Monoclonal antibodies to lampbrush chromosome antigens of Pleurodeles waltlii.
Germinal vesicles of oocytes from Pleurodeles waltlii were used for immunization of BALB/c mice to obtain hybridomas secreting monoclonal antibodies. The hybridomas were screened for reactivity of their antibodies against lampbrush chromosomes of oocytes, as revealed by indirect immunostaining. Antibodies labelling the lampbrush chromosomes were also tested on histological sections of oocytes, embryos, and larvae of Pleurodeles. Characterization of the antigens was accomplished through immunoblotting of two-dimensional electrophoretic gels of germinal vesicle proteins. The ten monoclonal antibodies giving a positive reaction were classed into five groups. Group 1, exemplified by antibody A33, recognizes all the lampbrush chromosome transcribing sites (loops). Moreover, it differentially labels the cell nuclei during embryonic and larval development. Group 2, antibody B71, also stains all the loops of the lampbrush chromosomes, but does not react with cell nuclei of embryos and larvae. Group 3, antibody A1, labels specific loops, some of which are heterozygous in the strain of P. waltlii used. These heterozygosities have allowed us to localize and to characterize a chromosomal segment on bivalent IV which is heteromorphic in the two partners of the bivalent. We suggest that this heteromorphism represents a morphological distinction between Z and W heterochromosomes. Moreover, this antibody reacts with only one transcription unit along a loop that contains several units. Group 4, antibody B24, stains the only two structures in the lampbrush chromosomes of P. waltlii that do not have a loop organization, the mass "M" and the spheres. Group 5, antibody A35, reacts with the chromomeres. The antigens corresponding to antibodies A33 and B24 have been identified as proteins, which have apparent molecular weights of 80 and 104 kilodaltons, respectively. They correspond to proteins abundant in the germinal vesicles. All the antibodies described here cross-react with the lampbrush chromosomes of five other species of Urodeles.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D002875:Chromosomes / Q000032:analysis / Q000648:ultrastructure*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D009865:Oocytes / Q000032:analysis* / Q000276:immunology; D010053:Ovary / Q000032:analysis / Q000276:immunology; D010999:Pleurodeles / Q000235:genetics*; D012455:Salamandridae / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface