Synthesis of different species of nucleolar ribonucleic acid

716

PRELIMINARY NOTES

greatly stimulates fl-galactosidase synthesis in comparison with the control culture where both protein and fl-galactosidase synthesis are very much depressed. However, when an energy source like glycerol was added, fi-galactosidase synthesis as well as protein synthesis were very much stimulated in the control culture. Addition of mitomycin C to such cells, however, reduced induced/3-galactosidase synthesis by about 50 %, while protein synthesis was little affected. Since catabolite repression of fl-galactosidase synthesis appears to take place through inhibition of the transcription of the lac gene 9, the effect of mitomycin C on the rate of /5-galactosidase messenger RNA synthesis was followed indirectly b y enzyme synthesis. Table I indicates that mitomycin C during the induction period causes appreciable reduction of the transcription of the lac gene only in the presence of glycerol, but stimulates nearly twofold in its absence. The translation process, on the other hand was little affected irrespective of the presence or absence of glycerol. It appeared that mitomycin C affects induced enzyme synthesis by inhibiting RNA formation in the presence of catabolites. The inability of mitomycin C to inhibit messenger RNA synthesis in general has been reported earlier 1°. This work was supported by grants from the East India Pharmaceutical Works Ltd., Calcutta and the Council of Scientific and Industrial Research, New Delhi. Mitomycin C was kindly given by Dr. A. R. Sa'ANELY, National Institutes of Health, Bethesda, Md., U.S.A.

Department o] Biochemistry, Calcutta University, Calcutta- 9 (India) I 2 3 4 5 6 7 8 9 IO

SANDIP K. BASu A . M . CHAKRABARTY S.C. RoY

S. CHEER AND T. T. TCHEN, Biochem, Biophys. Res. Cornmun., 9 (1962) 271. V. x~. IYER AND W. SZYBALSKI, Proc. Natl. Acad, Sci. U.S., 5 ° (1963) 355. A. WEISSBACH AND A. Llso, Biochemistry, 4 (1965) 196. Y. NAKATA, K. NAKATA AND Y. SAKAMOTO, Biochem. Biophys. Res. Commun., 6 (1961) 339, D. J. CUMMINGS, Biochim. Biophys. Acta, 95 (1965) 341. E. MCFALL, J. Mol. Biol., 3 (1961) 219. D. NAKADA, Biochim. Biophys. Acta, 55 (1962) 5o5 . B. D. DAvis AND E. S. MINGIOLI, J. Bacteriol., 60 (195 o) 17, D. NAKADA AND g . MAGASANIK, J. Mo1. Biol., 8 (1964) lO 5. I. SMITH-KIELLAND, Biochim. Biophys. Acta, 91 (1964) 360.

Received September I3th, 1965 Biochim. Biophys. Acta, lO8 (1965) 713-716

PN 91092

Synthesis of different species of nucleolar ribonucleic acid It is our purpose to report on the different species of RNA which are newly synthesized in the nucleolus of fully-grown larval salivary glands of the chironomid Smittia parthenogeneticaZ3. These species include both low and high molecular weight RNA's, of which only the latter has been described in nucleoli of other cells3. Synthesis of high molecular weight RNA was less clear in our previous experiments 1,*,4. Biochim, Biophys. Acta, lO8 (1965) 716-718

PRELIMINAJRY

717

NOTES

Salivary glands which are incubated in vitro 4,~ with appropriate radioactive precursors show the general intracellular distribution of RNA radioactivity given in Fig. Ia. On the other hand, in glands which are incubated in the presence of DRB and TRB the radioactivity is practically restricted to the nucleolus (Fig. Ib): this permits the study of radioactive nucleolar RNA without cell fractionation e.

