Physiological variations in satellite components of yeast DNA detected by density gradient centrifugation

BIOCHEMICAL

Vol. 23, No. 1, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PHYSIOLOGICAL VARIATIONS IN SATELLITE COMPONENTS OF YEAST DNA DETECTED BY DENSITY GRADIENT CENTRIFUGATION" Ethel Department

Received

of Genetics,

February

In order both

to study

lysates

satellites

corresponded

to the

ined.

The second

that

Diploid (Ephrussi

Sacch.

cerevisiae

described from

component

low

Prof.

had a higher "petite"

the

Preparation

mutant

-of Protoplasts. YEPD (1% Bacto

about 5~10~ were usually using

cytoplasmic

cytoplasmic

inoculate

a segregational

mannitol

cells/ml isolated

cerevisiae

genetic

determinant was obtained

mutants

density

than

exam-

the

main

and an acriflavine-

mutant

74A; haploid

mutant

N123P4

DPl-1C "p"

(kindly

mutant

(Sherman,

1964).

Routinely,

stationary

phase

extract,

2% Eacto

peptone,

shaken

cells

pressure,

and sorbose

used to with

phase

of cultures

osmotic

requirement.

* This work was supported by Public Health Grant No. Al-00328 1 On leave from Laboratoire Pasteur, Institut du Radium, Paris, 2 On leave from the John Innes Institute, Hertford, England.

56

were

good yields

from the cytoplasmic mutants and from stationary 74 were obtainable only by using a higher stabilizing this

A derived

Protoplasts phase cultures

protoplasts of strain

satisfied

which

2% Glucose)

at 30°C. However,

by

treatment.

1959) from log

stabilizer.

(Moustacchi

provided

deficient by acriflavine

, and the cultures were (Eddy and Williamson,

(25%,w/v)

DNA (Tewari

"petite"

(NCYC 74)

respiratory

(15%,w/v) as osmotic

One of these

AND METHODS

DPl-1CA yeast

growth.

of

accompanied

mutants.

carlsbergensis

Sacch.

strains

was usually

bouyant

centri-

a relatively

mitochondrial

"petite" -et al -* 5 1949) cytoplasmic N123 and a derived radio-resistant

P. Slonimski),

carries "p-"

Saccharomyces

to several

cytoplasmic

DNA during

gradient

DNA in

during

density

the two

in the

1962); haploid

and Marcovich,

Wash.

of yeast

density

component

in amount

MATERIALS Strains.

properties

approach

main nuclear

varied

recently

minor

molecular

of this

the

which

DNA and was present

induced

Seattle,

as a means of isolating

1965) and was absent

nuclear

of Washington,

we have explored

Application

revealed

by two minor et al.,

in the

and meiosis,

condition.

Saccharomyces

University

changes

growth

of protoplast

undegraded

'1 and D. H. Williamson'

28, 1966

vegetative

fugation

Moustacchi

France.

Vol. 23, No. 1, 1966

I:olation -___ccmntaining 0.005M ir

BIOCHEMICAL

and Analysis

washing, l-4 x109 protoplasts, --of DNA. After thorough ug DNA, were suspended in chilled saline-EDTA (0.2M NaCl,

50-100

EDTA, pH 8.0)

a few seconds.

containing

Duponol

(ea.

CsCl was then

added

to give

and preparative centrifuge tjons

for

UV-absorption

a suitable

1956)

(1945)

dilution

of the

centrifugation

H<:arst,

on extracts

Densitometric

wctre used to estimate and bouyant

the

relative

estimated

of the

using

Schmidt pooled

by CsCl density

the

and gra-

(Vinograd

of the

balculated

ex-

using

DNA were

W-absorption

L

5 frac-

were

by a modified

analysed

model

and about

E ultracentrifuge

proportions

were

a Spinco

fractions

containing

mixture

tracings

densities

of the

prepared

model

using

DNA contents

Fractions

resulting

Lysis was complete density of 1.690

was punctured

Dilutions

in a "Spinco"

1962).

nfints,

The tube

technique.

w/v).

a calculated

at 26Omv, and their

(Burton,

artd Thannhauser

0.5%, performed

-et --al 7 1957). ml of suspension.

per

d:phenylamine

d:ent

ulti-acentrifugation

(Meselson

collected

arlined

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

photographs

different

DNA compo-

position

of Pseudo-

monas aeruginosa DNA (density 1.727g/cm3; kindly supplied by Dr. B. McCarthy) -a:; reference (Schildkraut al 2 1962). The effect of heat on bouyant -et _-* dl?nsities was investigated by heating diluted fractions for 10 min at l)O°C. RESULTS 1).

DNJ Components.

w'z have ald wlich (0

observed

presumed

are shown increased

had a bouyant

density

from

density

on heating (5)

constituent

It

2). s?okn rich

that

During

phase

similar

(known

clearly

Cytological

varies

(content

with

‘IEPD containing

the

c(,

of l.699g/cm3,

and minor

component

It

additional samples

low density

grown

culture;

stationary

mitochondrial

studies

aerobically it

phase

comon heat-

have on media

is minimal

during

(Ephrussi

-et -*al 3 to determine

seemed of interest

therefore

in the relative

proportion

of the

DNA.

