Studies on the Chromatin of the Cellular Slime Mold Dictyostelium Discoideum

Author: Bakke, Antony Clifford

Year: 1978

Degree: Dissertation (Ph.D.)

Advisor: Bonner, James Frederick

Committee Member: Unknown, Unknown

Option: Biochemistry

DOI: 10.7907/rmzz-9k75

Abstract

This thesis concerns the chromatin of Dictyostelium discoideum which has been characterized chemically, physically, and biochemically. The chromatin was carefully purified by differential centrifugation using nuclease and protease inhibitors followed by a brief, controlled Staphylococcal nuclease digestion. It has a chemical composition in terms of mass of 1.0 part DNA to 0.18 part RNA to 0.98 part histone to 1.02 part nonhistone protein. Its ultraviolet absorption spectrum is typical of chromatins from other eukaryotes.

The histones have been extracted from chromatin with 0.4 N H2SO4 and characterized on three gel systems. Three are equivalent to histones in higher eukaryotes and have the following molecular weights: Hl is 20,000; H3 is 14,900, and H4 is 12,000. Another one is not found in higher eukaryotes, but may be present in lower eukaryotes other than Dictyostelium. It is termed H6 and has a molecular weight of 15,800. There are no equivalents of the higher eukaryotic histones H2a and H2b.

The slime mold histones were also analyzed by ion-exchange chromatography. Hl elutes with the weakly bound proteins, while H3, H4, and H6 elute with the tightly bound peak. No proteins elute with calf thymus histones Hl, H2a, or H2b indicating that slime mold Hl is less basic than calf Hl and confirming that H6 is not equivalent to H2a or H2b.

The chromatin may be separated into two fractions by shearing briefly with Staph. nuclease, centrifuging, reshearing the insoluble pellet, and centrifuging again. The first supernatant contains 5% of the total DNA and is the first sheared chromatin fraction. The second supernatant contains 80% of the DNA. Both fractions have the same histone compositions, but differ in their mass ratios of nonhistone chromosomal proteins (NHCP). The NHCP are characterized on gels and differences are pointed out.

A subset of NHCP are extracted and tentatively identified as actin-like and myosin-like proteins on the basis of solubility and molecular weight. The actin-like protein accounts for approximately 5% of the total NHCP mass.

The basic unit of structure in the chromatin is the nucleosome. In Dictyostelium it is 98.6 Å in diameter and has a sedimentation coefficient of 11.5S. It contains 187 b.p. of DNA consisting of a 137 b.p. core and a 50 b.p. linker. Approximately 50% of the chromatin is protected from Staph. nuclease digestion, but this decreases when protease activity is not inhibited. When the chromatin is melted, four transitions are observed at 54.5°, 66.7°, 74.9°, and 79.7°. The structure of Dictyostelium chromatin is very similar to that seen in higher eukaryotes.

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