The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.

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.

Expressed sequence tags from the midgut and an epithelial cell line of Chironomus tentans: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

Expressed sequence tags (ESTs) were generated from two Chironomus tentans cDNA libraries, constructed from an embryo epithelial cell line and from larva midgut tissue. 8584 5'-end ESTs were generated and assembled into 3110 tentative unique transcripts, providing the largest contribution of C. tentans sequences to public databases to date. Annotation using Blast gave 1975 (63.5%) transcripts with a significant match in the major gene/protein databases, 1170 with a best match to Anopheles gambiae and 480 to Drosophila melanogaster. 1091 transcripts (35.1%) had no match to any database. Studies of open reading frames suggest that at least 323 of these contain a coding sequence, indicating that a large proportion of the genes in C. tentans belong to previously unknown gene families.

Molecular characterization of Ct-hrp65: identification of two novel isoforms originated by alternative splicing.

Hrp65, a protein with two conserved RNA-binding domains, has been identified in Chironomus tentans as a component of nuclear fibers associated with ribonucleoprotein particles in transit from the gene to the nuclear pore. We have cloned two novel hrp65 isoforms and characterized the structure of the hrp65 gene. Comparison of the hrp65 gene to the hrp65 cDNAs revealed that the multiple hrp65 isoforms, hrp65-1, hrp65-2 and hrp65-3, are generated by alternative splicing of a single pre-mRNA. The hrp65-3 mRNA is only detected in C. tentans tissue culture cells of embryonic origin, whereas hrp65-1 and hrp65-2 mRNAs appear to be constitutively expressed. The hrp65 mRNAs are generated by differential 3' splice site selection at the last exon of the gene. Thus, the three hrp65 transcripts contain different 3' UTRs and encode proteins that vary in their C-terminal ends. Interestingly, the variant C-terminal region determines the subcellular localization of the hrp65 proteins. In transient transfection assays, hrp65-1 is efficiently targetted to the nucleus, whereas hrp65-2 and hrp65-3 localize mainly to the cytoplasm. Moreover, hrp65-3 is associated with cytoplasmic actin fibers. All together, our findings suggest that the different hrp65 isoforms serve specialized roles related to mRNA localization/transport in the different cell compartments.

The Ct-RAE1 protein interacts with Balbiani ring RNP particles at the nuclear pore.

RAE1 is an evolutionarily conserved protein that associates with both mRNPs and nucleoporins, and may bridge the interaction between mRNP export cargoes and the nuclear pore complex (NPC). However, the mechanism by which RAE1 functions in mRNA export is still unknown and the time point at which RAE1 interacts with the exported RNP has not been directly investigated. Here we have addressed this question in the Balbiani ring (BR) system of Chironomus tentans using immunoelectron microscopy. The RAE1 protein of C. tentans, Ct-RAE1, is 70% identical to human RAE1/mrnp41 (hRAE1) and is recognized by antibodies raised against hRAE1. As in vertebrate cells, Ct-RAE1 is concentrated at the nuclear envelope and also dispersed throughout the nuclear interior. Here we show that Ct-RAE1 does not bind to the BR particle either cotranscriptionally or in the nucleoplasm. Instead, the interaction between Ct-RAE1 and the exported BR particle occurs at the NPC. Moreover, the localization of Ct-RAE1 at the NPC is correlated with the presence of an exported RNP in the NPC. Finally, the anti-RAE1 antibody does not label the cytoplasmic side of BR particles in transit through the central channel, which indicates that Ct-RAE1 either remains anchored at the nuclear side of the NPC during translocation of the RNP through the central channel or becomes transiently associated with the RNP but is rapidly released into the cytoplasm.

Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm.

Using electron tomography, we have analyzed whether the Balbiani ring (BR) pre-mRNP particles in transit from the gene to the nuclear pore complex (NPC) are bound to any structure that could impair free diffusion through the nucleoplasm. We show that one-third of the BR particles are in contact with thin connecting fibers (CFs), which in some cases merge into large fibrogranular clusters. The CFs have a specific protein composition different from that of BR particles, as shown by immuno-EM. Moreover, we have identified hrp65 as one of the protein components of the CFs. The sequencing of hrp65 cDNA reveals similarities with hnRNP proteins and splicing factors. However, hrp65 is likely to have a different function because it does not bind to nascent pre-mRNA and is not part of the pre-mRNP itself. Taken together, our observations indicate that pre-mRNPs are not always freely diffusible in the nucleoplasm but interact with fibers of specific structure and composition, which implies that some of the posttranscriptional events that the pre-mRNPs undergo before reaching the NPC occur in a bound state.

