Balbiani Ring Genes Research Articles

POLYTENE CHROMOSOMES OF GLYPTOTENDIPES GLAUCUS MG. (DIPTERA, CHIRONOMIDAE) AS TEST – OBJEKT TO STUDY TOXIC EFFECTS OF LEAD IONS

Results of the analysis of cytogenetic effects of lead ions on polytene chromosomes of Glyptotendipes glaucus Mg are reported. The functional state of polytene chromosomes was determined via computation: chromosome compactness index (CR); coefficient of genetic activity of the nucleolar organizer (NOR) and coefficient of genetic activity of the Balbiani ring (BR). With increase of the lead nitrate solution concentration, the compactness index increases, that is, the chromosomes compactness decreases: medians of CR values (when measuring shoulder E) in the control are 6.05, for the maximum concentration studied they are 7.3. As a result of the of lead ions action, the activity of the nucleolar organizer genes decreases: medians of the NOR values decrease from 2.3 in the control to 2.0 for the highest concentrations used in the experiment. The activity of the Balbiani ring genes increases with the growing concentration of the lead nitrate solution: medians of the BR values in the control are 1.45, for the maximum studied concentration they are 1.95. The G. glaucus genome responds ambiguously to the change in the lead ions concentration in the medium: the coefficient of the nucleolar organizer’s genetic activity decreases, while the genetic activity of the Balbiani ring, on the contrary, increases.

Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo.

Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed.

The Balbiani Ring Story: Synthesis, Assembly, Processing, and Transport of Specific Messenger RNA-Protein Complexes.

Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.

The Chironomus tentans genome sequence and the organization of the Balbiani ring genes.

BackgroundThe polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes.ResultsThe genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150–250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations.ConclusionsWe show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-819) contains supplementary material, which is available to authorized users.

Nucleocytoplasmic mRNP export is an integral part of mRNP biogenesis

Nucleocytoplasmic export and biogenesis of mRNPs are closely coupled. At the gene, concomitant with synthesis of the pre-mRNA, the transcription machinery, hnRNP proteins, processing, quality control and export machineries cooperate to release processed and export competent mRNPs. After diffusion through the interchromatin space, the mRNPs are translocated through the nuclear pore complex and released into the cytoplasm. At the nuclear pore complex, defined compositional and conformational changes are triggered, but specific cotranscriptionally added components are retained in the mRNP and subsequently influence the cytoplasmic fate of the mRNP. Processes taking place at the gene locus and at the nuclear pore complex are crucial for integrating export as an essential part of gene expression. Spatial, temporal and structural aspects of these events have been highlighted in analyses of the Balbiani ring genes.

The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation

Many steps of gene expression take place during transcription, and important functional information can thus be obtained by determining the distribution of specific factors along a transcribed gene. The Balbiani ring (BR) genes of the dipteran Chironomus tentans constitute a unique system for mapping the association of specific factors along a eukaryotic gene using immuno-electron microscopy (immuno-EM). The chromatin immunoprecipitation (ChIP) technique has provided an alternative, more general method for studying the association of proteins with specific genomic sequences. The immuno-EM and the ChIP methods suffer from different limitations, and thus a combination of both is advantageous. We have established optimal conditions for ChIP on chromatin extracted from the salivary glands of C. tentans , and we have analyzed the association of the SWI/SNF chromatin remodelling factor Brahma (Brm) with the BR1 gene by combined immuno-EM and ChIP. We show that Brm is not restricted to the promoter of the BR1 gene but is also associated with sequences in the middle and distal portions of the gene, which suggests that Brm has additional roles apart from regulating transcription initiation.

The growing pre-mRNA recruits actin and chromatin-modifying factors to transcriptionally active genes

In the dipteran Chironomus tentans, actin binds to hrp65, a nuclear protein associated with mRNP complexes. Disruption of the actin-hrp65 interaction in vivo by the competing peptide 65-2CTS reduces transcription drastically, which suggests that the actin-hrp65 interaction is required for transcription. We show that the inhibitory effect of the 65-2CTS peptide on transcription is counteracted by trichostatin A, a drug that inhibits histone deacetylation. We also show that actin and hrp65 are associated in vivo with p2D10, an evolutionarily conserved protein with histone acetyltransferase activity that acts on histone H3. p2D10 is recruited to class II genes in a transcription-dependent manner. We show, using the Balbiani ring genes of C. tentans as a model system, that p2D10 is cotranscriptionally associated with the growing pre-mRNA. We also show that experimental disruption of the actin-hrp65 interaction by the 65-2CTS peptide in vivo results in the release of p2D10 from the transcribed genes, reduced histone H3 acetylation, and a lower level of transcription activity. Furthermore, antibodies against p2D10 inhibit run-on elongation. Our results suggest that actin, hrp65, and p2D10 are parts of a positive feedback mechanism that contributes to maintaining the active transcription state of a gene by recruiting HATs at the RNA level.

Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore

The nuclear poly(A)-binding protein, PABPN1, has been previously shown to regulate mRNA poly(A) tail length and to interact with selected proteins involved in mRNA synthesis and trafficking. To further understand the role of PABPN1 in mRNA metabolism, we used cryo-immunoelectron microscopy to determine the fate of PABPN1 at various stages in the assembly and transport of the Chironomus tentans salivary gland Balbiani ring (BR) mRNA ribonucleoprotein (mRNP) complex. PABPN1 is found on BR mRNPs within the nucleoplasm as well as on mRNPs docked at the nuclear pore. Very little PABPN1 is detected on the cytoplasmic side of the nuclear envelope, suggesting that PABPN1 is displaced from mRNPs during or shortly after passage through the nuclear pore. Surprisingly, we also find PABPN1 associated with RNA polymerase II along the chromatin axis of the BR gene. Our results suggest that PABPN1 binds to the polymerase before, at, or shortly after the start of transcription, and that the assembly of PABPN1 onto the poly(A) tail may be coupled to transcription. Furthermore, PABPN1 remains associated with the released BR mRNP until the mRNP is translocated from the nucleus to the cytoplasm.

Assembly and transport of a premessenger RNP particle.

Salivary gland cells in the larvae of the dipteran Chironomus tentans offer unique possibilities to visualize the assembly and nucleocytoplasmic transport of a specific transcription product. Each nucleus harbors four giant polytene chromosomes, whose transcription sites are expanded, or puffed. On chromosome IV, there are two puffs of exceptional size, Balbiani ring (BR) 1 and BR 2. A BR gene is 35-40 kb, contains four short introns, and encodes a 1-MDa salivary polypeptide. The BR transcript is packed with proteins into a ribonucleoprotein (RNP) fibril that is folded into a compact ring-like structure. The completed RNP particle is released into the nucleoplasm and transported to the nuclear pore, where the RNP fibril is gradually unfolded and passes through the pore. On the cytoplasmic side, the exiting extended RNP fibril becomes engaged in protein synthesis and the ensuing polysome is anchored to the endoplasmic reticulum. Several of the BR particle proteins have been characterized, and their fate during the assembly and transport of the BR particle has been elucidated. The proteins studied are all added cotranscriptionally to the pre-mRNA molecule. The various proteins behave differently during RNA transport, and the flow pattern of each protein is related to the particular function of the protein. Because the cotranscriptional assembly of the pre-mRNP particle involves proteins functioning in the nucleus as well as proteins functioning in the cytoplasm, it is concluded that the fate of the mRNA molecule is determined to a considerable extent already at the gene level.

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.

Promoter regions of four Balbiani ring genes in Chironomus tentans exhibit a common salivary gland-specific chromatin organisation, which is independent of the rate of transcriptional initiation.

The Balbiani ring (BR) gene family consists of four closely related genes which show some differences in transcriptional regulation. Here we analyse the chromatin structure of the promoter regions of the four BR genes. In cells in which the genes are inactive, digestion of chromatin with micrococcal nuclease suggests for the BR1 gene that precisely positioned nucleosomes are present from position +200 to -600. In salivary gland cells, a distinctly different organisation of the promoter chromatin leads to the appearance of two sites that are hypersensitive to both DNase I and micrococcal nuclease, located at the approximate positions -50 and -240. Nuclease digestion experiments suggest the presence of a nucleosome in the region between the two hypersensitive sites, and nucleosome disruption at the transcription initiation site. The positions of these hypersensitive sites in all BR genes is unaffected by extensive modulation of the rate of transcriptional initiation, although some alterations in the intensity of the hypersensitive sites were observed. We also show that total nucleosome displacement from the transcribing template is seen only when the RNA polymerase density exceeds one polymerase molecule per 100 bp.

Ku-related antigens are associated with transcriptionally active loci in Chironomus polytene chromosomes.

Antigens of Chironomus reactive with human sera containing anti-Ku antibodies and also with specific antibodies to each Ku subunit were characterized by immunoblot analysis. Three main antigen species were identified in nuclear-enriched extracts from salivary gland cells of Chironomus thummi, ranging in Mr from 55000 to 67000. The nuclear localization of Ku-related antigens in the dipteran Chironomus was studied by immunofluorescent labeling in polytene chromosomes of the salivary glands. Balbiani rings, loci highly active in transcription, were found to be strongly labeled by anti-Ku antibodies. Sugar-induced changes in the activity of the Balbiani ring genes were accompanied by the redistribution of Ku-related antigens as visualized by their absence in regressed Balbiani ring loci, and continued presence only in those that were transcriptionally active. A drastic change in the distribution of Ku-related antigens was also observed when C. thummi larvae underwent heat treatment as the immunofluorescent staining was restricted to previously described heat shock puffs. Anti-Ku sera reacted in addition with several chromosomal bands in which the presence of RNA polymerase II was also immunologically detected. The results show that Chironomus antigens reactive with anti-Ku antibodies are related to transcription in polytene chromosomes.