700

70C Q

60C

500

50(

500

~

Control

DRB +

TRB 400

400

I u 300

I

300

I

200

I ~2

! I

,oo

i

-L

.l..l..i. ,I. ,I. ,I

I00

0

Fig. I. Distribution of RNA radioactivity to components of individual cells. The glands in (a) were incubated first in synthetic medium b for I io min and then ilt the same medium with [5-SH] uridine (Radiochemical Centre, Amersham, U.K.; 22.4 C/raM; 9I/*C]ml) for 45 rain. Both incubations for glands in (b) contained DRB and TRB (4° / , g / m l each). Counts were obtained in each series from autoradiograms 5 of 3-4 glands made into squash preparations s. The preparative solutions 5 contained a 3ooo-fold excess of non-radioactive uridine. Most of the radioactivity is sensitive to ribonuclease. Exposure, 24 h. - - - , chromosomes ( t o t a l ) ; - - , nucleolus (total); . . . . ,cytoplasm (constant area equal to about one-fourth).

The sucrose gradient sedimentation profile of the RNA from glands incubated as for Fig. Ia shows typically a pattern of radioactive peaks ranging from 4 to > 28 S (Fig. 2), while the height of the intermediate peaks can differ between experiments. In glands treated with DRB and TRB, the radioactivity is largely confined to the 4-S and > 28-S peaks (Fig. 2), the relative height of which is variable. These two molecular species of newly synthesized RNA are therefore nucleolar, and r e p r e s e n t a t r u e n u c l e o l a r synthesis4, 6. T h e 4-S R N A s y n t h e s i s in t h e n u c l e o l u s i n c l u d e s t h e f o r m a t i o n of c o m p l e t e m o l e c u l e s of t r a n s f e r R N A , a c c o r d i n g t o s e v e r a l criteria1&4, 5. F u r t h e r c h a r a c t e r i z a t i o n of t h e n u c l e o l a r R N A ' s is u n d e r w a y . T h e w o r k is s u p p o r t e d b y t h e D a m o n R u n y o n M e m o r i a l F u n d for C a n c e r R e s e a r c h ( D R G - 5 7 9 ) . D R B a n d T R B are gifts of Merck, S h a r p a n d D o h m e , R a h w a y ,

N.J. (U.S.A.). Abbreviations: DRB and TRB, 5,6-dichloro- and 4,5,6-tXichl°r°- r - (fl-D-ribofuranosyl)benzimidazole, respectively.

Biochim. Biopkys. Acta, lO8 (1965) 716-718

718

PRELIMINARY NOTES o

S20, w

28

18

4

100 t 90b 80 l7C 6C

? g

;, / '~

4C

if

,/ ~,

3,:

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'

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101

I

5

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/"

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number

Fig. 2. Sucrose gradient sedimentation pattern of RNA radioactivity. Glands from a different batch were incubated as those in Fig. i, during the same day interval. Counts after 3° or 4° min are adjusted for I99 pairs of glands treated with DRB and TRt3 to 84 pairs in controls. The RNA was extracted from homogenates with cold phenol and i % sodium dodecyl sulphate, followed by phenol at 6o ° (ref. 3). The RNA was co-precipitated with i mg Xenopus RNA prepared by the same method. Recovery of radioactivity was approx. 80 %. The uridiae was incorporated almost entirely as UMP into the RNAL which rep*resents the bulk of the radioactivity (see legend to Fig. i). The sedimentation values correspond to carrier RNA.

A . R . C , Unit o/ A n i m a l Genetics, British Empire Cancer Campaign, M . R . C . EpigeneEcs Group, Institute of A n i m a l Genetics, Edinburgh (Great Britain)

J. L. SIRLIN J. J A c o B M. L. BIRNSTIEL

I •. L. BIRNSTIEL, J. L. SIRLIN AND J. JACOB, Biochem. J., 94 (1965) l o P . 2 J. JACOB, M, L. BIRNSTIEL &ND J. L. SIRLIN, Abstr. 2nd Meeting Fed. European Biochem. Soc., Vienna, 1965, p. 334. 3 R. P. PERRY, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2179. 4 M. L. BIRI~STIEL, J. JACOB AND J. L. SIRLIN, Symp. on Molecular Cytology, Brussels, 1964, Arch. Biol. Liege, in the press. 5 J. L. SIRLIS, J. JACOB AND C. J. TA~DJ.ER, Biochem. J., 89 (1963) 4476 J. L. SIRLn~ AND J. JAcoB, Nature, 204 (1964) 545.

R e c e i v e d A u g u s t 9 t b , 1965

Biochim. Biophys. Aeta, lO8 (1965) 716-718