The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles.

Specific premessenger ribonucleoprotein (pre-mRNP) particles, the Balbiani ring (BR) granules in the salivary glands of the dipteran Chironomus tentans, can be visualized in the electron microscope when they assemble on the genes, move through nucleoplasm, and bind to and translocate through the nuclear pores. As shown by BrUTP labeling and immunoelectron microscopy, newly synthesized BR RNP particles, released from the BR genes, appear early in all nucleoplasmic regions of the cell nucleus and they saturate the nucleoplasmic pool of BR particles after 2 h of labelling. It is concluded that within the nucleus the BR particles move randomly. Furthermore, estimates of minimum diffusion coefficients for the BR particles are compatible with the view that the particles diffuse freely in the interchromosomal space, although it is not excluded that the random movement could be slightly retarded. Once the particles get bound to the nuclear pore complexes, they seem committed to translocation through the nuclear pores.

The synaptonemal complex protein SCP3 can form multistranded, cross-striated fibers in vivo.

The synaptonemal complex protein SCP3 is part of the lateral element of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes. We have investigated the fiber-forming properties of SCP3 to elucidate its role in the synaptonemal complex. By synthesis of SCP3 in cultured somatic cells, it has been shown that SCP3 can self-assemble into thick fibers and that this process requires the COOH-terminal coiled coil domain of SCP3, as well as the NH2-terminal nonhelical domain. We have further analyzed the thick SCP3 fibers by transmission electron microscopy and immunoelectron microscopy. We found that the fibers display a transversal striation with a periodicity of approximately 20 nm and consist of a large number of closely associated, thin fibers, 5-10 nm in diameter. These features suggest that the SCP3 fibers are structurally related to intermediate filaments. It is known that in some species the lateral elements of the synaptonemal complex show a highly ordered striated structure resembling that of the SCP3 fibers. We propose that SCP3 fibers constitute the core of the lateral elements of the synaptonemal complex and function as a molecular framework to which other proteins attach, regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination.

Localization of the N-terminus of SCP1 to the central element of the synaptonemal complex and evidence for direct interactions between the N-termini of SCP1 molecules organized head-to-head.

The synaptonemal complex (SC) is a meiosis-specific, tripartite structure essential for synapsis of homologous chromosomes; it contains a central element positioned between two lateral elements and transversal filaments connecting the lateral elements. In mammals, a major constituent of the transversal filament is known: the SCP1 protein. It contains a long central coiled-coil motif and the molecules are probably organized as dimers, each forming a coiled-coil fiber. We have now developed a new sensitive procedure for immunoelectron microscopy of synaptonemal complex proteins and determined the exact localization of the two nonhelical ends of the SCP1 protein within the mouse synaptonemal complex. We found that the N-terminal end of the SCP1 protein is located within the central element of the synaptonemal complex, whereas the C-terminal end is close to or within the lateral element of the synaptonemal complex. This result supports the notion that SCP1 is an extended filamentous protein and that the two molecules of the putative SCP1 dimer are likely to have the same polarity. The observation that the N-termini are confined to the central element indicated that SCP1 dimers, anchored in opposite lateral elements, could establish contact with each other in the central element via their N-termini. To test this possibility we used the yeast two-hybrid system and found that the N-terminal end of the SCP1 protein indeed strongly interacted with itself, but not with other protein domains tested. We therefore suggest that a transversal filament consists of one or more pairs of SCP1 dimers, each pair being organized in a head-to-head arrangement with the C-termini anchored in the lateral elements and the two N-termini being joined in the central element.

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.

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.

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.

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.

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.

Cochlear hair cell and vascular changes in the guinea pig following high level pure-tone exposures.

Noise is thought to exert metabolic and/or mechanical stress on sensory and vascular tissues of the cochlea, the relative influence of the stressors being influenced by the intensity of the noise. Guinea pigs exposed to either of two pure-tone frequencies, 1.33 or 3.85 kHz for 6 hours at intensity levels ranging from 102 dB to 120 dB SPL, were studied for pathological changes in two spiral lamina vessels--the vessel of the basilar membrane (VSBM) and the vessel of the tympanic lip (VSTL). In general, animals sustaining mild to severe degrees of hair cell destruction one month after noise exposure showed little vascular change in the vessels studied. With respect to the vasculature, the concept of a 'critical level' seems to be dependent on exposure frequency, in that only above 117 dB SPL at 3.85 kHz was there any change in the pattern of damage to the spiral lamina vessels.

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.

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.

Multielectrode intracochlear implants. Nerve survival and stimulation patterns.

A series of neurophysiological and anatomical experiments have been performed on cats implanted on a long-term basis with scala tympani electrodes. Auditory nerve survival was assessed for both neomycin sulfate-deafened and previously normal-hearing cats three weeks to 30 months after electrode implantation. Acute neurophysiological experiments were performed on nearly all of the cats and the spatial response to electrical stimulation was determined. The results of these experiments suggest that the auditory nerve can withstand surgical implantation and the long-term interface with scala tympani electrodes. In addition, restricted portions of the auditory nerve can be discretely excited with bipolar stimulation. Monopolar stimulation produced a very broad excitation pattern. These findings have important implications for the application of multielectrode prosthetic devices to the profoundly deaf.