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.

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.

A cyclized peptide for studies of human parvovirus B19 infection.

Synthetic peptides corresponding to human parvovirus B19 sequences were evaluated for immunoreactivity with the sera of infected persons. A cyclized peptide deduced from the N terminus of viral protein VP2 and containing the amino acids 284-307 showed a high reactivity with IgM class antibodies when comparing seropositive and seronegative sera.

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.

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.

An on-line two-dimensional polyacrylamide gel electrophoresis protein database of adult Drosophila melanogaster.

An annotated two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein database of adult Drosophila melanogaster has been constructed, based on the protein patterns of heads, thoraces and abdomens of adult male and female Drosophila melanogaster. About 1200 major protein spots are catalogued. Common proteins, found in all body parts, as well as bodypart- and sex-specifically expressed proteins are reported. Of the major proteins, 91, or 7.5%, are differentially expressed in the two sexes or in different body parts, at least in part reflecting specific functional requirements. At the present time 43 proteins, or about 3.5% of the detected proteins, have been identified. These data can be accessed interactively from our World Wide Web (WWW) server through clickable inline gel images and hypertext links. Identified protein spots are cross-referenced, through hypertext links, to the SWISS-PROT annotated database of protein primary sequences and the Fly-Base database of Drosophila genomic data. Our reference gels can be used to gain immediate access to protein spot identify and to the pattern of differentially expressed proteins in Drosophila melanogaster. The work presented in this article ties together information from protein 2-D PAGE, molecular biology and genetics and offers a uniform way to access this large volume of data.

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.

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.

The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is tightly linked to the muscle-type glycogen phosphorylase (PYGM) gene in 11q13. This region of the human genome contains additional disease-related loci implicated in the development of insulin-dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome and translocation t(11;17) described in B-cell non-Hodgkin's lymphoma. We approached cloning of candidate disease genes from 11q13 by large-scale genomic sequencing. We obtained > 106 kb of sequence around the PYGM gene and established a transcriptional map that includes: (i) two genes previously localized to 11q13, PYGM and a zinc-finger protein (ZFM1) gene; (ii) the germinal center kinase (GCK, human B-lymphocyte serine/threonine protein kinase) gene; (iii) a novel human CDC25-like (HCDC25L) gene; (iv) a dystrophia myotonica protein kinase-like (DMPKL) gene; and (v) a novel ubiquitously expressed gene of unknown function (germinal center kinase- neighboring gene, GCKNG).