Quantitative cytophotometric and autoradiographic analysis of the nuclear changes in the myxomycete didymium iridis during growth and development of the plasmodium

Abstract

Microspectrophotometric and cytochemical examinations were made of the nuclear changes in basic protein, RNA, and DNA in the developing plasmodium of Didymium iridis, at the fourth, eighth, and twelfth day of its progression through the growth cycle prior to sporulation. Data was collected from cultures which had no indications of differentiation and there was no renewal of growth medium made during the course of the investigation. Relative determinations of basic protein and RNA were obtained using the double staining technique of Napthol Yellow-S/Azure B in conjunction with absorption microspectrophotometry. Cytophotometric measurements of the Feulgen-DNA nucleal reaction provided quantification of DNA. Autoradiography using tritiated thymidine and liquid-emulsion techniques provided information on periods of DNA synthesis. The results of the investigation include a net decrease in protein and RNA to a point of stabilization which correlates well with increased age of the organism and decreased nutrient supply in the cultures. No loss of DNA was observed under either of these conditions. These findings are in complete accord with macroscopic observations of -the initially rapid then decreasing growth rates until a point was reached where actual decrease in plasmodial mass was detected. Feulgen-autoradiography results showed the cultures of four and twelve day plasmodia in the replicated G2 condition. The 4C DNA content of the four day organisms indicates rapid growth and high metabolic activity but It has been determined that the replicated DNA of the twelve day organisms would not have been succeeded by a mitotic division until some later time, presumably following the Initial sporulat ion processes. Microspectrophotometric determinations of DNA in the eight day cultures showed a 2C DNA content, indicating no synthesis occurs during this period. Thls implies that a G1 period of some duration exists in the plasmodial slime mold which may extend up to the point of the final replication of DNA (G2 condition) prior to sporulation. The observed morphological growth patterns also support these conclusions.