The C-terminal DNA-binding domain of Chironomus BR gene products shows preferential affinity for (dA.dT)-rich sequences.

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midge Chironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived from BR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the large BRs (BR1 and BR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of the BR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of the BR genes.

Structure of the smallest salivary-gland secretory protein gene in Chironomus tentans.

The salivary gland secretion in the dipteran Chironomus tentans is composed of approximately 15 different secretory proteins. The most well known of the corresponding genes are the four closely related Balbiani ring (BR) genes, in which the main part of each approximately 40-kb gene is composed of tandemly arranged repetitive units. Six of the seven additional secretory protein genes described share structural similarities with the BR genes and are members of the same BR multigene family. Here we report the identification of a new secretory protein gene, the sp12 gene, encoding the smallest component of the C. tentans salivary gland secretion. The gene has a corresponding mRNA length of approximately 0.7 kb and codes for a protein with a calculated molecular weight of 7,619 Da. The sp12 gene was characterized in seven Chironomus species. Based on a comparison of the orthologous gene sequences, we conclude that the sp12 gene has a repetitive structure consisting of diverged 21-bp-long repeats. The repeat structure and the codon composition are similar to the so-called SR regions of the BR genes and the sp12 gene may represent a diverged member of the BR multigene family.

A new member of the balbiani ring multigene family in the dipteran Chironomus tentans consists of a single-copy version of a unit repeated in other gene family members.

The known Balbiani ring (BR) multigene family members in the dipteran Chironomus tentans encode salivary gland secretory proteins in the size range between 38 and 1,000 kDa. The proteins interact to form protein fibers used by the aquatic larvae to spin feeding and protective larval tubes or pupation tubes. Here, we describe a new BR multigene family member, the sp17 gene, which codes for an 89-amino-acid-long protein with a relative mobility of 17k. The gene has a high content of charged amino acid residues and consists of two structurally different halves. Five regularly spaced cysteine codons are present in the 5' half while the 3' half contains five proline codons. These two different halves exhibit similarities to the C and SR regions, respectively, which form the tandemly repeated units in the about 40-kb-long BR genes and which also, in different versions, are the building blocks of all genes in the BR multigene family. In this multigene family, encoding interacting structural proteins, the long BR genes with their 125-150 tandemly arranged repeat units as well as the short sp17 gene with its single-copy version of such a repeat unit, have therefore evolved from a common ancestor.

Ultrastructural analysis of Balbiani ring genes of Chironomus pallidivittatus in different states of Balbiani ring activity

Patterns of gene activity on Balbiani ring (BR) transcription units (TUs) of polytene chromosome IV of Chironomus pallidivittatus larvae were ultrastructurally characterized by electron microscopy. In Miller chromatin speads, the 9 μm long TUs of Balbiani ring 1 and 2 (BR1 and BR2), respectively, were unambiguously identified on the basis of the size and structure of their stalked ribonucleoprotein (RNP) granules. Because of the mild spreading procedure the morphology of these 50 nm BR RNP particles remained quite reminiscent of their in situ configuration. BR TUs were found as clusters of sister copies, looking almost homogeneous in structure and organization, a number of them appearing as loops. TUs of normal and hyperactive BRs (the latter resulting from stimulation of the larvae with pilocarpine) were compared. The patterns of transcriptional activity of BR TUs were rather similar but not absolutely identical on all homologous sister chromatin strands. In normal as well as in hyperactive BRs, the great majority of TUs were highly transcriptionally active, i.e. densely packed with RNP fibres all along the deoxyribonucleoprotein (DNP) axis. Mean RNP fibre frequency of BR TUs of normal larvae was 30/μm while hyperactive BRs showed a fibre frequency of 31μm. The average number of RNP fibres per BR TU is in the range of 200 to 300. Assuming a transcription time of 20 min per BR mRNA molecule, the number of RNA polymerase molecules starting transcription of the 37 kb gene could be estimated to be 10 to 15 per minute. A number of BR genes were only partially active, exhibiting short, middle or long arrays of RNP fibres. Such arrays occurred in any segment of the genes showing normal RNP fibre density and a nucleosomal configuration in the inactive part of the gene. The variations in RNP fibre frequencies suggest that transcription can be modulated at the level of the individual BR gene. However, the large increase in the rate of BR mRNA synthesis upon pilocarpine stimulation may mainly be due to activation of hitherto inactive sister copies of the BR gene.

