Chromosomal architecture in giant premeiotic nuclei of the green algaAcetabularia

Abstract

Giant primary nuclei of the unicellular green algaAcetabularia contain 40 small lampbrush chromosomes which have proved difficult to visualize in the light microscope in vivo by conventional fluorescent DNA staining techniques. We report here that immunofluorescence staining with the monoclonal anti-phosphoepitope antibody MPM 2 is the method of choice to study the architecture of whole chromosomes within primary nuclei fixed in situ or after hand isolation. Using confocal laser scanning microscopy, we have been able to produce images ofAcetabularia lampbrush chromosomes of hitherto unsurpassed structural detail. Particularly striking is the visualization of abundant loops extending from the periaxial chromosome core region for variable distances into the nucleoplasm. Staining of the loops can be abolished by pretreatment of isolated nuclei with alkaline phosphatase, whereas the chromosomal core remains unaffected. At meiosis, when transcription ceases and the extended chromosomes condense, MPM 2 staining is lost indicating that maintenance of the loops depends on the phosphorylation status of the MPM 2 antigen. Anti-histone antibodies, on the other hand, exclusively stain the chromosomal core in the extended lampbrush configuration as well as in the condensed metaphase configuration. This indicates that MPM 2 and anti-histone antibodies recognize different molecular components on the chromosomes. We postulate that the loops stained by MPM 2 represent actively transcribing regions of the chromosomes and that the MPM 2 antigen could be a molecular component involved either in the structural maintenance of the loops or in a process associated with transcription.