NONHISTONE CHROMOSOMAL PROTEINS OF DROSOPHILA MELANOGASTER: IMMUNOCHEMICAL CHARACTERIZATION AND IN SITU IMMUNOFLUORESCENT LOCALIZATION OF SELECTED PROTEINS USING MONOCLONAL ANTIBODIES

Hybridomas were produced by fusion of mouse myeloma P3X63/Ag8.653 with spleen cells from mice immunized with purified Drosophila melanogaster chromatin. One hundred ten independent hybridoma cell lines demonstrating anti-chromatin activity by an indirect alkaline phosphatase enzyme-linked immunosorbent assay were produced by this procedure. Fusion-related observations were as follows: By my procedure I was able to obtain greater than 90% fusion efficiency, with 10-50% of those demonstrating anti-chromatin activity. Extensive immunization schedules favored IgG-synthesizing hybridomas whereas shorter immunization schedules favored IgM-producing hybridomas. Five hybridoma cell lines were cloned and cultured for further analysis of their hybridoma proteins, they were DM(DELTA)12, DM(DELTA)19, DM(DELTA)41, DM(DELTA)55 and DM(DELTA)71. The apparent molecular weights of the protein antigens found in purified chromatin which immunoreacted with each monoclonal antibody were as follows: DM(DELTA)12 - 10 kd, DM(DELTA)19 - 12 kd, 34 kd and 39 kd, DM(DELTA)41 - 72 kd, DM(DELTA)55 - 13 kd (HMG A13) and 13.4 kd (histone H2b) and DM(DELTA)71 - 23 kd, 42 kd and 120 kd. All specificities were determined by indirect immunoperoxidase staining of Western blots of 2-dimensional polyacrylamide gels (Triton DF16-Acetic Acid-Urea PAGE versus SDS PAGE). Preferential release of these protein antigens from intact nuclei by nucleolytic digestion with DNase 1 or RNase A as evaluated by indirect immunoperoxidase staining of Western blots of polyacrylamide gels containing electrophoretically separated protein samples that had been obtained by EDTA extraction of nuclei following nucleolytic digestion. Similarly, release of each protein antigen from intact nuclei by extraction with elevated salt concentrations or by acid extraction was determined in an attempt to characterize these proteins. The in situ locations of these protein antigens on polytene chromosomes of Drosophila melanogaster were determined by indirect immunofluorescent staining. Monoclonal antibody, moAb(DELTA)12, moAb(DELTA)19, moAb(DELTA)55 and moAb(DELTA)71 demonstrated general chromosomal fluorescence with a preference given to phase dark chromosomal regions as judged by their more intense fluorescence. In contrast, moAb(DELTA)41 demonstrated preferential phase light region staining superimposed upon a general chromosomal fluorescence. By virtue of their chromosomal distribution, these protein antigens appear to function as general structural proteins. In addition, moAb(DELTA)55 reacted with two distinct proteins, HMG A13 and H2b, which do not appear related by amino acid composition. (Abstract shortened with permission of author.