Vol. 69, No. I, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

PHYSICAL

MAPPING

Saccharomyces

IN K.

S.

OF MITOCHONDRIA

Sriprakash,

K.

and

A.

B. W.

rRNA

GENES

cerevisiae

Choo,

P.

Nagley

Linnane

Department of Biochemistry, Monash University, CLAYTON, Victoria. 3168 Australia

Received

December 29,

1975

SHMMARY

We chondrial

have established previously a physical map of mitogenetic markers for antibiotic-resistance i n Saccharomyces cerevisiae (i) . Using physically and genetically characterised cytoplasmic petite mutants we now physically map the mitochondrial ribosomal RNA (mt-rRNA) genes on the circular grande mitochondrial genome defined to c o n t a i n i00 units of mtDNA° The gene for 15S-rRNA maps within 13 D N A - u n i t s near t h e parl l o c u s a n d t h e g e n e f o r 2 1 S - r R N A lies within 15 D N A - u n i t s between capl a n d olil c l o s e t o the eryl l o c u s . The minimum distance between t h e t w o r R N A g e n e s is a t l e a s t 27 u n i t s .

INTRODUCTION

Molloy resistance

et

markers

mutants. map

concluded after for

genome in

by

by

two

rRNAs

position markers

the using

characterised the

fraction

the

petite

correlation

five

et

al. the

for

(i) . mt-rRNAs

a quarter

for

a set

of well

both

the

the

the

grande

Copyrtght©1976byAcademicPress, lnc. Allrightso]reproductioninany/ormreserved.

two

In

the

rRNAs

defined

85

and

of

genome

present

the

physical

et

was al.

the

apart

the

genes

in we

other

which

markers

as w e l l

(3)

obtained

communication, to

of

deletion

markers

that

strains

retain

mtDNA

fragments

mtDNA

relative

they

are

of

petite

a circular

Sanders

antibiotic-resistance mtDNA

on

antibiotic-

co-retention

same

to m t D N A

are

least

map the

Recently

treatment

at

of

which

this

endonuclease

genes

of

mutants

between

carlsbergensis.

for

mapped

frequency

hybridization

hybridizing

Saccharomyces

genetically

scoring

DNA-DNA

restriction

the

by

Sriprakash

by

have

cytoplasmic

A good

obtained

observed

(2)

(capl, eryl, olil, mikl, parl)

markers

mitochondrial the

al.

as

were and for the

Vol. 69, No. 1, 1976

amount The

of

sequence

technique

for m a p p i n g

MATERIALS

overlaps

described

other

AND

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

between this

the

paper

various

can

be

petite

used

as

mtDNAs

(i) .

a general

one

genes.

METHODS

Strains: A l l t h e Saccharomyces cerevisiae c y t o p l a s m i c p e t i t e m u t a n t s t r a i n s e m p l o y e d in the p r e s e n t s t u d y e x c e p t for s t r a i n 9 8 7 ~ 1 9 w e r e s p o n t a n e o u s i s o l a t e s and w e r e d e s c r i b e d e a r l i e r (i). The p e t i t e 9 8 7 - 1 9 w a s d e r i v e d a f t e r a b r i e f e t h i d i u m b r o m i d e t r e a t m e n t of a g r a n d e s t r a i n c a r r y i n g t h e r e s i s t a n c e m a r k e r s capl eryl olil mikl and parl. the r e t e n t i o n a n d loss of t h e m i t o c h o n d r i a l m a r k e r s in the p e t i t e s t r a i n s u s e d are s h o w n in T a b l e i. T h e g e n e t i c s t a b i l i t y of the p e t i t e s (i.e. the p e r c e n t of s u b c l o n e s t h a t r e t a i n e d all the m i t o c h o n d r i a l m a r k e r s p r e s e n t in the p a r e n t p e t i t e s ) w a s g r e a t e r t h a n 80% (cf. S r i p r a k a s h et al. (i) ) e x c e p t for Y8 a n d 9 8 7 - 1 9 w h i c h s h o w e d a g e n e t i c s t a b i l i t y of 15% a n d 70% r e s p e c t i v e l y . Y8 and o t h e r s p o n t a n e o u s l y a r i s i n g p e t i t e s w h i c h r e t a i n t h e eryl, capl, parl l o c i are i n t r i n s i c a l l y unstable; e v e n on r e p e a t e d s u b c l o n i n g no h i g h l y s t a b l e p e t i t e s c o n t a i n i n g eryl, capl, parl w e r e o b t a i n e d . E x t r a c t i o n and p u r i f i c a t i o n of n u c l e i c a c i d s : DNA from whole c e l l s w a s e x t r a c t e d as d e s c r i b e d e a r l i e r (i) . The rRNAs were p r e p a r e d f r o m the m i t o c h o n d r i a l r i b o s o m e s i s o l a t e d f r o m the g r a n d e s t r a i n s (4) a n d p u r i f i e d a c c o r d i n g to F o r r e s t e r et al. (5). L a b e l l i n g the r R N A w i t h 125I: T h e p r e p a r a t i o n of [125I] r R N A w a s s i m i l a r to t h a t d e s c r i b e d by G e t z et al. (6) w i t h s e v e r a l modifications. T h e r e a c t i o n m i x t u r e (0.2 ml) c o n t a i n e d 0 . 1 M s o d i u m a c e t a t e (pH 4.8); 25 ~M KI; 500 ~Ci 125I (The R a d i o c h e m i c a l C e n t r e Ltd., A m e r s h a m ) ; 4 0 - 5 0 ~g r R N A a n d 2 m M t h a l l i u m c h l o r i d e (K a n d K L a b o r a t o r i e s Inc., P l a i n v i e w , N . Y . ) . B o t h KI and thallium chloride solutions were made freshly, and used within a minute. T h e r e a c t i o n m i x t u r e w a s a s s e m b l e d in the cold; RNA a n d t h a l l i u m c h l o r i d e w e r e a d d e d l a s t a n d the m i x t u r e w a s i m m e d i a t e l y h e a t e d to 60 ° a n d h e l d at t h a t t e m p e r a t u r e for i0 min. The r e a c t i o n m i x t u r e w a s c h i l l e d , T r i s - H C 1 (pH 9.0) w a s a d d e d to a f i n a l c o n c e n t r a t i o n of 0 . 4 M a n d the s o l u t i o n was r e h e a t e d to 60 ° f o r i0 m i n , r e c o o l e d a n d t h e n m i x e d w i t h 150 ~g of w h o l e r a t l i v e r RNA. T h e l a b e l l e d m t - r R N A w a s p u r i f i e d by p a s s i n g the w h o l e m i x t u r e t h r o u g h a S e p h a d e x G25 ( P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , S w e d e n ) c o l u m n (8 x 200 mm) s u p p o r t e d on a p a d of l0 g l a s s f i l t e r s (GF/C, W h a t m a n ) . The Sephadex was presaturated by r u n n i n g a b o u t 150 ~g of w h o l e r a t l i v e r t h r o u g h the c o l u m n . The g l a s s f i l t e r s r e m o v e c o n t a m i n a t i n g m a t e r i a l s w h i c h are l i k e l y to g i v e r i s e to h i g h b a c k g r o u n d c o u n t s in the h y b r i d i z a t ion experiments (7). A p p r o x i m a t e l y 6% of the r a d i o a c t i v i t y in the r e a c t i o n m i x t u r e w a s r e c o v e r e d in the c o l u m n ' s e x c l u d e d volume which contained ~125I]rRNA. The [ 1 2 5 I ] r R N A w a s f u r t h e r p u r i f i e d b y a d s o r p t i o n to a c o l u m n (1.5 ml) of h y d r o x y l a p a t i t e (Bio--Rad. L a b o r a t o r i e s , C a l . ) . T h e c o l u m n w a s e l u t e d w i t h 9 ml of i0 m M p o t a s s i u m p h o s p h a t e (pH 6.8) f o l l o w e d by 4.5 ml of 250 m M p o t a s s i u m p h o s p h a t e (pH 6.8). The l a b e l l e d r R N A w h i c h phosphate solution was treated

e l u t e s in t h e w i t h 50 ~ g / m l

86

250 m M p o t a s s i u m of R N a s e - f r e e P r o n a s e

Vol. 69, No. 1, 1976

TABLE

i:

BIOCHEMICA L AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

Hybridization different

Strain

of

15S-

genetically

Mi~o~hon~ria~l

capleryl

and

21S-rRNA

marked

" ~arkers

olil

to

mtDNA

Re lativekybridizatio

mikl

parl

of

strains.

15S~rRNA

n 21S-rRNA

Grande 770-7B

+

+

+

+

+

i00

i00

863-2C

+

+

+

+

+

i00

i00

U3

-

-

-

+

-

0

U4

+

+

+

+

-

0

i00

U5.1

+

+

+

-

-

0

85

U5.2

+

+

-

-

-

6

94

U5.3

-

+

+

-

-

l0

170

U6

+

+

-

-

-

0

124

YI.2

-

-

+

+

-

0

YI.3

-

-.

+

+

+

193

30

Y1.4

-

-

-

+

+

220

18

Y2

-

-

+

+

-

13

8

Y6

.

+

243

22

Y9

-

-

-

+

-

Y8

+

+

-

-

+

346

98

987-19

+

+

-

-

+

430

170

Petite

.

.

.

.

The mitochondrial described by Molloy et al. locus; ~ denotes loss of procedure was as described 987-.19 in which the amount respectively.

0

0

4

18

genetic markers were determined (2). + denotes retention of the the locus. The hybridization in the Methods except for Y8 and of mtDNA was 1 ~g and 0.7 ~g

as

In the controls in which no DNA was added 0.2% of the input counts was retained on the filter, while the controls with 2 pg of DNA from a rho-o s t r a i n which lacks mtDNA bound 1% of the input counts. The hybridization values are expressed as percentages relative to the counts hybridized to the grande mtDNA less the correction for the counts bound in the rho-o controls. The mtDNA from the grandes hybridized about 300 and 500 cpm above the controls for 15Sand 21S-rRNA respectively.

87

Vol. 69, No. 1, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

(Pronase was preincubated at 500 ~g/ml for 1 h r a t 37 ° ) f o r 30 min at room temperature. The solution was deproteinised by extracting t h r i c e w i t h an e q u a l v o l u m e o f a m i x t u r e of 1 volume of phenol (presaturated i n 2 x SSC) a n d 1 v o l u m e o f c h l o r o f o r m isoamylalcohol ( 9 9 : 1 , v/v) . The rRNA was precipitated from the aqueous phase with absolute ethanol in the presence o f 2% p o t a s s i u m acetate as d e s c r i b e d earlier (6); the precipitated [125I]rRNA was finally dissolved in 2 x SSC. RNA-DNA hybridization: The hybridization mixture (0.2 ml) c o n ~ained 0 . 2 " 0 . 2 5 ~g, u n l e s s o t h e r w i s e stated, of denatured mtDNA and 0.i -0.2 ~ g of [ 1 2 5 I ] r R N A in 30% formamide and 2 x SSC. After incubation f o r 2 h r at 37 ° a n d s u b s e q u e n t digestion with RNase for 1 hr at room temperature the RNA-DNA hybrids were collected on nitrocellulose filters as d e s c r i b e d b y N a g l e y e t al. (4). The radioactivity was measured in a Philips Automatic Gamma Analyser. The input radioactivity in hybridization mixtures w a s 104 c p m f o r 2 1 S - [ 1 2 5 I ] r R N A and was 2 x l04 cpm for 15S-[125I] rRNA. RESULTS Table various

DNA

1 summarises preparations

preparations activity

bound

to

absence

of

2 ~g

It was

excess

of

mtDNA

DNA

from

also

affected

not

two

grande

strains

were

not

extent

mitochondrial

(data

the

not

in

These

the

the

amount

between

the

the

cytoplasmic of

a ten-fold RNA

The

whole-cell

the

rRNA

to

observations

interaction

by

presence

to b o t h

RNA

radio-

indicating

rRNA

shown).

of

The input

of mitochondrial

hybridized to

the

1 legend)

hybridized counts

shown).

of

the

even

hybridization

21S-rRNA.

1%

(Table

proportional

a specific

and of

hybridization

strains

the

DNA

that

the

Moreover,

(data was

observed

was

grande ~g

of

of

15S-rRNA

the

nuclear

DNA

i) .

there

to

nuclear

(Table

0.5

of

results

with

only

contamination

RNA.

the

the

the

species

DNA

from

of mtDNA

up

established mtDNA

and

to

the

to that

the

[1251]rRNAs. A set were the is

selected rRNAs

those

petites

is

that

observations observed the

eryl

in

the

the

that close

and

to

have

of

those

results

to

eryl

retained

the

(4,

8,

9).

Some

YI.3,

YI.4,

locus.

