We have now demonstrated that the particle in transition can be described as an RNP ribbon with the same thickness and the same minimal width as the ribbon of the nucleoplasmic granules. The ribbon in the pore is, however, somewhat longer and it lacks the broad regions of the ribbon in the nucleoplasmic granule. We conclude that when entering the pore the bent ribbon of the Balbiani ring granule is being straightened and also somewhat drawn out, while its width is accommodated to the maximal size of the pore, i.e., about 25 nm. On the cytoplasmic side the ribbon is unpacked, and the elementary fiber can be seen extending into cytoplasm, probably being available for polysome assembly.

Animals↗
PubMed1991

The glucocorticoid receptor in homodimeric and monomeric form visualised by electron microscopy.

The purified glucocorticoid receptor (GR) from rat liver has been visualised by electron microscopy. The specimens were prepared by spreading on thin carbon support and negatively stained using uranyl acetate. Two forms of GR, the monomeric and the dimeric forms, were identified based on size, chromatographic distribution, and DNA binding properties. The GR monomer consists of two globular domains of slightly different size with a thinner connecting domain in between. In the absence of DNA the dimeric GR has a characteristic four-leaf clover structure. The size and appearance of this structure is consistent with two GR subunits arranged in a side-by-side fashion. Monomeric and dimeric GR specifically bound to DNA are also shown.

Animals↗
PubMed1990

Higher order structure of Balbiani ring premessenger RNP particles depends on certain RNase A sensitive sites.

Specific premessenger ribonucleoprotein (RNP) particles, the Balbiani ring (BR) granules from Chironomus tentans salivary glands, were treated with RNase A to study the effect of RNA strand breaks on the higher order structure of the particles. Isolated, radioactively labeled BR granules, known to sediment at 300 S, were digested with RNase A and centrifuged in sucrose gradients. The fractionated particles were subsequently analyzed using electron microscopy and caesium chloride centrifugation. At a low RNase concentration, most of the 300 S particles disintegrated completely, and no metastable degradation products were observed. At intermediate RNase concentrations, no 300 S particles were left, but a minor fraction of the BR granules had unfolded and sedimented at 160 S. These granules could represent particles modified during the RNase treatment or represent a more slowly degrading subfraction of the particles. At a high RNase concentration, no RNP particles at all remained in the gradient. The rapid disintegration of the majority of the BR granules was investigated further by electrophoretic analysis of RNA in the remaining particles. During the RNase treatment BR granules, still sedimenting at 300 S, accumulated strand breaks; in fact, as many as 50 to 100 nicks in the 37 kb RNA could be tolerated. It was concluded from RNA analyses that the disintegration of the BR granules was not dependent on any single nick in the RNA, nor on the accumulation of a certain number of nicks, but rather on one or a few critical strand breaks. We propose that there are organizing sequences essential for particle integrity; once these sequences are nicked, the premessenger RNP particles are rapidly and completely degraded.

Animals↗
PubMed1990

Presence of histone H1 on an active Balbiani ring gene.

We have investigated whether histone H1 is present on active Balbiani ring genes in the salivary glands of Chironomus tentans using immunoelectron microscopy. The genes were studied in two activity states: maximally activated genes with a fully extended template and repressed genes in a 30 nm fiber conformation. Histone H1 was recorded on the gene in both conformations; the immunosignal was considerably stronger in the transcriptionally active state, probably reflecting the increased accessibility of histone H1 to the antibody in unfolded versus compacted chromatin. We conclude that during transcription the DNA template is extended and the nucleosomes are disrupted at the RNA polymerases, but histone H1, and most likely also the core histones, remains bound to the template.

Animals↗
PubMed1990

Isolation and initial characterization of a specific premessenger ribonucleoprotein particle.

