Appearance of polyadenylated RNA species during sporulation in Bacillus polymyxa

Vol. 86, No. 2, 1979

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 30, 1979

Pages 331-339

APPEARANCE OF POLYADENYLATED RNA SPECIES SI'ORULATION IN BACILLUS ----E‘OLYI'ZXA Yatwant Kaur Department School of Madurai Madurai Received

December

DURING

and Kunthala Jayaraman of Molecular Biology Biological Sciences Kamaraj University 625 021, India

11,1978

SUMIMARY Two main features of altered transcriptional programming during sporulation are reported; 1. The occurrence of the polyA RNA species exclusively during sporulation. 2. The localisation in the membranes of regions of DNA that may be enrimhed in spore genes. INTRODUCTION Sporulation tified

specific

either

capacity

transcriptional

by way of appearance

of mEtNAs appearing

by the

possible

appearance sporulation

of

alteration new polypeptides

(4,5).

onset

of sporulation phenomenon

sporulation

of DNA for (6).

This

the

the

of the

RPase

appearance

during

of mem-

activity

at

and had suggested increased

transcriptional

transcriptional

or

by the

with

transcriptional

of i.polymyxa was a result

(1,2,3)

RNA polymerase

associated

iden-

hybridization

sporulation

We had reported

DNA (mDNA) associated

of regions

the

has been

of altered

during of

brane

this

activity

the

that

amenability

machinery activity

during

was present

The initial hybridization studies reported here were carried out in the laboratory of Dr X.H.Doi at University of California, Davis during the tenureship of Fulbright Senior Fellowship to KJ. Abbreviations: mDNA - membrane DNA; RPase - RNA polymerase; mRNA - messenger RNA ; SDS - sodium dodecyl sulphate 0006-291X/79/020331-09$01.00/0

331

Copyright All rights

@,! I979

by Academic Press, Inc. in anyform reserved.

of reproduction

Vol. 86, No. 2, 1979

BIOCHEMICAL

as a tightly

bound

complex

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

tant

to rifamycin,

while

vity

was sensitive

to rifamycin

These

results

indicated

of DNA associated present populations

studies

the

with are

the

mDNA and was highly

soluble

transcriptional

acti-

(6j. special

membrane

concerned

of mRNAs during

resis-

involvement

during with

the

of regions

sporulation.

the

growth

differences

The in

the

and sporulation

stages.

MATERIALS

AND METHODS

Microorganisms: The organism used in the course of these --studies was Bacillus polymyxa obtained from Pfizer and Co. USA. The RiKSpdO mutant was isolated as described earlier (6). Media: --

The rich

medium

of Schaeffer

(7)

was used.

DNA extraction: DNA was extracted from whole cells membrane framons after phenol deproteinization. fractions were prepared as described earlier (6).

and the Membrane

Extraction of labelled RNA: Por hybridization studies,the ---RNA was labelled by pulsing the growing cultures of B. 01 m xa at the vegetative phase with 3 - 5 mCi of 3HZ&Y&+ or 3 min and at the T phase with 15 mCi for 15 min. Par the extraction of po 2 yA RNA species, the cells were labelled by growing in the presence of 100 uCi of %adenosine in 50 ml of medium and harvesting them at the vegetative phase and the T phase (2 hrs after the end of growth). The cells were r 2 suspended in 10 ml of RNA extraction buffer containing Tris-HCl, IO mM. uH 7.4, 10 mM magnesium chloride and l-unit/ml of heparin: Lysozyme (lmg/lml) and DNase (30 u&ml) were added to the mixture and incubated at 37OC for-30 Gin.SDS at a final concentration of 0.2% was added and the lysed cells were immediately deproteinized with an equal volume of phenol:chloroform:isoamyl alcohol (50:48:2) mixture containing 0.1% 8'hydroxyquinoline. The deproteinised aqueous phase was precipitated with 95% ethanol and left overnight at -lO°C, centrifuged and dissolved in the same buffer. DNA:RNA hybridization: This was carried out according to the method of Nygaard and Hall (8). The hybridization was carried out under conditions of RNA excess, The reaction mixture, in addition contained 5% formamide (v/v) and citrate buffer (0.15 iV sodium chloride and 0.015 M sodium citrate. pH 7.0) at a final concentration of 6X, and was

332

Vol. 86, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

incubated for 24 hrs at 37OC. At the end of the incubation period the mixture was cooled in ice and filtered on nitrocellulose filters, presoaked in 2X citrate buffer. The filters were washed with 2X citrate buffer (75 ml) and incubated with stirring in the presence of 20 ug/ml pancreatic RNase in 3 ml of the 2X citrate buffer for 30 min at 3OOC. The filters were washed with 50 ml of 2X citrate buffer on both the sides, dried and counted in a liquid scintillation counter. In the control assays, DNA was omitted from the reaction mixtures. Competition:-The labelled an equal amount of cold

DNA:RNA hybrids RNA.

