Abstract.Anti-Sm antibodies recognize a group of small, nuclear RNA-protein complexes (snRNPs) containing U 1, U2, U4, U5, and U6 snRNAs. Anti-RNP antibodies only react with U I snRNA-containing complexes.The intranuclear distribution of snRNP particles was studied by double immunofluorescence staining of human fibroblasts. Mouse monoclonal anti-Sm antibodies and polyclonal patient sera reacting with different peptides in the snRNP complexes were used. The immunofluorescence patterns obtained with fluorescein isothiocyanate-conjugated anti-mouse Ig and tetramethylrhodamine isothiocyanate-conjugated anti-human Ig second antibodies were examined using computer analysis of digitized images. With this approach the similarity of different patterns could be visualized and estimated with mathematical methods. It was found that human anti-Sm serum as well as three different anti-RNP sera produced speckled patterns overlapping with the anti-Sm monoclonal pattern. Thus, Sm antigenic intranuclear domains also reacted with anti-RNP antibodies, suggesting a high degree of co-localization of the antigenic structures. A partial overlap was found between speckles detected by mouse anti-Sm antibodies and a human La-antiserum. No significant co-localization occurred between speckles detected by mouse anti-Sm antibodies and speckles detected by human antisera reacting with Scl-70 and centromeric antigens.As the U 1 snRNP complex is believed to play a role in the splicing of RNA polymerase II transcripts, it appears that the speckles detected by Sm and RNP antibodies may be regions of hnRNA synthesis and m.RNA processing. Although no function has been demonstrated for the U2, U4, U5, and U6 snRNPs, the co-localization with the U 1 RNA complexes shown in this report indicate that they too participate in some aspect of mRNA processing. The results suggest that computer-assisted analysis of nuclear immunofluorescence patterns will be a useful tool in studies of the spatial and functional organization of the interphase nucleus. IMMUNE staining of mammalian cells with autoimmune sera frequently results in a speckled nuclear pattern. The two major specificities that produce speckled patterns are known as Sm and RNP. These autoantibodies occur primarily in systemic lupus erythematosus, mixed connective tissue disease, and less frequently in other autoimmune diseases (3,31,35).The Sm and RNP antigenic structures are thus concentrated in certain nuclear domains. Immunochemical and biochemical studies have elucidated the nature of the antigens (1, 11-13, 16, 18, 20, 25, 33, 34, 37, 38). Sm-antibodies were shown to immunoprecipitate RNA-protein complexes containing a class of small nuclear RNAs designated U l, U2, U4, U5, and U6 snRNAs) RNP-antibodies, on the other hand, immunoprecipitated only U 1 RNA-containing complexes (18). Proteins were required for antigenicity (18). The fact that small, U3 is nucleolar and not precipitated by Sm/RNP antibodies. nuclear RNA-protein complex (snRNP) 2 particles containing only U l RNA can be immunoprecip...

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK(-) and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

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