Apparent changes in yeast whole-cell ribonucleic acid base composition during growth

IMICA ET BIOPHY$ICA ACTA

Y E A S T W H O L E - C E L L RIBC ?OSITION DURING GROWT

ACID

D. I. E D D Y

boratory Pty. Ltd*, Sydney, N.S.W. (A~ (Received S e p t e m b e r 2oth, I963'3)

SUMMARY

I. A non-growing and non-dividing cell population relation of Sacc; ; an average whole-cell RNA molar purine/pyrimidir midine ratio c eristic of that physiological state. 2. However, growing and dividing populations of ccells have ~rage whole-cell RNA purine/pyrimidine ratios lower lby up to, ', above value only with cessation of growth and/or multiplicati, mt tionary phase.

revisiae 5, char~tically, cling to Vinto a

INTRODUCTION

It has ]as been reported, with evidence from a variety of sources, sou that irrespective of the th~ conditions ~ditions of growth obtaining, the molar ratios of the bases ] in whole-cell RNA aarc re relativel" ~tively invariable for a given organism 1. Confirmatior nation of this can be found in the th~ ext ~eriments of LOMBARD AND CI-IARGAFF~ using Esch¢,ichia coli, BOURDETs usingLg Sacc :charomyces cerevisiae and THOMAS4 using a torula yeast ~ (Torulops, s (Candida ~ndida)

utilis). While it thus seemed probable nlo_roll "I~ATA a ¢ o Nwnr~t~¢ifi,~n n whole-cell RNA hbase com t ~osition non examine the base composition ition of a invariability of the stationlary phase iological ages.

that cells might attain al a relatively invariabk riable ,-,~l,~+{,~n c,rn~, completion ~off growth, it was of interest also to tc population during growth to see whether the th~ organism's RNA was reflected at earlier phys'S-

EXPERIMENTAL

The organism used was Saccharor, ~ccharomyces r0~ ~ cerevisiae (strain DGI-Io3, Dansk Gaerings LgsIndustri, Copenhagen, Denmark namark) sampled from the final stage of a multi-stage lin dustrial "differential" fermentati mentation, involving the continuous addition of carbocarbo hydrate as blackstrap molasses .sses in a predetermined approximately exponential fashior ~shion under strongly aerobic conditions nditions. Lons. Samples were taken at 3o-min intervals from time o to 16.5 h, centrifuged, and the sedimented cells washed at the centrifuge with tap water. On resuspension of the cell, :ells A b b r e v i a t i o n : P u / P y , purine/p~ arine/pyrimidine. " Subsidiary of Mauri Brothers ~thers a n d T h o m s o n L t d .

Biochira. Biophys. Acta, 8o (1964) 373-37~8

D. I. E D D Y

ntaining 6o-15o mg of dry yeast , for pxd immediately resedimented. )n of the R N A of non-growing an~ ag cells ing cells, similar quantities of eac tples of yeasts from factories in New Zeal; , Great mstralia were also examined. ns with io ml of cold IO o/ acid at /o tric] I to remove acid-soluble material : is. The ion with acid was left overnight i ~thanol te at o-4 °, to remove excess trichl id. The ,portions of yeast d r y matter and trichloroacetic acid were chos. almost nplete removal of acid-soluble phosphorus-containing compouI minary ~eriments it had been found that three successive Io-ml Io-r. portiol ~moved 6 and 2 ~o of the total acid-extractable phosphorus from i ut only 300 ~f I0 o/ 'east. An aci 9 and 4 % from some 2.5 times this q u a n t i t y of yeas /o s used because of the observation 5 that this was the o p t i m u n ion for ,. extraction of acid-soluble polyphosphates from ye mast. Lipid materials were extracted with successive Io-ml I, voh thanol, thanol-chloroform (I :I, v/v) and n-butanol at room temp 3 ° rain :h, followed b y two ether washings to remove excess exc, n-but ling to ,RTIN AND ~¢[ORTON 9. This was found to be superior to successi, ns with ml volumes of ethanol, ethanol-chloroform ( 3 : I , v/v) tu 1-ether I, v/v) twice and ether twice at 0-4 ° for 3o min each, both in time consumed and nd the extraction of 2o °/o more non-nucleic acid phosph( ~horus. Lipid extraction after removal of acid-soluble sub:stances has, in some hands nds losses:n. Quadruplicate samples of OCC~asioned losses of R N A m, and in other hands no losses ressed yeast were subjected to four different extraction extractic procedures in this laboraa pressed v brief washing in ethanoltoryy (a) asreported under EXPERIMENTAL, (b) as in (a) but with in ether er (3 :I) and ether in place of lipid extraction, (c) as iin (b) but with hydrolysis m :tmn e: and ((t) extraction 0.3 N K O H at 35 ° for 6o min (see ref. 12) instead off KC1 extraction, of whole whole cells with an equal volume of 9 ° °/o phenol at room r. temperature for 60 mln min lowed b' by ethanol precipitation of RNA. The P u / P y • values va obtained ( ± S . D . ) were, followed respectively, I. I 1-4-o.o3, I..zi-¢-o.o3, i I -t--O.O o.c o . 8 1 ± o . o 6 and z.13±o.o2. KC1 extracts of the residues from (c) and (d) gave ve R N A with ratios 1.13 + o . 0 8 and 1.13 ! o . o 2 , respectively7. No selective loss of R N A as a result of lipid removal was revealed b y these results, 'ent raneous phosphorus compounds in method c is apparent but the interference of extraneous (see also, e.g., ref. 14). .Sslum ~rs were twice extracted with 13 % KC1 in o.1 M potassium The ether-dried powders acetate-acetic acid buffer (pH 4.0) at 95 °. The extracts and single water washin~g ime were made to ioo ml wi~h water. It had previously been found that the total timt nlof extraction rather than the volume of extracting solution (above a given mixnimal volume) was more important. Consequently, the original 3o- and I5-min eextraction periods 9 were increased •eased to 90 min each. moder-dried yeast with 0.3 N K O H at 37 ° for 60 min (a mod. Extraction of the ether-dried ISON ification of the SCHMIDT AND THANNHAUSER13 method suggested b y HUTCmSOI~ A N D 1MUNRO 12) w a s found to be unsatisfactory when applied to yeast, because of oJ •action of phosphorus from replicates, and extraction b)Y erratic and excessive extraction Biochim. Biophys. Acta, 8 o (1964) 3 7 3 37~8 ~