However,

the The

these

mtDNAs

locus

and

locus

and

confirming

Y9

which

do do

to

l) .

only

to

thus

the

hybridized

to

all

previous

hybridization

observations

88

(Table

hybridized

parl

petites

to h y b r i d i z e

mtDNAs

21S-rRNA

limited Y6

characterised

grande

15S-rRNA

retained

parl.

their

of

that

had

strains

physically

abilities

compared

from

petites gene

genetically

and

were

evident

rRNA

of

was not

not

also

contain vitiate

It

15Sthe

VoI. 69, No. 1, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

15S-rRNA

21S-rRNA

Grande 0t Genome par1

20

410

810

6i0

1~10

mik"lo~il eryl cap1 L

R

U4

Y1-3 ................

Y1.2 U5"1 - U E~3---

U 6 - Figure

l:

Mapping

of

15S

and

21S m i t o c h o n d r i a l

rRNA

genes.

A l i n e a r r e p r e s e n t a t i o n of t h e c i r c u l a r m i t o c h o n d r i a l g e n o m e of S. cerevisiae s h o w i n g the e s t a b l i s h e d r e l a t i v e p o s i t i o n s of the f i v e a n t i b i o t i c - r e s i s t a n c e m a r k e r s (1) a n d the m a p p o s i t i o n s of 1 5 S - and 2 1 S - r R N A g e n e s . T h e f u l l l i n e s a n d the d o t t e d l i n e s r e p r e s e n t r e s p e c t i v e l y , the h o m o l o g o u s a n d n o n h o m o l o g o u s s e q u e n c e s to the U4 m i t o c h o n d r i a l g e n o m e . L and R c o r r e s p o n d to l e f t and r i g h t e n d s of U4 g e n o m e a r b i t r a r i l y d e f i n e d for convenience. T h e l e n g t h s of the g e n o m e s are d r a w n to s c a l e . G r a n d e m t D N A c o n t a i n s 100 u n i t s b y d e f i n i t i o n (i) .

the

results

segment

of

account

for As

as the

the

grande

36

four

units

of

The

mtDNA

the

left

the

end

grande

mtDNA

and YI.2

to Y I . 3 ,

it d i d

not

hybridize

15S~rRNA

is w i t h i n in

the

obtained

The rRNA

with

the YI.3

in the this

study

al.

and

was

markers chosen

the

i) ;

to Y I . 2 . near

but

not the

the DNA

1 depicts of over-

U4 w h i c h

and

corresponds

our

reference

petite.

40

units

while

mapped

in t h e other

could

strains.

sequence

and

27

15S-rRNA

Therefore,

the

a

fraction

petite

respectively

(Fig.

of

as

these

(i) ~ F i g u r e

The

of

ergl l o c u s in

the p e t i t e s ,

retain

13 u n i t s genome

et

retention

parl

the

to

to

hybridized gene

locus

YI.2

genome.

petite

genomes

for

which The are

mapping.

occurrence genes

genome

observed

genomes.

extend

U4

small

containing

in

they

petite

the

consistent

the

retained

of

results

not

antibiotic-resistance

of Y I . 3

is p r e s e n t

the

Sriprakash

sequences

particular

possibilities,

hybridization

by

markers

mtDNA

between

gene

limited

established

antibiotic

retains

several

21S-rRNA

the

the

laps

among

of p e t i t e s

together

in

which

retain

the m i t o c h o n d r i a l

89

both

the

genomes

large is

and

rare;

Vol. 69, No. 1, 1 9 7 6

to d a t e have

there

tested

is

and

as w o u l d ~Table

eryl,

capl

one

ability

i).

petite

of m t D N A

predicted However,

and parl

and M e t h o d s ) .

On

after

s~ch

strain

from

loci

the

mild

are

other

ethidium

to

genetically

hand,

the

a regions

reponsible

spontaneous

petites

retaining

the

987-19.

In

the

strains,

we

the

hybridization

order

used

to

large

mixtures

same

both

for

the

which

deleted

for

the

low

of m t D N A

(Table

1 legend).

was

instability

was

compensate

the retain

a moderate

loci

amounts

the

(see M a t e r i a l s

987-19, has

We

species,

which

unstable

strain

perhaps

rRNA

petites

treatment,

stability;

strain

two

(i0) .

retain

retained

spontaneous

bromide

which

the

thesepetites

the

reported

petites

parl l o c i to h y b r i d i z e

n o w be

genes

derived

only

the

eryl, capl a n d

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

from

in

of

the

stability

these

the

of

petites

in

DISCUSSION The

In

genes

for

contrast,

we

genome

the

is

two

rRNAs

separation

of

bergensis

(3)°

the m o d e

of We

15S~rRNA the

the

lincomycin carrying these

the

three

ied.