The hrp23 protein in the balbiani ring pre-mRNP particles is released just before or at the binding of the particles to the nuclear pore complex.

Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine-rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression.

Immunocytochemical evidence for a stepwise assembly of Balbiani ring premessenger ribonucleoprotein particles.

In the active Balbiani ring (BR) genes of the dipteran Chironomus tentans, the assembly of a specific pre-mRNP particle can be analyzed in situ, and the incorporation of hnRNP proteins into the nascent pre-mRNP can be directly visualized by immunoelectron microscopy. In the present study we have shown that hrp36, one of the major hnRNP proteins in Chironomus tentans, is continuously added to the nascent BR pre-mRNP particle throughout transcription and is localized along the entire BR RNP fiber. Interestingly, hrp36 becomes concealed during the structural transition that occurs during the formation of the mature BR RNP particle. This conclusion is based on the observation that hrp36 can be revealed by a monoclonal antibody during the initial assembly of the BR RNP fiber but becomes almost undetectable in the final packaging stage. The hrp36 protein, however, is not removed from the BR RNP particle since the ability of the monoclonal antibody to reveal hrp36 is restored by artificial relaxation of mature BR RNP particles. Another major hnRNP protein, hrp45, is also incorporated in a continuous manner into the nascent pre-mRNP fiber but remains accessible in mature BR RNP particles. Our results provide immunocytochemical evidence for drastic structural changes occurring in the final stage of BR pre-mRNP packaging, and suggest that different hnRNP proteins might be differently involved in the pre-mRNP assembly process.

A protein of the SR family of splicing factors binds extensively to exonic Balbiani ring pre-mRNA and accompanies the RNA from the gene to the nuclear pore.

We report on the molecular cloning and intracellular localization of a heterogeneous nuclear ribonucleoprotein (hnRNP), Ct-hrp45, one of the major components of pre-mRNP particles in Chironomus tentans. It is shown that hrp45 belongs to the SR family of splicing factors and exhibits high sequence similarity to Drosophila SRp55/B52 and human SF2/ASF. The distribution of hrp45 within the C. tentans salivary gland cells is studied by immunocytology. The hrp45 protein is found to be abundant in the nucleus, whereas it is undetectable in the cytoplasm. The fate of hrp45 in specific pre-mRNP particles, the Balbiani ring (BR) granules, is revealed by immunoelectron microscopy. It is observed that hrp45 is associated with the growing BR pre-mRNP particles and is being added continuously concomitant with the growth of the transcript, indicating that hrp45 is bound extensively to exon 4, which comprises 80-90% of the primary transcript. Furthermore, hrp45 remains bound to the BR RNP particles in the nucleoplasm and is not released until the particles translocate through the nuclear pore. Thus, hrp45 behaves as an hnRNP protein linked to exon RNA (and perhaps also to the introns) rather than as a spliceosome component connected to the assembly and disassembly of spliceosomes. It seems that hrp45, and possibly also other SR family proteins, is playing an important role in the structural organization of pre-mRNP particles and is perhaps participating not only in splicing but also in other intranuclear events.

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.

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.

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.

Rearrangements of intranuclear structures involved in RNA processing in response to adenovirus infection.