A specific type of premessenger ribonucleoprotein (RNP) particle, Balbiani ring (BR) granules, has been isolated from heterogeneous nuclear RNP (hnRNP) in the salivary glands of the dipteran Chironomus tentans. A BR granule contains a single 75S RNA molecule coding for a large secretory protein (Sp1). The isolation procedure is based on the abundance and exceptional size of the BR granules: in EDTA-containing sucrose gradients they sediment as a sharp 300S peak ahead of the remainder of the hnRNP population. The isolated BR granules were identified on the basis of both ultrastructural and biochemical criteria: large spherical particles that contain 75S RNA and BR sequences. A three-dimensional reconstruction of isolated particles by electron microscope tomography further supported the identification of the isolated particles as BR granules. In contrast to the entire hnRNP population, the BR granules exhibited a sharp peak in CsCl gradients with a buoyant density of 1.45 g/cm3. This result indicates that a BR granule consists of 40% RNA and 60% protein by weight, corresponding to a 75S RNA molecule of 12 megadaltons and a total protein content of 18 megadaltons, or about 500 average-sized protein molecules.

Animals↗
PubMed1989

The ultrastructure of upstream and downstream regions of an active Balbiani ring gene.

When active, the 37 kb Balbiani ring genes are known to form transcription loops with an almost fully extended chromatin axis. Here we examine the upstream and downstream regions of such transcription loops by electron microscopy. We demonstrate that a loop starts and ends in tightly packed chromatin; the two anchoring sites are clearly separated from each other in space. The upstream, nontranscribed region consists of a thin, extended, apparently flexible and nucleosome-free fiber corresponding to about 0.5 kb DNA. The downstream, nontranscribed region appears as a 200 nm long nucleofilament loosely coiled into a short, thick chromatin fiber and estimated to contain about 3 kb DNA.

Animals↗
PubMed1988

Terminal repeats in long repeat arrays are likely to reflect the early evolution of Balbiani ring genes.

Balbiani ring (BR) genes in Chironomus tentans are 35 to 40 kb (1 kb = 10(3) bases or basepairs) in length and encode secretory proteins of exceptional size. Each gene contains a large homogeneous core block consisting of approximately 100 tandemly arranged, highly homologous repeat units. The repeat unit has a constant (C) region and a subrepeat (SR) region. The various BR genes exhibit similar C regions, while the SR regions differ as to sequence, length and number of subrepeats. To study early steps in the evolution of the coding repeat arrays of the BR genes we have analyzed the 3' ends of the four BR genes in C. tentans: BR1, BR2.1, BR2.2 and BR6. In each gene the very end of the core block consists of two or three repeat unit variants; in each variant repeat the C region is linked to a Cys region, replacing the SR region. Sequence comparisons between the C regions of the closely related BR1 and BR2 genes show that during evolution the terminal repeat unit variants have to a large extent been isolated from the remainder of the core block and have probably been more conserved than the interior repeat units. Detailed analysis of the structure of the variant repeat units further supports this latter notion and suggests that the BR core blocks have evolved from an array of a simple 36 base-pair long sequence; larger, more complex repeat units containing subrepeats were gradually formed and spread in the block, mainly by homologous unequal recombination events. During this evolution the interior of the core blocks evolved as a homogeneous repetitive structure, while ancestor repeat units remained as sequence relicts in the terminal parts.

Animals↗
PubMed1986

Inhibition of transcription does not affect the total amount of ubiquitinated histone 2A in chromatin.

Using a polyclonal anti-ubiquitin antibody in Western blotting experiments, we detected three antibody-binding components in a HeLa cell extract: ubiquitin, a ubiquitin-histone 2A conjugate (uH2A) and a 17 kD protein, probably corresponding to an additional ubiquitin conjugate. Since ubiquitination of histone 2A (H2A) has been invoked in the transcription process, the amount of uH2A was studied after inhibition of ribosomal RNA (rRNA) synthesis with actinomycin D and of heterogeneous nuclear RNA (hnRNA) synthesis with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The amount of uH2A did not change, suggesting that the overall level of ubiquitination of histone 2A is not directly coupled to on-going transcription of either rRNA or hnRNA. Since the uH2A content of protein coding genes constitutes a considerable portion of total chromatin uH2A, it seems also likely that there is no major change in the degree of ubiquitination on the templates of the protein-coding genes themselves upon cessation of transcription. It is proposed that the pattern of ubiquitination of histone 2A is established on a long-term basis and that it is related to the overall organization and distribution of the chromatin material in the interphase nucleus.