altered We

observed

pose

from

within

some

our

The

of

physical between

recently

that

which

of

several

the

is y e t

also

an o p e n

cross

The

resistant

ribosomes

antibiotics due

to

ribosomal

are p u r s u i n g

(14). a change

component the

question. to

isolated

also

been

Although

Mutants

spiramycin, from shown the

is

90

consist-

(4,

8,

and

that

9). 15S-rRNA

carry

carbomycin

resistant

resistance

to be

effect(S)

which

to be

is

ribosomes.

for

strains

mitochondrial

on

and

to

to

ribosome

definitively

of parl m u t a t i o n

on

parl l o c u s

the

grande

in the yet

carls-

gene

gene

laboratories

for

of

rRNAs.

15 u n i t s

21S-rRNA

genes

questions

include

within

of y e a s t

extent

in S.

intriguing

(11,12).

map

the

A similar

studies

13 u n i t s

mapping

other

of m i t o c h o n d r i a l

encompassed

findings

the

distance

synthesis

eryl-r l o c u s h a v e

is

and

mitoehondrial

to e a c h

eryl and parl l o c i are p a r t of 2 1 S - r R N A

(13).

erythromycin the

are

was

the

is

work

in the

close

cerevisiae.

findings of

rRNAs are

minimum

genes

21S-rRNA

respectively

eryl-r l o c u s

this

the

conclude

earlier

small cells

in S.

eryl l o c u s .

the

Whether genes

These

encompassed for

from

that

two

and

HeLa

units

these

further

gene

include

27

regulation

is

ent with

and

estimate

mitochondrial

and

large

of Neurospora

genomes

(15),

identifthe

Vol. 69, No. 1, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

REFERENCES i.

S r i p r a k a s h t K.S., Molloy, P.L., N a g l e y , P., L u k i n s , H.B. and L i n n a n e F A.W. (1976) M a n u s c r i p t s u b m i t t e d for p u b l i c a t i o n .

2,

Molloy, P.L., 122., 7-18.

3.

Sanders, Biophys.

4.

Nagley, P. , Molloy, P.L., Lukins, H.B. and Linnane~ Biochem. Biophys. Res. Commun. 57, 232-239.

5.

F o r r e s t e r , I.T., Nagley, P. FEBS L e t t e r s ii, 59-61.

6.

Getz, M.J., A l t e n b e r g , L.C. and S a u n d e r s , Biochem. Biophys. A c t a 287, 485-595.

7.

Scherberg, 2143-2150.

8.

Faye, G., Fukuhara, H., G r a n d c h a m p , C., Lazowska, J., Michel, F., Casey, J., Getz, G.S., Locker, J., R a b i n o w i t z , M . , B o l o t i n F u k u h a r a , M., Coen, D., Deutsch, J., Dujon, B., Netter, P. and S l o n i m s k i , P.P. (1973) Biochemie 55, 779-792.

9,

Faye, G., Kujawa, 88, 185-203.

Linnane,

A.W.

and Lukins,

J.P.M., Heyting, C. and Borst, Res. Commun. 65, 699-707.

N.H.

H.B. P.

and Linnane,

and Refetoff,

S.

(1974)

C. and F u k u h a r a ,

H.

(1975)

(1975)

A.W. G.F.

Biochem. A.W.

(1974)

(1970) (1972)

J. Biol.

(1974)

J. Bact.

Chem.

J. Mol.

249,

Biol.

i0.

Nagley, P., Molloy, P.L., Lukins, H.B. and Linnane, A.W. (1975) In "The E u k a r y o t e C h r o m o s o m e " (W.J. P e a c o c k and R.D. Brock, eds.) pp 155-167, A.N.U. Press, Canberra.

ii.

Kuriyama,

12.

Robberson, D., Aloni, Y., Attardi, J. Mol. Biol. 64, 313-317.

13.

Trembath, M.K., Bunn, C.L., Lukins, H.B. (1973) Mol. Gen. Genet. 121, 35-48.

14.

S p i t h i l l , T.W. and Maxwell, R.J. (1975) Proc. Soc. 8, 67. Also p e r s o n a l c o m m u n i c a t i o n .

15.

Grivell, L.A., Reijnders, L. FEBS L e t t e r s 16, 159-163.

Y.

and Luck,

D.J.L.

(1973)

J.Mol. Biol. 73,

G. and Davidson,

and de Vries,

91

N.

and Linnane,

H.

Aust. (1971)

425--437. (1972) A.W. Biochem.