We have studied in HeLa cells at the electron microscope level the response to adenovirus infection of the RNA processing machinery. Components of the spliceosomes were localized by in situ hybridization with biotinylated U1 and U2 DNA probes and by immunolabeling with Y12 anti-Sm monoclonal antibody, whereas poly(A)+ RNAs were localized by specific binding of biotinylated poly(dT) probe. At early stages of nuclear transformation, the distribution of small nuclear RNPs was similar to that previously described in non-infected nuclei (Visa, N., Puvion-Dutilleul, F., Bachellerie, J.P. and Puvion, E., Eur. J. Cell Biol. 60, 308-321, 1993; Visa, N., Puvion-Dutilleul, F., Harper, F., Bachellerie, J. P. and Puvion, E., Exp. Cell Res. 208, 19-34, 1993). As the infection progresses, the large virus-induced inclusion body consists of a central storage site of functionally inactive viral genomes surrounded by a peripheral shell formed by clusters of interchromatin granules, compact rings and a fibrillogranular network in which are embedded the viral single-stranded DNA accumulation sites. Spliceosome components and poly(A)+ RNAs were then exclusively detected over the clusters of interchromatin granules and the fibrillogranular network whereas the viral single-stranded DNA accumulation sites and compact rings remained unlabeled, thus appearing to not be directly involved in splicing. Our data, therefore, suggest that the fibrillogranular network, in addition to being the site of viral transcription, is also a major site of viral RNA splicing. Like the clusters of interchromatin granules, which had been already involved in spliceosome assembly, they could also have a role in the sorting of viral spliced polyadenylated mRNAs before export to the cytoplasm. The compact rings, which contain non-polyadenylated viral RNA, might accumulate the non-used portions of the viral transcripts resulting from differential poly(A)+ site selection.

Intranuclear distribution of poly(A) RNA determined by electron microscope in situ hybridization.

We used a biotinylated poly(dT) probe to localize poly(A) RNA in HeLa cells at optical and electron microscope levels. We established that the fluorescent speckled staining pattern corresponds at the ultrastructural level to the labeling of perichromatin fibrils, at least part of the population of perichromatin granules, and clusters of interchromatin granules. Coiled bodies and the interchromatin granule-associated zones, a recently described subcompartment containing U1 but not U2 snRNA, were not labeled. The density of the labeling of interchromatin granule clusters exceeded by three to five times that of the surrounding extranucleolar area. These results are discussed in relation to the role of perichromatin fibrils in splicing of pre-mRNA and to the possible involvement of interchromatin granules in the assembly of mature spliceosomes as well as in sorting and/or coordination of RNA molecules to be transported to the cytoplasm.

Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells.

We have examined the intranuclear distribution of U1 and U2 small nuclear RNAs (snRNAs) in HeLa cells by electron microscope in situ hybridization using biotinylated DNA probes reacting at the surface of thin sections of Lowicryl-embedded cells. U1 and U2 snRNAs colocalized on perichromatin fibrils, clusters of interchromatin granules and coiled bodies. The perichromatin granules were just occasionally labeled. In addition, we identified a novel nuclear domain associated with the clusters of interchromatin granules which contains U1 but not U2 snRNA. This new compartment termed "interchromatin granule-associated zone" has a fibrillar texture, does not contain DNA and might be the equivalent of the A snurposomes described in germinal vesicles of amphibians (Wu et al., J. Cell Biol. 113, 465-483 (1991)). We propose that the interchromatin granule-associated zones might be sites of the final maturation of the U1-pre-snRNP particle before its transfer to interchromatin granules and its subsequent assembly in the spliceosome.

Progressive redistribution of alcohol dehydrogenase during vitellogenesis in Drosophila melanogaster: characterization of ADH-positive bodies in mature oocytes.

The use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S7), ADH appears to be located mainly in the nurse cells. From stage S9 onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S13-14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or beta-spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.

The Adh in Drosophila: chromosomal location and restriction analysis in species with different phylogenetic relationships.

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.

Comparative analysis of four parameters involved in puffing activity along chromosome arm 2L of D melanogaster.

Autoradiographic and immunofluorescent techniques have been used to analyse the relationship between puffing, transcription and occurrence of DNA/RNA hybrids in D melanogaster salivary gland chromosome 2L. Experiments of 3H-uridine incorporation have indicated that similar rates of RNA synthesis are observable in well developed puffs as well as in some diffuse bands and interbands. On the other hand, puffs of similar size incorporate 3H-uridine at quite different rates. The presence of RNA polymerase II seems to follow a coincident pattern with that of 3H-uridine incorporation. Our results indicate that the rate of transcription does not determine either the formation of a puff or its potential size. Instead, we have found a positive correlation between the amount of DNA/RNA hybrids and puff size, independently of the transcription rates. Transient accumulation of transcribed RNAs in their chromosomal compartment could therefore play a relevant role in the determination of puff size.