Biomedical subjects

B Daneholt

Publications and source records attributed to B Daneholt.

At least 37 records · Page 2Linked to original sources

PubMed1996

A pre-mRNA-binding protein accompanies the RNA from the gene through the nuclear pores and into polysomes.

In the larval salivary glands of C. tentans, it is possible to visualize by electron microscopy how Balbiani ring (BR) pre-mRNA associates with proteins to form pre-mRNP particles, how these particles move to and through the nuclear pore, and how the BR RNA is engaged in the formation of giant polysomes in the cytoplasm. Here, we study C. tentans hrp36, an abundant protein in the BR particles, and establish that it is similar to the mammalian hnRNP A1. By immuno-electron microscopy it is demonstrated that hrp36 is added to BR RNA concomitant with transcription, remains in nucleoplasmic BR particles, and is translocated through the nuclear pore still associated with BR RNA. It appears in the giant BR RNA-containing polysomes, where it remains as an abundant protein in spite of ongoing translation.

Amino Acid Sequence↗

PubMed1996

A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export.

In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

The central region of the synaptonemal complex revealed in three dimensions.

The synaptonemal complex plays a key role in pairing of homologous chromosomes during meiosis. Its gross structure was already known by conventional electron microscopy, but only recently has it been possible to reveal the synaptonemal complex in three dimensions at higher resolution by electron microscope tomography. As the molecular analysis of meiosis is developing rapidly, a more thorough understanding of the principal organization of the synaptonemal complex is essential.

Journal Article↗

PubMed1995

Structural interaction between the nuclear pore complex and a specific translocating RNP particle.

The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.

Animals↗

PubMed1995

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication.

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

3T3 Cells↗

PubMed1994

Assembly and disassembly of spliceosomes along a specific pre-messenger RNP fiber.

Transcriptionally active Balbiani ring (BR) genes in the salivary glands of the dipteran Chironomus tentans were studied by immunoelectron microscopy to establish the distribution of spliceosome components along a specific pre-messenger ribonucleoprotein (pre-mRNP) fiber. The BR genes are 35-40 kb in size with three introns close to the 5' end and one close to the 3' end; a very large middle portion lacks introns. As a rule the 5' introns are spliced concomitant with transcription in the promoter proximal third of the gene, while the 3' intron is spliced post-transcriptionally. The BR genes with growing pre-mRNPs were visualized in situ, while completed and released pre-mRNPs were isolated from the nucleoplasm and studied unfolded on a grid surface. An anti-snRNP antibody (Y12) bound mainly to the promoter proximal third of the BR gene (86%) and only to a minor extent to the middle and distal thirds (7 and 7% respectively). An antibody to an hnRNP protein reacted with the proximal, middle and distal regions to an increasing extent (17, 38 and 45% respectively), reflecting the increase in size of the growing transcription product. In the nucleoplasmic pre-mRNP particle only one end of the RNP fiber was labeled by Y 12, presumably the 3' end; the anti-hnRNP antibody decorated the entire RNP fiber. Thus, the snRNPs do not associate along the whole pre-mRNP fiber but rather bind to the 5' and 3' ends, i.e. the regions containing the introns. The results also imply that the spliceosomes both assemble and disassemble rapidly on the pre-mRNP fiber.

Animals↗

PubMed1993

Solitary and synaptonemal complex-associated recombination nodules in pro-nurse cells during oogenesis in Drosophila melanogaster.

An oocyte in Drosophila melanogaster originates from 1 of 16 cells comprising an ovarial syncytium. The two pro-oocytes proceed into the pachytene stage of meiosis, but only one develops further into a mature oocyte while the other reverts to a nurse cell. It is known that pro-nurse cells also enter meiosis, as they contain incomplete synaptonemal complexes (SCs). We now show that these cells also harbour recombination nodules (RNs). In cells that only occasionally contain SC segments, the RNs are typically not located close to distinct tripartite SC structures. Instead, these RNs are frequently associated with a spherical body of amorphous material and two to three, more or less parallel fibres, possibly representing SC material. The significance of the solitary RNs is discussed in relation to the present knowledge of the assembly and disassembly of the SC.

Animals↗

PubMed1993

The central region of the synaptonemal complex in Blaps cribrosa studied by electron microscope tomography.