~,"~TJ,f~~te,~,",~~~~~ss~~~ binding 0.01

ters acid

were chased

by

w~~i~i~~~l~ea,rr:~d,o"u~b~ft~~e

buffer (0.05 YL Tris-BCl, 0.2 M sodium chloride, The fil!Y EDTA) and was bound to the polyU filters. were washed extensively with 2.5% cold trichloroacetic and ethanol.

dT oligo dT cellu&ose chromatoEa h : To one gram of oligo cellulose (generous gift of F.-&y-ilt, University of California, Berkeley, USA) about 100 0.D units of RNA was loaded. The polyA containing RNA was bound to the column with a high salt buffer (10 mM Tris-HCl, pH 7.4, containing 0.5 M sodium chloride, 0.5% SDS and 1 unit/ml of heparin) and for subsequent elution Tris-HCl buffer containing 0.5% SDS and 1 unit/ml of heparin was used (10). Fractionation of total RNA ---on sucrose density gradient:---_A continuous sucrose density gradient of 5 - 15% (w/v) in 0.05 M sodium chloride and 0.01 M sodium acetate, pH 5.0 3H-adenosine labelled samples of RNA (0.2 ml) was made. were loaded on to the gradient and centrifuged in the swing out rotor in a Janetzki VAC 601 ultracentrifuge for 18 hrs at 90,000 g.

RESULTS

AND DISCUSSION

Hybridization

studies

tions

with

the

brane

fractions

total

hybridised

contrast,

poorly

the

with

pulse-labelled

DNA and the

indicated

of sporulation In

with

that

for

vegetative

T2 mRNA in

rate

and extent

the

DNA associated

messengers

with

mRNA populations That

there

mDNA was evident

of hybridization

with

mDNA (Fig.la). hybridised

were increased by the

of T 2 messengers

333

mem-

at T2 stage

preferentially

mDNA (Fig.lb).

logy

mRNA popula-

higher with

homo-

Vol. 86, No. 2, 1979

BIOCHEMICAL

veO-m

10

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

RNA

20

40

30

=I+-LABELLED

b

RNA(PI)

Fig. 1 Kinetics of hybridization of mRNA species with total and mDNA: Differen% quantities of Pulse labelled mRNA isolated from the cells at the vegetative phase and at the T2 phase were hybriIn the dized to mDNA,and total DNA(1 ug/assay). control assay DNA was omitted from the reaction mixture. a - T 2' * b - vegetative. 334

BIOCHEMICAL

Vol. 86, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table dpecificity

hybridization

of

i

of

T2

messengers

Total vegetative vegetative

RNA t unlabelled RNA

3kI vegetative

RNA + unlabelled

vegetative

3H T2 RNA + unlabelled

tiNA

T2 RNA

3h-adenosine labelled RNA (15 stages was hybridized to 5 ug were added at the beginning of values were subtracted in each

b - Not

compared

to

total

3560

700

3000

500

200

ug/assayj from vegetative and the T2 of totaL+mDNA. Cold KNIST$lSb;l~~;ssay) the chase experiment. e case.

cellular

of hybridization

wa.s further

riments

in which

the

by the

vegetative

considerable

firming

could the

vegetative most

gers

hybrids

mRNA (Table

earlier genes

out

reports during

the

high

well

known that

polyA

bind

to the (11).

could However,

blank

values

nitrocellulose

filters we decided

335

expe-

be competed

there

occured

total

DNA, most

RNA, thus

con-

transcription stages

experiments

RNA populations

fience,

with

extensive

sporulating

chase

not

by vegetative of

of our hybridization

is

1).

specificity

by the

of T 2-mFBAs

be chased

we had observed

concentrations

mDNA-T2

This

DNA.

confirmed

hybridization

of which

It

ND

detectaole

mDNA as

In

ND b

400

'I2 RiuA

with

(1,2).

and lb).

stretches at

of

T2 messen-

(Fig.la with

a

mDNA

2970

1‘ 2 RNA + unlabelled

a-

DNA

NB

'H T2 RNA 'ii

with

2200

RNA

3Ii vegetative

mDNA

CM4 of ;KNA hybridized --------_-------------------

RNA

'ii

the

with

higher

to look

of salt

into

the

Vol. 86, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Occurrence of the polyA the T2 stages of growth

II

stretches in the in g.polymyxa

RNA populations

Percentage of total _------_-------__--_-----------P0lyU filters

Y tage

Strain

Wild

type

Vegetative

01

(Hifr,

Sp00)

possibility

that

a polyA

of

Nil

Stationary

1.8

Nil

the

'H-adenosine.

tract

RNA populations

by polyU total

The data

presented

in

populations

alone That

revealed

oligo

its In that

this

sensitivity

filter

Table

cells

bind

the

presence

column.

indicated by the

that presence

manifestation

T2 RNA of was

isolated

to polyU

the

polyA

and frac-

dT cellulose

confirmation

T2 RNAs came from

at vege-

assays

of RNA populations

to either

on

of the

of RNA with

binding

2 clearly

Further in

in

and extent

RNA on oligo

inability

dT cellulose. tract

the

examined

with

RNA species

was a spore-specific

SpoO mutant

of polyA

the

were distinguished

by the

a Rif',

may be endowed

all

by growing

of the

polyA.

labelled with 'B-adenosine were polyU filters and their retention are expressed as the percentage the input RNA.