YEAST RNA BASE COMPOSITION

(c]. ref. 15) interfering wit

Lt esti-

~RENSON AND DE DEKEN TM imprc

HMIDT

lymers

sorbing the ribomononucleotides Lkaline e) at p H 7.8, washing the resin ng the
RESULTS AND DISCUSSION

The results of the experiment Lent on growing cells are shown in Fig. I. A decline in whole-cell R N A P u / P y ratio from 0.82, followed by a rise after 4 h to a final value of Biochim. Biophys. Acta, 8o (1964) 373-378

D.

;

'

I. EDDY

'

'

' '~'

, '

'

'

;5'

Time (h)

ge whole-cell RNA Pu/Py ratio ( © - © - O ) of aerobically g r o w n yeast. P u / P y ratio w a s e s t i m a t e d v i a t h e acid NA. of b u f f e r e d K C l - e x t r a c t e d lZNA

(P)z60 mg ~) ribose

weight 2 occurred. Truly stationary conditions were not a,.chieved i tease of 5.5 %/h continued even at harvest. Light of as discoun Differential extraction of distinct RNA species w wa •ic acid npleteness of extraction as judged b y Az,0m~ meassurements :tion of facts of the residues from KC1 extraction, and of the ol)bservatio] 6 h did [A from acid-extracted and delipidated yeast with II(0 ~0 NaC] 'olysed, of d : significantly alter base ratios in comparison with those t] ~xtracted ~RNA3. Fig. I. Changes with time in values of e(P) of the extrac.cts are als midine, ese are quite compatible with the known lower molar absorptiv ~ (ref. 23.) From Fig. I the npared with purine, mononucleotides at p H 4•and 26om# 260 ~ntent of extracts on either acid~lihood of error in basing estimates of RNA content l]Kel] RNA ~arlson with a sample of RI~A dle ribose or A,e0m~ measurements, even in compari: labile little error is introduced byY "ived from the same organism, is apparent. However, derived u se )horus/o.o97 at any physiological age becaus~ culating RNA content as R N A phosphorus/o.o97 m calculatin ) m with changes in base comthe small variation in phosphorus content of RNA of position. 'easts are shown in Table I. I The results of the examination of commercial ye ',lls. :tle invariabilit' invariability in P u / P y ratio was apparent in these supposedly " m a t u r e " cells Little centag of cells in the sample: ples bearing buds, as indices of oJ ,wever. plotting ~lottin¢ the percentag oercenta~e However, NA RN2~ the average physiological ages of the populations, against average whole-cell R ical rrelation of coefficient --0.62 (P < 0.005). Physiologica P u l P y ratios showed a correla 'as state or age thus has a l;profound influence on the average base composition (a, de I] P u / P y ratio) of the RNA off a population of cells (Fig. 2). This is shown also in Table ainimal in the cases of the dried y,easts from factories A, M and N, grown to have minima] )le :ells, and of "seed" yeasts from factory M grown in ample final contents of budding cells, nutrient supply with lowv generation times. Thus there appears to) be a tendency, with cessation of growth and entry into [ue ~verage whole-cell R N A P u / P y ratio to approach a value stationary phase, for the avera characteristic for S. cerevis;iae. Seven samples of yeast of diverse cultivation history:73 led total R N A P u / P y ratio o'I 1.244-o.o 4. Pooling of these these were found to show a pooled )r S. cerevisiae RNA in eight at-81 other papers, and those those values with those given for o values lying within one st~mdard error of estimate of x (o.17) from the x intercept of 'I the regression of x on y inn Fig. 2, gave the value 1.264-o.o6 as the average of 71 estimates. ;-378 Biochim. B~ophys. Acta, 80 (1964) 373-37~

