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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.The nuclear precursors of mRNA, often designated hnRNA (heterogeneous nuclear RNA), are associated with proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs) (8, 10, 11). The hnRNP proteins constitute a major fraction of the nuclear proteins; in growing cells, they are as abundant as the histone proteins (18). They bind to the RNA concomitantly with transcription (12, 27) and are generally assumed to be confined to the cell nucleus (17, 35), although some of them have recently been shown to shuttle between the nucleus and the cytoplasm (34). The hnRNP population is complex; for example, there are about 20 major and a large number of minor hnRNP proteins in humans (33; see also reference 16). The hnRNP proteins contain a modular structure with one or more RNA-binding domains and at least one auxiliary domain that is probably involved in protein-protein interactions (for a recent review, see reference 11).hnRNP proteins have a general RNA-binding ability, but more and more information indicating that the proteins bind preferentially to distinct RNA sequences is accumulating. For example, the binding of several hnRNP proteins to different ribohomopolymers was studied, and it was found that various hnRNP proteins show different relative affinities for the ribonucleotide homopolymers (42). The hnRNP C proteins recognize a binding site consisting of five uridines in pre-mRNA polyadenylation substrates (28,46). The polypyrimidine tract...

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotidelong sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA, produced other products longer than 17 nucleotides. Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3¢-ends. However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3¢-end complementarity, therefore yielding an RNA-primed RNA-template. The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10% to 100%. Further, two type B DNA templates were extended by two nucleotides at the 3¢-end, to produce RNA transcripts theoretically unable to form 3¢-end base-paired duplexes. The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions. Keywords: DNA template; in vitro transcription; RNAprimed RNA extension; RNA-primed template; T7 RNA polymerase.Bacteriophage T7 DNA-dependent RNA polymerase is one of the best characterized single subunit RNA polymerases [1]. It is a highly specific enzyme able to recognize a particular promoter sequence through specific interactions with both promoter strands [2]. It initiates a new RNA chain from a single nucleotide (nt) and terminates transcription when it reaches either a terminator sequence or the 5¢-end of a linear template. Transcription by T7 RNA polymerase proceeds through three main stages: (a) initiation, during which abortive transcripts up to 6-8 nt are synthesized; (b) promoter clearance, representing the transition between initiation and elongation (which takes place between the 6-8-mer and 11-12-mer RNA stage); and (c) processive elongation, which begins after the synthesis of 12-mer RNA [3-6].T7 DNA-dependent RNA polymerase is able to transcribe both supercoiled and linear DNA templates and is highly effective with both double and single stranded synthetic templates containing the doubl...