Chromatin↗
PubMed1986

Low degree of ubiquitination of histone 2A in the dipteran Chironomus tentans.

A polyclonal antibody to ubiquitin has been prepared and shown to react with both ubiquitin and ubiquitinated histone 2A (uH2A). Applying this antibody in Western blotting experiments, we have observed that the salivary glands of Chironomus tentans contain an unusually low amount of uH2A (1% of histone 2A), while the amount of free ubiquitin is as abundant as in other animal cells, e.g. HeLa cells. The same low content of uH2A was also found in diploid epidermal cells of Chironomus origin suggesting that the low amount is not a characteristic of the polytene state of chromatin in salivary gland cells but rather a property of C. tentans as a species. The significance of the low degree of ubiquitination is discussed in relation to the information available on the organization of Chironomus chromatin into unusually large chromomeric entities.

Animals↗
PubMed1984

Conserved and nonconserved structures in the secretory proteins encoded in the Balbiani ring genes of Chironomus tentans.

The large, repetitive Balbiani ring (BR) genes, BR 1, 2, and 6, in Chironomus tentans originated from a short ancestral sequence and have all evolved according to analogous amplification schemes. We analyzed the structures of the BR-encoded secretory proteins and defined the parts that have been conserved during the evolutionary process. The BR products show striking similarities, with the BR 1 and BR 2 products being more similar to each other than to the BR 6 product. In the constant (C) region of the repeat units, 7 of the 30 amino acid residues are strictly conserved; 4 of these are the cysteine residues. The subrepeat (SR) regions of all the BR products are dominated by repeated tripeptide elements rich in proline and charged amino acid residues. Most of the amino acid replacements in both regions are conservative. Secondary structure predictions suggested that the C regions of the BR 1 and BR 2 products have several elements of secondary structure: an alpha-helix, a beta-strand, and one or two reverse turns, as in "globular structures." The prediction for the C region of the BR 6 product is similar but lacks a beta-strand. The predictions for the intervening SR regions appear less conclusive, but are clearly different from those for the C regions, and suggest regular structures not differing in their conformational elements. The SR regions evolved from an ancestor sequence similar to the C region; thus, the BR products seem to represent an example of evolution from one structure to two differently folded products.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Packing of a specific gene into higher order structures following repression of RNA synthesis.

Transcription of the Balbiani ring (BR) genes of the dipteran Chironomus tentans was inhibited by teh nucleoside analogue DRB (5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole). The BR genes were emptied of RNA polymerases and the subsequent packing of the genes was monitored by transmission electron microscopy. The thin chromatin axis of the transcriptionally active genes condensed into a thick (20-25 nm) chromatin fiber, which was recorded as a linear structure, an open loop or a supercoiled loop. The compacted genes were finally packed into dense clumps of chromatin. It was proposed that upon repression of RNA synthesis the BR gene template attains the following consecutive stages with increasing compaction: transcription loop----linear thick fiber----open thick fiber loop----supercoiled thick fiber loop----dense chromatin. Within the chromatin blocks structures that resembled the supercoiled loops were discerned, suggesting that the final packing of the template might be accomplished by a close alignment of supercoiled loops.

Animals↗
PubMed1983

Visualization of the formation and transport of a specific hnRNP particle.

The growth and maturation of the transcription products on the Balbiani ring (BR) genes in Chironomus tentans has been characterized by electron microscopy. The BR transcript is packed into a series of well defined ribonucleoprotein structures of increasing complexity: a 10 nm fiber, a 19 nm fiber, a 26 nm fiber, and a 50 nm granule. The basic 10 nm element was revealed in Miller spreads. The in situ structure of the transcription products and RNA compaction estimates suggested that the 10 nm fiber is packed into the 19 nm fiber as a tight coil. The transition of the 19 nm fiber into the 26 nm fiber is accompanied by a major change of the basic 10 nm fold into a noncoiled structure. Finally, the 26 nm fiber makes a one and one-third left-handed turn forming the final product, the BR granule. Upon translocation through the nuclear pore the BR granule becomes rod-shaped, which most likely corresponds to a relaxation of the highest-order structure into a straight 26 nm fiber.

