FRACTIONATION OF BIOLOGICALLY ACTIVE NUCLEIC ACID EXTRACT OF VOLVOX AUREUS

The colonial flagellate Volvox has been under study recently because its pattern of differentiation is simple and because it synthesizes chemical substances that induce or influence specific stages in its development (Darden, 1970; Starr , 1970). Volvox shows cellular differentiation and division of labor but has a maximum of four cell types, making Volvox\u27s cellular diversity among the least complex of multicellular organisms . Chemical induction systems for males, females, and parthenospores are known for several species. Male differentiation in Volvox aureus M5 is controlled by a male inducing substance (MIS) obtained from filtrates of sexual cultures (Darden, 1966). MIS has been concentrated and purified to an eighteen fold increase in its specific activ ity . There are indications that MIS is a glycoprotein (Ely and Dard en, 1971). Recently, it has been reported that the differentiation of male colonies can be induced in Volvox aureus M5 by a combination of Volvox aureus nucleic acid extract and either histone or ribonuclease (Darden, 1971). Both MIS and the active extract are influencing cellular differ­ entiation. Since both male and vegetative colonies are derived from a haploid clone, this appears to be a system of genetic control that is as yet unknown . Discovering what the inducer is composed of must precede any any further work on the system of genetic control. This may also shed some light on the action of histones in genetic systems, as the addition of histone to the extract causes it to become active in producing males . With this system , histone action may be studied both in vitro and in vivo. Finding out how histone operates in this case may indicate how it functions in genetic systems as a whole . It has been shown by Darden (1971), that pronase completely destroys the activity of the extract while treatment with deoxyribonuclease only slightly reduces the activity of the inducer. This, however, is probably due to contamination of the enzyme. This project was to purify the Volvox nucleic acid extract and determine what caused the male inducing activity . The chemical and physical properties of the extract were also to be compared to MIS to determine whE ther the active fraction was similar to MIS in composition. Efforts were made to obtain the biologically active fraction of the Volvox extract to which histone had been added by using G-25, G-75, and DEAE Sephadex chromatography. The results from this pre- liminary work were not successful. The fractions obtained from these columns were either inactive or only very slightly active in inducing differentiation of male colonies. Since this method of purifying the Volvox extract was unproductive, it was decided that disc gel electrophoresis should be attempted . Two strains, DS and V-1, have been described which yield a nucleic acid extract capable of forming the histone-nucleic acid complex, but are not active in male induction. It appeared likely that an electrophoretic comparison of these to the standard M5 strain might shed considerable light on the nature of the complex. It was also thought that a comparison of bands might show something missing in the two deficient strains which was present in the M5 strain, this possibly indicating the biologically active fraction. This was an effort to determine what part of the nucleic acid extract combined with the histone and resulted in the extract becoming biologically active . This paper describes efforts made to purify the extract and the electrophoretie analyses of the extract from three strains of Volvox aureus