Oocytes of Xenopus synthesize a specific type of S5 RNA, which is not made in somatic cells. The DNA containing the genes for this RNA (S5 DNA) has been isolated from X. laevis and X. mulleri. The proposed program is to take advantage of what is known about the structure of these genes in a study of the mechanism by which they are regulated in development. The first phase involves completion of a current study of the organization of X. mulleri S5 DNA. The repeating units of this DNA will be characterized by analysis, using electron microscopy and gel electrophoresis, of the products of digestion of the purified S5 DNA by restriction endonucleases. Single chromosomal loci will be investigated in more detail by incorporation of restriction fragments, carrying several repeats, into bacterial plasmids, followed by cloning and amplification of individual fragments for analysis. The second and more extensive phase consists of a study of the chromatin containing the S5 genes, and its relationship to the regulation of S5 RNA synthesis. Information on the chromatin structure will be sought by determining the susceptibility of S5 DNA in chromatin to staphylococcal nuclease, and to restriction enzymes having known modes of attack on deproteinized S5 DNA. Chromatin from cells in which the oocyte-type S5 genes are active will be compared with those in which it is not. If the action of restriction enzymes on DNA in chromatin approximates that on free DNA, this property and a chromatin fractionation technique based on DNA molecular weight will be combined in a procedure for purifying S5 chromatin. Its chemical and template properties will be investigated.