(36), but VP1 is essential for formation of infectious AAV type 2 (AAV-2) particles (17, 42, 50). VP2 cotransports VP3 into the nucleus before capsid assembly (18,36). VP3 alone also forms capsids but only when targeted to the nucleus (18). Encapsidation of the AAV-2 genome likely occurs in the nucleoplasm in areas where capsids, Rep proteins, and DNA colocalize (52). Detailed analysis of the protein-protein interactions of Rep and VP proteins favors a model by which Rep-tagged DNA initiates packaging by interaction with capsid proteins (11). Several of the above-mentioned studies of the AAV-2 capsid assembly process were aided by using monoclonal antibodies (MAbs) directed against the capsid proteins.AAV-2 infects a broad range of cells by binding to its primary receptor, heparan sulfate proteoglycan (47). Two types of coreceptors, ␣ v ␤5 integrin and fibroblast growth factor receptor

Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38‐containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re‐populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one‐ and two‐dimensional gel electrophoresis, co‐migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone‐binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo‐oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the ‘nucleoplasmin family’. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre‐rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly.

Abstract, The formation of daughter nuclei and the reformation of nucleolar structures was studied after rnicroinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionaUy resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.

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Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50-200 bp in length and cloning these into the 5' terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.

Small nucleolus-related bodies which occur in the nUcleoplasm of " micronuclei " lacking nucleolar organizers have been studied by immunofluorescence microscopy . These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers . Our data show that, in the absence of rRNA genes , the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei . The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.

Abstract. After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK z ) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed . These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

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