Subunit structure of a naturally occurring chromatin lacking histones F1 and F3.

Abstract

Macronuclei of the ciliated protozoan Tetrahymena pyriformis contain at least five classes of histones, including two with properties like those of histones F3 and F1 of higher eukaryotes. Micronuclei isolated under identical conditions contain little or no detectable F3 or Fl. Digestion of both macronuclei and micronuclei with staphylococcal nuclease results in DNA fragments of discrete sizes. The electrophoretic mobilities of the larger fragments suggest that they are oligomers of the smallest ones. These resu s indicate that the periodic subunit structure observed in the chromatin of higher organisms also occurs in protozoans, and that this structure does not depend on the presence of either histone F1 or F3, even in an organism which has the genetic information for synthesizing these proteins.Recent studies indicate that the chromatin of higher organisms has an underlying subunit structure which can be visualized directly by electron microscopic techniques (1-5). The existence of this-repeating structure can also be inferred by digesting isolated nuclei or chromatin with relatively nonspecific (endogenous or exogenous) nucleases. When used under conditions which completely degrade free DNA, these nucleases digest only about 50% of the DNA in chromatin to acid-soluble fragments (6). The majority of the undigested DNA is found in a nucleoprotein particle (7), and can be isolated as fragments containing approximately 50-200 base pairs (7-11). If nuclei or chromatin are only partially digested under conditions in which 10-30% of their DNA content becomes acid-soluble, the remaining DNA is found in an oligomeric series of nucleoprotein particles (10, 12). As first reported by Hewish and Burgoyne (13), the DNA molecules of such partial digests have discrete sizes. They also form an oligomeric series, the size of the larger fragments being integral multiples of the smallest fragment, which contains 100-200 base pairs (10,12,13).Based on results of the type described above (10) (14). One possible test of this postulated role for F3 and F2A1 in chromatin would be to examine the structure of chromatin lacking one (or both) of these histones.The macro-and micronuclei of the ciliated protozoan Tetrahymena pyriformis offer an opportunity to assess the role played by histone F3 in the structure of a naturally occurring chromatin. In ciliates, macro-and micronuclei are formed from daughter products of a mitotic division during the sexual process of conjugation and, in Tetrahymena, 80-90% of the DNA sequences in the two nuclei are the same (18). In this report, we present evidence that macronuclei isolated from Tetrahymena pyriformis contain histone F3, but that micronuclei do not. Nonetheless, when micronuclei are digested with staphylococcal nuclease, a series of DNA molecules of discrete sizes is obtained which is similar to that produced when macronuclei or calf thymus nuclei are digested under identical conditions. MATERIALS AND METHODS

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

“…This analysis is based on two reasonable assumptions: 1) the lighter edge of the 3H-peak does not contain rDNA, and 2) the density dis- Isolated nuclei, RSB soluble nucleosomes, dialyzed nucleosomes and core particles were digested with DNase I at room temperature in RSB. At indicated time intervals, the percentages of radioactivity soluble in 5% TCA-1 M NaCl-5 mM inorganic pyrophosphate were determined by counting aliquots of the reaction mixture on triplicate filters as described previously (47). Staphylococcal nuclease digestions were performed in RSB containing 0.1 mM CaCl2.…”

An intervening sequence in an unusual histone H1 gene of Tetrahymena thermophila.

“…Tetrahymena macro-and micronuclei have partitioned between them some of the processes in which H1 is proposed to function: macronuclei are transcriptionally active but divide amitotically, while micronuclei are transcriptionally inert and divide by mitosis (38). Both nuclei exhibit typical nucleosomal chromatin structure (39)(40)(41). In Tetrahymena thermophila, macronuclei contain a single type of H1 with typical solubility properties and composition (42,43).…”

Micronuclei of Tetrahymena contain two types of histone H3.

“…Electron microscopic (4) and biochemical (3,4) evidence demonstrates that micronuclear chromatin is organized in nucleosomes that are similar to those in macronuclei. Therefore, the absence of H3 in micronuclei is problematic.…”

“…On the other hand, H2A, H2B, and H4 in micronuclei are largely if not entirely unmodified. Furthermore, our previous studies suggested that micronuclei contain little, if any, H1 and H3 (3)(4)(5)(6).…”