670 SHORT COMMUNICATIONS

disappearance of CO 2 from the gas phase shortly after the warm period indicate that this CO s is utilized by the cells. This conclusion will be explored further by use of radio- active isotopes.

In summary, temperature-synchronized A. longa appear to require the presence of CO 2 in the media either as the gas or as H C Q - and that C02 is specifically required 3o-6o min before the cells divide.

This investigation was supported by Public Health Service Postdoctcral Fellow- ship No. CA-2o318-o2 from the National Cancer Institute.

Research sponsored by the U.S. Atomic Energy Commission under contract with Uuion Carbide Corporation.

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. (U.S.A.)

P. A. VAN DREAL O. M. PADILLA

1 C. SOROKIN, Arch. Microbiol., 44 (I962) 219. 2 G. M. PADILLA AND T. W. JAMES, Exptl. CellRes., 20 (I96o) 4o1. 3 T. W. JAMES AND N. G. ANDERSON, Science, 142 (1963) 1183. 4 ]~. W. WILSON AND T. W. JAMES, Exptl. CellRes., 32 (1963) 305.

Received September 4th, 1964

Biochim. Biophys. Acla, 93 (1964) 668-670

sc 23o58

Mechanism of action of y-hexachlorocyclohexane in Neurospora crassa

The antagonism between 7-hexachlorocyclohexane (y-HCH) and mesoinositol has been studied in many organisms 1-3. A survey of the literature in this subject reveals that contradictory results have been obtained by many investigators for the existence of a competitive relationship between y-HCH and mesoinositol a-5. Following the demonstration by VLOTEN et al. 6 that it is the 3-isomer of HCH whose configuration corresponds to that of meso-inositol, the simple theory of antagonism due to stereo- chemical resemblance had to be abandoned. FULLER et al. a demonstrated two kinds of inhibition by y-HCH with inositol, one reversible and another irreversible. However, these experiments were criticised by SCHOPFER 7 who reported a variable antagonism between y-isomers of HCH, and inositol, quercitol and mytilitol in the inositol-less mutan t of Neurospora crassa when grown in liquid medium. Nevertheless, a com- petitive relationship between y-HCH and meso-inositol was demonstrable in Corcyra cephalonica st. a. The aim of this investigation was to study the mechanism of action of y-HCH. Evidence is presented for the induction of two types of inhibition by y-HCH. One type interferes with the uptake of exogenous inositol and could be re- versed with inositol and is present in the inositol-less mutant of N. crassa. The second type of inhibition is found in the wild type and is refractory to the action of inositol.

In this work two strains of N. crassa were used, wild type EM 5297 A and inositol-less mutant 37401. Both the strains were kindly supplied by Dr. H. K.

Abbreviat ion: y-HCH, 7-hexachlorocyclohexane.

Bioehim. Biophys. Acta, 93 (t964) 67o-67 '

S H O R T C O M M U N I C A T I O N S 671

MITCHELL of California Inst i tute of Technology, Pasadena, Calif. (U.S.A.). The organ- ism was grown in Ioo-ml conical flasks containing 20 ml of the basal medium 9 of pH 5.4-5.46. Mycelia of 72 h growth mats were used for dry weight determinations. For the radioactive experiments, 4/~C of randomly labelled [3H~inositol was added to each flask. ([3H~Inositol was a gift from Dr. B. W. AGRONOFF, Laboratory of Neurochemistry, National Insti tute of Neurological Diseases and Blindness, N.I .H., Bethesda Md., U.S.A.) The radioactive, lipid-bound, inositol was extracted from the mycelial mats (of 72 h growth) and the radioactivity was determined in a gas-flow proportional counter attached to a Berkley scaler. The counts were corrected for self- absorption and background.

The results, which are presented in Table I, indicate that 7-HCH inhibited the growth of N . c r a s s a 374o1 to a greater extent than that of the wild strain, EM 5297 A. In yeast 9o % of the inositol was found to be lipid bound1°, 11. Therefore, the measure of radioactivity in lipid, would represent both the uptake and utilization of exogen- ous inositol in the present experiment. The results presented in Table I indicate that 7-HCH interfered with the uptake of [3H!inositol by N . c ras sa 37401. In the wild strain no appreciable uptake of I3Hlinositol was observed. The results presented in Table I I demonstrate that inositol could reverse the toxic effects of 7-HCH on the mutant . However, in the wild strain inositol, even at a level of 25oo/zg per flask, could not reverse the growth inhibitory effect of 7-HCH.

T A B L E I

THE EFFECT OF T - H C H ON THE GROWTH OF, AND UPTAKE OF [SH i n o s i t o l b y , N . crassa 3 7 4 O l AND WILD STRAIN E M 5297 A

All f l a s k s w h e r e i n t h e m u t a n t s t r a i n w a s g r o w n c o n t a i n e d 4 ° / i g of i n o s i t o l p e r f lask . V a l u e s fo r r a d i o a c t i v i t y u p t a k e a r e t h e a v e r a g e o f t w o s e t s o f e x p e r i m e n t s . R e s u l t s in v e r t i c a l C o l u m n 4 r e p r e s e n t s t h e u p t a k e i n c o u n t s / m i n b y t h e m u t a n t s t r a i n . T h e n u m b e r s in p a r e n t h e s i s i n d i c a t e t h e p e r c e n t a g e i n h i b i t i o n o v e r c o n t r o l . All v a l u e s a r e t h e a v e r a g e o f 6 s e t s of e x p e r i m e n t s a n d

e a c h s e t r e p r e s e n t s t h e a v e r a g e o f 3 v a l u e s .