Animals↗
PubMed1982

Rapid reformation of the thick chromosome fiber upon completion of RNA synthesis at the Balbiani ring genes in Chironomus tentans.

We have studied the ultrastructure of the Balbiani ring genes in Chironomus tentans during treatment with the RNA synthesis inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). This nucleoside analogue blocks transcription at or near the initiation site but does not interfere with the elongation and termination processes. In the ordinary active state the Balbiani ring genes display a 5 nm chromosome fiber, carrying densely distributed, growing ribonucleoprotein particles (Andersson et al., 1980). When the transcriptional activity declines, a 10 nm fiber can be observed between sparsely distributed RNA polymerases. Furthermore, after passage of the last RNA polymerase the 10 nm fiber can be seen as well as its gradual packing into a 25 nm thick fiber. Thus, the active chromosome fiber is rapidly packed into higher order structures when the fiber is not directly involved in transcription. The formation of the thick fiber does not require that the gene along its entire length is devoid of active RNA polymerases. The thick fiber can again be mobilized for transcription, since in reversion experiments the BR genes appear as ordinary active genes with an extended nucleofilament and densely packed nascent transcription products. The dynamic behaviour of the chromosome fiber during transcription is discussed as well as the packing and unpacking of a gene into higher order structures.

Animals↗
PubMed1982

A hierarchic arrangement of the repetitive sequences in the Balbiani ring 2 gene of Chironomus tentans.

One cloned cDNA sequence, pCt63, was used to characterize the repeated structure of the Balbiani ring 2 gene in Chironomus tentans. Although small in size (0.63 kb), the cDNA insert corresponds to a large portion (25 kb) of the BR2 gene (37 kb). Southern blotting experiments suggested that a large part of the BR2 gene consists of tandemly repeated units, each about 215 bp. Sequence analysis of the cDNA confirmed the repeated nature of the BR2 gene and revealed the internal structure of the repeat unit. Each such unit is composed of two regions of approximately equal length; one is highly ordered and built from about six 18 bp repeats, each consisting of a slightly diverged 9 bp duplication. The recorded hierarchic arrangement of the repetitive sequences in the BR2 gene and a specific pattern of base substitutions along the gene have enabled us to propose how a major part of the giant BR2 gene has evolved from a short primordial sequence, 110-120 bp in length.

Amino Acid Sequence↗
PubMed1982

Evidence for a common ancestor sequence for the Balbiani ring 1 and Balbiani ring 2 genes in Chironomus tentans.

The Balbiani ring (BR) 1 and BR 2 genes in Chironomus tentans are functionally related and are only expressed in the salivary gland cells. Here we reveal the principal structure of the BR 1 gene and analyze the structural and evolutionary relationship between the BR 1 and BR 2 genes. The properties of the BR 1 gene, 37 kilobases in size, are derived from the analysis of a cloned cDNA sequence, pCt 21. A considerable part of the BR 1 gene consists of one or a few blocks of a tandemly repeated 246-base-pair (bp) major repeat unit. About half of this major repeat unit is in turn built from four tandem repeats of a 33-bp sequence. This hierarchic arrangement of repetitive sequences within the BR 1 gene suggests that the gene has evolved through two major amplification steps, starting from a short primordial sequence. A similar evolutionary model has been put forward for the BR 2 gene [Sümegi, J., Wieslander, L. & Daneholt, B. (1982) Cell 30, 579-587]. The two putative primordial genes contain a similar, 102-bp-long sequence (86% nucleotide sequence homology), indicating that the BR 1 and BR 2 genes most likely arose from the same ancestor sequence. During the course of evolution the two genes diverged, mainly due to differences in the length and sequence of the gene segments involved in the two amplification steps. Moreover, at least one of the BR genes was translocated to another chromosomal locus.

Journal Article↗