Isolation of nuclei of Neurospora crassa

MICA ET BIOPHYSICA ACTA

t Communications

f nuclei of Neurospora crassa mold Neurospora crassa has been studied intensively [y with resp~ cycle, its hereditary and physiological properties. Therefore, Therefor, isolatic Lclei Of organism might be of interest for diverse biochemical and biol¢ ments. :his note we report a method for obtaining suspension ~enslons of such rather form, free of significant contamination with other cellular cc or~ The experimental protocol (the 9-in rotor of a refri~gerated ] trifuge used throughout) was as follows: N. crassa, osmotic mutant, s~ (wild can be used equally well) was incubated in stationar~y culture replete lium 3 with 1 % sucrose as carbohydrate source. After 6o-h gro, celium washed extensively with distilled water, then sque, ueezed dry d with I M mannitol. It was then pressed between sheets of blottil ltil no her moisture could be removed, chopped into small fragments fl ~°, and m d in a mortar at this temperature with three times its weight of washed sand. en a homogeneous mass had been obtained by persis ~ersistent gentle grinding, 3 vol. of mannitol aannitol were added and the paste stirred until smootLh. The resulting quite thick thre layers of cheesecloth, and sust ~ension was poured into a large funnel lined with three filtration ation permitted to proceed for approx. 30 min. 56 g of mycelium, wet wt., to which ch had been added 15o ml mannitol solution, yieldo delded on filtration 90 ml of an opaque, que, orange fluid. The filtrate was almost completely free of mycelial fragments and sand. This homogenate could be used for the isolatiol on of mitochondria and riboleoprotein particles, for the flotation of lipid material materi; which is present in subnucleo stantial ltial amounts, as well as for the concentration of nuc] clei. The crude homogenate was centrifuged at 500 x g for 5 min. The sedimented material possessed an opaque, [ue, gelatinous central portion which was resuspended in its own supernatant, and an outer, white ring, which was discarded. The next centrifugation, 2o00 x g for: 7 min, yielded a larger pellet whose appearance resembled that obtained at 500 x g. Once again, the central and superficial portions were resuspended, this time in mannitol, and combined with the pellet obtained on centrifuging the supernatantit f, for a further 30 min at 5000 x g. The resultant suspension was rich in nuclei, most: of which (approx. 85 %) could be sedimented in relatively pure form by centrifugation 3n at 3200 x g for 7 min. The pellet, on staining with crystal violet, showed small .11, spherical nuclei (approx. I/~ in diameter) clustered in grape-like bunches. The yield d at this stage could be increased only slightly by extending the period of centrifugation m to 22 min, but at the cost of significantly reduced homogeneity of the pellet. Itit is noteworthy that a portion of the DNA (approx. IO %) can no longer be readily sediment ente( in these low centrifugal fields. This probably edimented results from the lysis of some me nuclei in the course of their resuspension. A portion of the final pellet was fixed with osmium tetroxide (I % in 0.35 M Biochim. Biophys. Acta, 53 (1961) 574-575

HORT COMMUNICATIONS

DID

TABLE I ATION

OF

DNA

Volume (too

IN

NEUROSPORA

HOM(

Total DNA present (ug)

89

247o

86 82 78

2500 2050 86o

46

16oo

in pellet ~al DNA)

~ts comt)lned a n d r e s u s p e n d e d in al r e s u s p e n s i o n ~rnatant of 3200 × g, 7 m i n ~rnatant of 3200 × g, 22 m i n

248 I69

mitol) and embedded in methacrylateL After sectioning, al was mined in the electron microscope, where some morphol 'phological fe~ ; nuclei d be observed. The presence in the pellet of tiny (0.5 tLin diam. articles ciated with the nuclei rendered impossible the prep~ )aration oJ factory sections. Thus, the two layers of the characteristic nuclear nbrane d not be resolved. However, the uniform diameter of the , ,rtictes, esponding to that expected from their appearance 111 in Iintacl I I L C t ( ~ L I I I y L ; U I I U . I I I -L, , their LI|UII of DNA in the aing reaction with crystal violet, and the coincident concentration c ~t left little doubt that these were, in fact, nuclei. The Th pellet was seen to contain :ontain pellet )lasm and an occasional mitoin adddition small amounts of adherent granular cytopla,, ldrion and hexagonal crystal bodyL chondrion inal homogenate is shown in the The yield based on the DNA content of the original i ~mpanying table. Owing to the fact that substances interfering with the deteraccom to be present in intact kn miraation of DNA b y the diphenylamine reaction are known tg mycelium :elium 4, no a t t e m p t has been made to compute the yield based on the starting material. erial. However, this is estimated to be very low, of the t[ order of IO % or less. The ed, if desired, b y using a larger volume of suspending fluid fluid yield could easily be increased following grinding. Nuclei isolated in the manner described can be used as a convenient source of high-molecular-weight DNAk of N. crassa. aowledge the interest of Dr. E. L. TATUM in this work. I t is a pleasure to acknowled ational ed in part b y Research Grant RG-6972 from the National This work was supported Advisory Health Council, [ LS. Public Health Service.

;enetics, The Rockefeller Institute, Laboratory of Biochemical Genetics New York, N . Y . (U.S.A.)

EDWARD REICH SEIZO TSUDA

I A. J. SHATKIN AND E. L. TATUM TOM, J. Biophys. Bigchem. Cytol., 6 (1959) 423. S. TSUDA AND E. L, TATUM, J].. Biophys. Biochem. Cytol., in t h e press. T, N. FRIES AND D. BONNER, A m . J. Botany, 37 (195o) 38. E. L. TATUM, R. W. BARRATT ~ID B. STRAUSS, Arch. Biochem. Biophys., 80 (1959) 442. 4 Z. MINAGAWA, B. V~rAGNER AND

Received May I5th, 1961 Biochim. Biopt~ys. Acta, 53 (1961) 574-575 74-575-