The synaptonemal complex (SC) in the beetle Blaps cribrosa contains a highly organized central element (CE), two flanking lateral elements (LEs), and a number of regularly spaced transverse filaments (TFs) crossing the central region. The CE is built like a ladder with two longitudinal components running in parallel and a number of regularly spaced transverse CE components, bridging the two longitudinal components. The CE is multi-layered with the ladders of the individual layers more or less in register. Essentially every TF originates in one of the LEs, crosses the CE through a transverse CE component and reaches the opposite LE; every transverse CE component in a given layer corresponds to one, and only one, TF. In a CE layer, short irregular pillars form the junctions between the transverse and longitudinal CE components. Adjacent pillars are connected to each other by fine fibrous bridges: the two pillars in the same transverse CE component are linked, and so are the pillars along each longitudinal component, and also more occasionally adjacent pillars in separate CE layers. It is proposed that a TF with the two associated short pillars represents the structural unit in the central region. The ordered structure of the CE is accomplished by linking adjacent pillars to each other into the well-defined three-dimensional organization of the CE.

Animals↗

PubMed1993

The three-dimensional structure of the central region in a synaptonemal complex: a comparison between rat and two insect species, Drosophila melanogaster and Blaps cribrosa.

The highly ordered central region of the synaptonemal complex (SC) in Blaps cribrosa has recently been studied by electron microscope tomography (EMT), and a simple three-dimensional model presented. Using the same experimental approach we have now compared the central region in Blaps with the central regions in Drosophila melanogaster and rat. In all three species, the SCs exhibit a central element (CE) flanked by two lateral elements (LEs). The central region between the two LEs is crossed by transverse filaments (TFs). The Blaps CE element is the most ordered one with a well-defined ladder-like structure with two longitudinal components bridged by a number of regularly spaced transverse components, the rungs of the ladder. At the junctions between the longitudinal and transverse components there are prominent dense structures. The CE is multi-layered with the ladders of the separate layers in approximate register. In Drosophila the transverse CE components are as distinct and well organized as in Blaps, while in rat they are present but are less frequent and less well ordered. The longitudinal CE components in Drosophila are often fragmented and even more so in rat. The tomographic analysis revealed that in all three species the central region contains the same structural units: a single TF associated with two short pillars (or globules), which correspond to the junction structures. A fibrous lattice connects the two pillars/globules on the same TF forming the transverse CE component and those on adjacent TFs forming the longitudinal CE component; fibers between pillars/globules also link consecutive CE layers together. In the longitudinal component the number of fibrous bridges between the pillars/globules is related to the conspicuousness of the longitudinal component, i.e. Blaps has most, Drosophila almost as many, and rat considerably fewer bridges. We conclude that the central region in rat, Drosophila and Blaps contains the same basic structural unit but the degree of order and concentration of the units differ: a higher density seems to be accompanied by a higher order within the CE.

Animals↗

PubMed1993

The Balbiani ring particle: a model for the assembly and export of RNPs from the nucleus?

Balbiani rings are exceptionally large puffs on the polytene chromosomes in the dipteran Chironomus tentans. These puffs are particularly well suited for studies of the structure of active genes and the synthesis and transport of specific RNA-protein (RNP) particles. The Balbiani ring RNP particle consists of a ribbon bent into a ring-like structure, the ribbon being built from a tightly folded 7 nm RNP filament. The assembly of the particle takes place concomitant with transcription and occurs in a stepwise fashion. As the particle is transported through the nuclear pore the ribbon straightens out, with the 5' end of the transcript leading. During translocation the tightly packed RNP filament is gradually unfolded, and on the cytoplasmic side the mRNA is immediately engaged in polysome formation.

Journal Article↗

PubMed1993

The elementary RNP fiber--not the higher order structure--determines the all-or-none disintegration behaviour of Balbiani ring pre-messenger RNP particles upon RNase A treatment.

Balbiani ring premessenger ribonucleoprotein (RNP) particles are built from a 7-nm RNP fiber which is tightly folded into a ring-shaped RNP ribbon. Isolated particles are known to disintegrate in all-or-none fashion upon RNase A treatment. In the present study we investigated whether this mode of disintegration is dependent on an intact particle structure or is inherent in the 7-nm fiber. When treated at low ionic strength, the Balbiani ring (BR) particles lost their higher order structure and the 7-nm fiber was unpacked, as evidenced by sucrose gradient sedimentation and electron microscopy. When treated with RNase A, unfolded as well as intact particles disintegrated in the all-or-none fashion, with similar kinetics and without apparent intermediates. Proteinase K treatment, however, obliterated this pattern: the protein-free particle RNA degraded progressively. As the typical disintegration pattern of the particles was not altered by unfolding, but was lost by deproteinization, the all-or-none mode of disintegration is likely to be a property of the 7-nm RNP fiber.