Two Secretory Protein Genes in Chironomus tentans Have Arisen by Gene Duplication and Exhibit Different Developmental Expression Patterns

The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 × 10 4 and 1 × 10 6. Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.

Conserved and variable repeat structures in the Balbiani ring gene family in Chironomus tentans

The four Balbiani ring (BR) genes, BR1, BR2.1, BR2.2, and BR6 in the midge Chironomus tentans constitute a gene family encoding secretory proteins with molecular weights of approximately 10(6) daltons. The major part of each gene is known to consist of tandemly organized composite repeat units resulting in a hierarchic repeat arrangement. Here, we present the sequence organization of the 5' part of the BR2.2 and BR6 genes and describe the entire transcribed part of the two genes. As the BR1 and BR2.1 genes were also fully characterized recently, this allows the comparison of all genes in the BR gene family. All four genes share the same exon-intron structure and have evolved by gene duplications starting from a common ancestor, having the same overall organization as the BR genes of today. The genes encode proteins that have an approximately 10,000-amino acid residue extended central domain, flanked by a highly charged, approximately 200-residue amino-terminal domain and a globular 110-residue carboxy-terminal domain. Exons 1-3 and the beginning of exon 4 encode the amino-terminal domain, which throughout contains many regions built from short repeats. These repeats are often degenerate as to repeat unit and sequence and are present in different numbers between the genes. In several instances these repeat structures, however, are conserved at the protein level where they form positively or negatively charged regions. Each BR gene has a 26-38-kb-long exon 4, which consists of an array of 125-150 repeat units and encodes the central domain. The number of repeat units appears to be largely preserved by selection and all repeat units in the array are very efficiently homogenized. Occasionally variant repeats have been introduced, presumably from another BR gene by gene conversion, and spread within the array. Introns 1-3 at the 5' end of the genes have diverged extensively in sequence and length between the genes. In contrast, intron 4 at the 3' end is virtually identical between three of the four genes, suggesting that gene conversion homogenizes the 3' ends of the genes, but not the 5' ends.

Sequence organization of the Balbiani ring 2.1 gene in Chironomus tentans.

Balbiani rings are giant chromosomal puffs, containing related genes that provide unique possibilities for in vivo analysis of gene expression at the chromatin and ribonucleoprotein levels. Here, the 5' end of the Balbiani ring 2.1 gene in the dipteran Chironomus tentans has been isolated and the sequence organization of the entire Balbiani ring gene is presented. The gene contains five exons, one being extremely small, only 6 base pairs, and one being extremely large, approximately 30 kilobase pairs. Three introns are located at the 5' end and a fourth one is located at the 3' end. The central 30-kilobase-pair exon is entirely built from tandemly organized repeats. All repeats are virtually identical except for a few variant repeats at both ends of the repeat array. The number of repeats may vary between alleles and the length of the gene therefore changes between 30 and 35 kilobase pairs.

Balbiani ring 1 gene in Chironomus tentans: Sequence organization and dynamics of a coding minisatellite

Balbiani ring (BR) genes in diptera encode large secretory proteins and are classical model systems for studies of gene expression. In Chironomus tentans, four closely related BR genes, BR 1, BR 2.1, BR 2.2 and BR 6 form a gene family. The BR genes have been partially characterized and are known to contain long arrays of tandemly arranged repeat units with an hierarchical repeat organization. Here, we report the sequence organization of the complete transcribed part of the BR 1 gene in C. tentans. The gene contains five exons and four introns. Three of the introns are located at the 5′ end and the fourth at the 3′ end of the gene. Exon 4 is approximately 35,000 bases long and is built completely from tandemly organized repeats. We show that this long repeat block contains two types of related repeat units, β and γ. Each type forms a large uninterrupted array, a 5′ β array and a 3′ γ array with a sharp border between them. In the hierarchical repeat structure in each repeat array, all repeats are virtually identical at one level of repetition, but shown differences at the next level. The whole repeat block in the BR 1 gene fluctuates in size between different alleles, but not by more than 10%. In contrast, within the block, the β and γ arrays vary in length between 8000 and 29,000 bases in an inverse fashion, together keeping the overall length requirement. We propose that the length of exon 4 is conserved by selection of cross-over products of a given length, and that the internal hierarchical sequence organization in the BR 1 gene is a consequence of the combined action of several different sequence turnover mechanisms, all dependent on the unequal pairing of homologous sequences at different, competing levels of repetition.