YEAST R N A

BASE COMPOSITION

TABLE I

D D E F F G G H H H

2b 2b 3 4 4 4 4 5 5 5

I

5

J K K L L M M M M M N

5 5 5 5 & 6 5 & 6 5 & 6 5"* 5"* 6"* 6" 6"

(P)z60 m# OF WHOLE-CELL RNA AND PER N COMMERCIALLY GROWN YI~AST

~UDDING

ate e s t i m a t e s on 3oo-4oo m g of d r y yea, KCl-extracted from commercially grow

tandard

4oIar u[Py

ding cells

RNA (%)

*(P)

92- o.o4 ~2- o.o2 52-O.Ol 0.892-0.02 o.912-o.o 3 1.142-o.o2 0.762-0.02 1.o82-o.o2 0.842-0.02 1.2°2-°.°4 1.262-O.Ol 1.272-°.°4 1.292-o.o2 1"362-°-°3 1.372-°.°4 0.982-0.02 1.222-°.°3 o.89~o.oI o.83io.o2 1.332-o.o 3 °.912-o.°3 0.742-0.o2 o.872-0.02 1.292-o.o2 1.29~o.o 4

84602- 9o 78202- 65 6 9 3 o I 35 8 9 0 0 ± 90 85002- IOO 803o2- 4 ° 7 5 I O + 9° 7 5 7 o ± 7° 87OO2- 18o 81102- 4 ° 82502- 9 ° 8380 ± 4 ° 84702- 60 838o2- io 84002- 5 ° 86502- 12o 827o2- 3 ° 839o2-18o 840o2- 4 ° 8620-}- 85 78702- 7 ° 67802- 3 ° 7 2 9 o ± 25 8I 502-

..

20

8 0 2 0 ± 60

2.4~ 4.4 ~ 3.4~ 2.7~ 3.14 4-42 3.8~ 4.I] 3.6~ 3.6( 5.o~: 2.91 2.8( 3.2; 3 . 2~ 2.2~ 4.8( 2.6: 3.6.= 4-3( ~.I9 8.1( 8.32 4.75 3.56 2.60

(%) I 12

12 9 15 20 IO

8 I4 7 3 6 4 6 4 7 12

8 12 -15 3 I

* Dried yeast. ** Seed yeast.

It has recently been shown that in Salmonella typh bhimurium increase in growff ,wth o cellular RNA that is riboratee is accompanied by an increase in the proportion of 0aal, as opposed to soluble, from about one-third at 0.2 o.: doublings/h to about 80 °% somal i _ , 21 i1_ [_^£ ~ _\ 1 " 1 :a. . . . . . . . . . . 2 1 _ 1 _ ..... at 2.4 doublings/h (ref. 32). ). If it were possible to draw an analogy between this or. or_._1L.

12__

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3

21

2¢

""o",b,...

I 0.70

0.80

I 0.90

•

I IL)O

I

l

1.10

1.20

"~t, 1.30

el 1MO

Pu/Py molar ratio Fig. 2. The relationship b e t w e een n aver~ average whole-cell R N A P u / P y ratio and average physiologica ical age as indicated b y percentage of b u d d i n g cells, r ~ --0.62 ( P < o.oo5). The solid line represent )resents the regression y = 24.3--i4.5 x, and the b r o k e n line the regression x = 1.31--o.o27y. P u / P !y ratio was estimated in the same m a n n e r as for Fig. L

Biochim. Biophys. Acta, 80 (1964) 373-371z8

I). I. EDDY suits presented the experiment mpositional

here might be inte: of Fig. I was o.2

changes

predominan

an t h a t in dry gh

not

lble RNA. ACKNOWLEDGEMENTS

:icism during the preparation

ade to the Directors of Mauri Brol t h i s p a p e r . T h e a u t h o r is i n d e b t e c

lomson Le l a t e )

r. A . L . H U N T AND D r . R . A . BOT'

helpful

of the manuscript. RE FERENCES

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15 16 17 18 19 20

21 22

23

24 25

~6 27 28 29

30

al 32

Biochim. Biophys. Acta, 8o (1964) 373-378