Animals↗

PubMed1993

The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with conventional techniques using electron microscope tomography.

A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption-stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic-embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption-stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7-nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption-stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic-embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA-embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt-series to be taken for electron microscope tomography.

Adsorption↗

PubMed1992

Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography.

A specific premessenger ribonucleoprotein (RNP) particle in the salivary glands of the dipteran Chironomus tentans was studied with electron microscope tomography during translocation from the cell nucleus to the cytoplasm. The RNP particle consists of a thin RNP fiber tightly folded into a ribbon, which is bent into a ring-like structure. Upon translocation through the pore, the particle is first orientated in a specific manner at the pore entrance, and subsequently the bent ribbon is gradually straightened and transported through the pore with the 5' end of the RNA in the lead. Concomitantly, the elementary RNP fiber constituting the ribbon is gradually unpacked and will appear more or less extended on the cytoplasmic side of the pore complex. The ordered nature of the process suggests a specific recognition of the RNP particle at the nuclear pore.

Animals↗

PubMed1992

Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle.

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.

Animals↗

PubMed1992

The transcribed template and the transcription loop in Balbiani rings.

The active Balbiani ring (BR) genes in the giant puffs in Chironomus salivary glands can be visualized in three dimensions within the cell nucleus. The chromatin template exhibits a dynamic structure: at the RNA polymerases the template is fully extended, while in between polymerases it can be packed into a nucleosome, a nucleosome filament, or even into a thick chromatin fiber, if the spacing between consecutive polymerases is large enough. Even in its extended state, the template harbours histone H1, and most likely also the core histones, indicating that the nucleosomes unfold and refold during transcription. Apart from the transcribed template, a transcription loop contains a thin, extended, apparently flexible and nucleosome-free fiber upstream of the gene, and a loosely coiled nucleosome filament downstream of the gene.

Animals↗

PubMed1991

The complexity of 75S premessenger RNA in balbiani ring granules studied by a new RNA band retardation assay.

Under normal growth conditions, Balbiani ring granules constitute premessenger ribonucleoprotein (RNP) particles synthesized in two chromosomal puffs, Balbiani ring (BR) 1 and 2, in the larval salivary glands of Chironomus tentans. At least three genes encoding 75S RNA are present in these two BRs: one in BR1 and two in BR2 (BR2.1 and BR2.2). The complexity of BR granule 75S RNA was studied by agarose gel electrophoresis under non-denaturing conditions. We recorded three main bands, designated I, II and III. Experiments with denaturing gels demonstrated that the differences in migration reflected mainly, but not exclusively, conformational differences. Northern blotting experiments showed that band I contained BR1 sequences, band II contained BR2.1 sequences, and band III contained BR2.2 sequences. To study whether additional genes contributed to the BR granule 75S RNA, an RNA band shift assay was developed. When an oligodeoxyribonucleotide complementary to repetitive BR1 and BR2.2 sequences was hybridized to 75S RNA prior to electrophoresis, bands I and III were retarded but not band II. An oligonucleotide complementary to a repetitive BR2.1 sequence only shifted band II. Since no detectable 75S RNA remained unchanged in these experiments, and all bands were identified by Northern blotting, all the BR granules are likely to originate from the BR1, BR2.1 and BR2.2 genes; no additional genes have to be invoked. Possible applications of the new RNA band shift assay are discussed.

Animals↗

PubMed1991

Analysis of a murine male germ cell-specific transcript that encodes a putative zinc finger protein.

A cDNA species, corresponding to a gene with testis-specific expression (TSGA), was isolated from a testis cDNA library. The temporal and spatial expression of TSGA was studied by in situ hybridization as well as RNA filter hybridization. In tissue sections, the TSGA sequence was confined to cells within the seminiferous tubules. For filter hybridization, RNA was isolated from testis of prepubertal rats of different ages as well as from enriched populations of various germ cell types. It was found that TSGA is expressed only in male germ cells and that the steady-state level of TSGA transcripts reaches a maximum during the meiotic and the postmeiotic stages of germ cell development, suggesting a meiotic or postmeiotic function for the encoded protein. TSGA encodes a putative protein having 1,214 amino acids and contains a zinc finger, a structure that previously has been shown to mediate binding to nucleic acids.

Amino Acid Sequence↗