The presence

was analysed

tionation

12.0

1.2

and T2 stages

tative

4.0

Vegetative

We had labelled

tail.

Oligo dT columns

15.0

Total RNA isolated from cells for the extent of binding to oligo dT columns. The values total TCA-precipitable cpm of

filters of the

demonstration

from or

presence of

to T2 RNAse.

order

to understand

are

polyadenylated,

the

nature

of the

we had separated

336

species the

and

RNA bound

1.2

*2

RS

of the vegetative

of RNA 3H-adenosine

to

Vol. 86, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3 2

__

-m-v---*--

--_

_________

i r 0 5 i L 3 c ; L ?

v-------,

k

‘P

1 .

labelled Density gradient profiles of 3H adenosine total RNA from vegetative&T2 stages: Samples of labelled RNA (0.2 ml) were loaded on a 5 ml 5-15% (w/v) sucrose density gradient and centrifuged for 18 hrs at 90,000 g. The individual fractions were monitored for their total acid precipitable CPM ( +-- + ) and their polyU filter binding capacity a - vegetatives b - TIC2 (v--v ). labelled in

RNA species

a sucrose

checked

density

for

their

filter

binding

tracts

are

lating

stage

The results another

from

vegetative

gradient.

Individual

acid-precipitable capacity.

present

in

It 5-9s

counts is

2a and 2b).

of these

investigations feature

evident

RNA species

(Figs.

interesting

and sporulating

only

have

of specialised

337

that

stages

fractions

were

and their

polyU

the at

brought

polyA

the

sporu-

forth

transcriptional

yet

Vol. 86, No. 2, 1979

programming rence

BIOCHEMICAL

during

of

cells.

has been

reported

in

eucaryotic

lising

the

nucleus role

to

is

In view

of

The presence

messenger the

messengers

The role

unclear

pool

localising

the

here

(14).

for at the

enriched ease

of RNA,

as of studying

species

during

feature

occurof

spo-

of polyA

stretches

to be that

of stabi-

their

passage

from

Whether

or not

a similar

p01yA RNA species

the

in

sporu-

moment. homology of

of mDNA regions

separation

we now have

spore-specific

as well

the

the

RNA in procaryotes

speculated

RNAs enroute

cytoplasm

and the

total

of polyA

(12,13).

RNA has been

the

We report

RNA as a specific

can be attributed

lation

the

sporulation.

polyadenylated

rulating

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

regions turnover

of

with

polyA

RNA from

a powerful

tool

in

cellular

the

of the

T2

polyA

of DNA

RNA

sporulation.

ACKNO-VLEDGEMENT

The skilful technical assistance of Miss S.Padmavathy is most gratefully acknowledged. SK was a recipient of the Junior Fellowship from CSIR. This research was supported by the grant-in-aid from the Department of Science and Technology. dote added in proof: The presence g.brevis has been reported recently m.rlgley , I;: and iaulus $1. diochemistry.

of polyii R&L in (Sarkar,X., 17, 3468 (19783

REFERENCES 1

2

Sumida-Yasumoto,C. and R.H.Doi. 1974. 3. Bacterial. J-Q, 775 ;;;y"Jy ,C . , M.Manca de Nadra and J.Szulmajster. . Biochem. 629 53

338

1976.

Vol. 86, No. 2, 1979

3 4

5 6 7 8 9 10 11

12

13 14

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DiCioccio,R.A. and Strauss,N. 1973 J. Mol. Biol. 77, 325 Fukuda,R., G.Keilman, E.McVery and R.H.Doi. 1975. In: Spores VI (Ad. Gerdhart,I., R.B.Costilow and KL.Sadoffj. American Society for lY1icrobiology, Washington Linn,T.G., A.L.Greenleaf and R.Losick. 1975. J. Biol. Chem. 70, 490 R.Balakrishnan and K.Jayaraman. 1978. Satwant Kaur., Biochem. Biophys. Res. Commun. 8l, 50 Schaeffer,E. 1967. Polia Microbial. 12, 291 Nygaard,A.P. and B.D.Hall. 1964. J. Mol. Biol. 9, 125 C.Jurale and J.Kates. Sheldon,R., 1972. Proc. Natl. Aca.d. Sci. USA. @, 417 Cabada,M.O., C.Darnborough., P.J.Ford and P.C.Turner. 1977. Develop. Biol. 22, 427 Cavalieri,R.L., E.A.Havell., J.Vilcek and S.Pestka. 1977. Proc. Natl. Acad. Sci. USA 74, 4415 -and M.Bdmunds. 1975. Na.kazato,H. , S.Venkatesan Nature. 256, 144 M.Sanders and A.Newton. 1975. Ohta,N., Proc. Natl. Acad. Sci. USA. 72, 2343 Brawerman,G., 1976. In: Progress in Nucleic Acid Research and Molecular (tid. Waldo.E.Cohn) Academic Press, Eology . Vol.17 New York.

339