7-HCH added to the basal medium

(t*g per flask)

Inhibition of growth over control (%)

Wild strain Mntantstrain

Radioactivity uptake (counts/rain per g of mycelium)

Nil - - - - 2000 200 7.0 20 .0 17oo (I5) 5 0 0 20 .0 4 9 . 0 lO4O (48 )

IOOO 30 .0 66 .0 280 (86) 2 0 0 0 35 .o 9 7 . 0 24o (88)

I t is obvious, from the results presented in Tables I and II , that 7-HCH produces inhibition of growth in both the strains of N . c ras sa . However, the mechanism by which 7-HCH inhibited the growth of the two strains appears to be entirely different. 7-HCH produced the inhibition of growth in the inositol-less mutan t strain 374o1 which required exogenous inositol for growth, by interfering with the uptake of inositol from the medium (Table I). This inhibition was completely reversed by the addition of inositol (Table II). In a similar type of experiments FULLER et al. 3 have observed that the addition of inositol could not completely reverse the growth rate of N . c r a s s a

374o1 inhibited by 7-HCH when grown on a solid agar medium. From their results they have suggested that there are two types of inhibition in N . c r a s s a 374Ol, one

B i o c h i m . B i o p h y s . A c t a , 93 (1964) 6 7 0 - 6 7 2

672 SHORT COMMUNICATIONS

type tha t could be reversed by inositol and the other tha t could not be reversed by inositol. The la t ter type of inhibi t ion was observed in N. crassa wild strain.

However , the present exper imenta l results clearly point out tha t , in N. crassa 374Ol when grown on l iquid medium, y -HCH produces only one type of inhibit ion l:y inter-

fering with the up take of exogenous inositol. This inhibit ion was completely reversed

by the addi t ion of inositol. The second type of inhibit ion refractory to the action of

inositol as found in the wild strain seemed to be quite independent of inositot me-

tabolism.

TABLE lI

INFLUENCE OF INOSITOL ON GRO~,VTH-INHIBITORY ACTION OF ;*-HCH IN N . c r a s s a 37401 (I~IUTANT STRAIN)

The values are the average of six sets of experiments and each set consisted of the average of three values.

Supplements added to the basal medium

4 °/~g of inositol 4 ° / lg inositol + IOOO fig of 7-HCH 8o/~g inositol + IOOO Fg of 7-HCH

12o #g inositol + IOOO l*g of 7-HCH 16o #g inositol + IOOO #g of y-HCH 32o F,g inositol + IOOO l,g of 7-HCH

Il eight of mat % reversal (rag)

42.0

15. 4 32,8 57-3 38.o 74-3 39.2 78-3 44.6 Ioo.o

These results could probably explain the conflicting observat ions repor ted earlier

in the l i tera ture tha t only in some cases y -HCH inhibi ted the growth and inositol

could reverse only in some organisms. Thus the mechanism by which y -HCH acts

on inosi tol-dependent organisms such as N. crassa 374Ol, Saccharomyces cerevisiae and N. gossvppi is by prevent ing the uptake of exogenous inositol. Such an inhibit ion of

growth by y -HCH could be counterac ted by mesoinositol. The second type of inhi-

bi t ion of y-HCH, which inositol cannot reverse, exists in inosi tol- independent organ-

isms and appears to be independent of inositol metabol ism.

The financial assistance of the Rockefeller Founda t ion is gratefully acknowledged. These results form par t of the Ph.D. thesis submi t t ed to Madras Univers i ty in 1962

by S.R.

Department of Biochemistry, Indian Institute of Science, Bangalore (I.ndia)

S. RAJALAKSHMI

D. S. R. SARMA P. S. SARMA

1 s. I~IRKWOOD AND P. H. PHILLIPS, J. Biol. Chem., 163 (1946) 251. 2 H. VV. BUSTON, S. C. JACOBS AND A. GOLDSTEIN, Nature, I58 (1946) 22. a R. C. FULLER, R. W. BARRATT AND E. L. TATUM, J. Biol. Chem., 186 (195o) 823 4 VV. H. SCHOPFER AND M. L. BEIN, Experientia, 4 (1948) 147. 5 C. FROMAGEOT AND M. CONFINO, Biochim. Biophys. Acta, 2 (1948) 142. 6 G. W. VLOTEN, A. I~RUISSINK, B. STRIJK AND J. M. BIJVOET, Nature, 162 (1948) 771. 7 W*. H. SCHOPFER, Bull. Soc. Chim. Biol., 33 (1951) 1113. 8 S. RAJALAKSHMI, V. SRINIVASAN AND P. S. SARMA, Proc. Soc. Exptl. Biol. Med., lO 4 (106o) 97. 9 G. W. BEADLE, J. Biol. Chem., 156 (1949) 683.

10 H. F. YARBROUGH AND V. M. CLARK, J. Bacteriol., 73 (1957) 318. 11 W. N. BARNES AND P. SAGAR, J . Gen. 2~Iicrobiol., io (1954) 475.

Received J u l y I3th, 1964

Biochim. Biophys. Acta, 93 (1964) 67o-672