Absence of Histones from the Chromosomal Proteins of Fungi

Abstract

Interphase chromosomes were isolated in good yield from four species of fungi. In no case does the chromatin contain histones such as are characteristic of the chromosomes of other eukaryotic organisms.That histones are characteristic chromosomal proteins has been demonstrated for a wide variety of eukaryotic creatures. Thus, the chromosomes of higher plants and animals not only contain histones but, in addition, these proteins are similar in number, chemical properties, and even, in some cases, in primary structure (1, 2). Histones chemically similar to those of higher plants and animals have been found in the green alga Chlorella (3) and in the protozoan Tetrahymena (4,5), as well as in a wide variety of invertebrates.There have been several reports that the nuclei and/or chromatin of fungi lack histones, but contain instead chromosomal proteins of a less basic nature (6-9). We have examined the basic chromosomal proteins of several fungi, using what we believe to be the most rigorous of techniques for both chromatin isolation and histone characterization.We describe methods for obtaining purified fungal chromatin that result in the recovery of at least 70% of the DNA present in the homogenate. The possibility of proteolytic degradation of histones (if present) is unlikely. We find that histones analogous to those cf higher eukaryotes are missing in the fungi we have examined. MATERIALS AND METHODS Chromatin isolationAttempts to isolate chromatin from the fungus M4icrosporum gypseum by the methods suggested for liver and pea (10) were not successful. Fungal nuclei are sheared by these methods, and centrifugal forces sufficient to pellet the chromatin result in gross RNA contamination of the nuclear fraction. A more viscous grinding medium should afford greater protection to the nuclei during the cell breakage step. Stern's glycerol grinding medium admirably satisfied this requirement (11).Sporulation and spore purification procedures for Microsporum have been described previously (12, 13). 2-liter Erlenmeyer flasks, containing 600 ml of glucose (1% w/v) and neopeptone (Difco, 1% w/v) (pH 6.5), were inoculated with 106 conidia per ml and shaken at 350 rpm on a New Brunswick controlled environment dry-air shaker (30"C) for 4 days.The mycelia were harvested by suction filtration. The mycelial mat (150 g wet weight) was washed 4 times with 1-liter amounts of ice-cold physiological saline (pH 6.5).The mycelial mat was resuspended in 350 ml of grinding medium (glycerol 50% w/v, 0.5 M sucrose, 0.001 M CaCl2, and 0.05 M Tris, pH 8). The slurry was poured into an aluminum container and frozen by the addition of liquid nitrogen. Several additions of liquid nitrogen were necessary to completely freeze the material. The frozen slurry was ground to a coarse powder and placed into a Waring blendor. When the temperature of the grinding solution reached -30'C, the blendor was turned to full speed (110 V). Homogenization was continued until the temperature reached 0C. The homogenate was again poured into an aluminum c...

Biochemical genetics of Neurospora crassa conidial germination.

“…Chromatin has been isolated from both mycelia (45) and conidia (43) of the wild-type strain of N. crassa and from a cell wall-less mutant strain, slime (83). Histones were not found in the chromatin from either mycelia or conidia (43,45,106). However, two slightly lysine-rich histones were isolated from chromatin preparations from the slime mutant (83).…”

The Karyotype in Systematics

“…Except in fungi(76), the chromosomes have histones intimately associated with them 3. Kinetochore and primary constriction are interchangeable terms for centro mere.…”

Recombination and chromosome structure in eukaryotes

“…However, it does not A. PFTRAMENT seem unreasonable to assume that the same species of non-histone protein can also be associated with several non-identical segments of DNA. This seems to apply also to fungi, although in their chromosomes no histones were found (Leighton et al 1971).…”