Moreover, of the

occurred

such a study

the

increased

et al. (1965). -and enzymatic

of yeast

A third

DNA.

which

to the

age of the

during

1962).

nLedium and adequate

corresponded by Tewari

fluctuations

that

of 1.683g/cm3,

Growth.

to be mitochondrial)

kas hoped

components

designated

density

A second

described

and maximal

1~156; Yotsuyanagi, if

component,

had a bouyant

seems to be a native

density

the mitochondrial

in glucose

the log

three

0. material,

and therefore

recently

Variation

the

The major

1.

origin,

by 0.015g/cm3.

had a bouyant

by 0.015g/cm3.

depicting

It has not so far been obtained of IL.706g/cm3. but has been observed to increase in

contaminating

p,nent,

tracings

in Fig.

to be of nuclear

on heating

free

iig

Densitometer

component might glucose during

of the

throw

light

(5.4%, the

w/v,

early

57

cell's

on the nature total) growth

B it y DNA.

was used as growth stages

were

obtained

Vol. 23, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

(0) A

p -A---u PS

Y

L

a

05 L

(cl

35 L Figure

1.

of yeast

Microdensitometer

(Pseudomonas, 1.699

g/cm3,

24 hrs

culture.

plasmic

petite

by using that fell

tracings

The peak on the

DNA.

density 1.683

1.727 g/cm3

(b)

density

gradient

in each tracing

g/cm?).

Strain

preparations

is the

reference

DNA

"cx'l, Vati and "ylt had densities

ma 1.706 g/cm3 respectively. 74, 6 hrs

culture.

(a)

(c)

Strain

of

Strain

74,

74A,

a cyto-

mutant.

a heavy

inoculum

(0.6x

10'

cells/ml).

From Fig.

2 it

is

seen

during the first 8 hrs of growth, the proportion of the 6 component to about 3% of the total DNA. Later on, it increased, reaching about

20% of the total DNA in the varied in a similar fashion, than

from

right

1% of the total

stationary the

DNA, while

much as 11% of this

minimal the

about

also

for

true

the the

level

stationary

(24 hrs).

The y component

observed phase

(8 hrs)

being

DNA contained

less

as

component.

Comparison of normal strains 3). -___-Each of the three DNA components N123P4 in

phase

same proportions

haploid

strain

and respiratory occurred

in the

as in the DPI-IC,

58

deficient haploid

diploid

a segregational

mutants. strains

strain

N123 and 74.

respiratory

This de-

was

Vol. 23, No. 1, 1966

BIOCHEMICAL

SATELLITE TOTAL

20

0

AND BIOPHYSICAL

DNA DNA

RESEARCH COMMUNICATIONS

o,.

468

24

HOURS

Figure

2.

Changes curve

determined (a),

and course

been

of satellite from

ug x 10m6.

data

in

DNA during

growth.

counts.

in

(a)

The course

strain

Total

assuming

74. (b)

(a)

Growth

6 DNA per ml of

a total

DNA content/

of synthesis

between

8 and 24 hrs

the

cytoplasmic

studied.

mutant

contrast,

of DNA synthesis

by haemocytometer

calculated

of 0.045

has not ficient

curve

in the proportion (0)

culture cell

Growth

which

the two

20 detectable

cytoplasmic

amounts

as much of the

nevertheless

Wpetite"

of the

y component

carries

mutants

B component,

as their

"p"

DPl-1CA

though

parental

In

and 74A contained

they

strains

factor.

both

carried

(Fig.

about

1).

DISCUSSION The DNA of many types nay comprise

of cell

contains

up to 10% of thle cell's

total

minor

density

and the

occurrence

universal rule

(Nass -et -*al 3 1965). was established by Tewsri

organelles

a discrete

native

of DNA in

which

DNA (Chun and Littlefield,

Shun -et -*al 3 1963; Leff et al., 1963; Luck and Reich, In many cases these are located in sytoplasmic 1965). mitochondria,

components

the latter

The conformity

of yeast

1964;

organelles bodies

1963;

Suyama and Preer, such as

seems to be

mitochondria

to this

et al., (1965), who isolated from these -DNA having a bouyant density of 1.685g/cm3.

59

Vol. 23, No. 1, 1966

This

BIOCHEMICAL

material

strain

corresponds

74 in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to our

stationary

phase

low

density

contains

B component.

about

0.045

Knowing

~10~~

that

ng DNA per

cell

(cf.Ogur

al 3 1952), and assuming about 50 mitochondria/cell (Avers -et _* our analysis of the stationary phase cells indicates an -et -*al y 1965)) -10 average B DNA content of about 1.6 xl0 ng (9.6 x 107 daltons) per mitochondrion. This figure is in good agreement with the estimate of Tewari which was based on a different experimental approach. The --et a1.(1965) function of this DNA is not certain. However, the accumulated evidence concerning

cytoplasmic

(Ephrussi,

1953;

cytoplasmic whose

genetic

behavior

could

Our observation

that

mutants,

the

the

which

1953),is

amount

teins

the

of

1962;

not the

synthesized

were

synthesizing

specific

rate,

increase

consequence happens count

that

mitochondrial

consistent

with

R DNA.

clear,

it

cells

that

to that

of the

early

in

stages

a mean doubling of synthesis

respiratory

during

of growth time

total

the

and our that this

B DNA was 3 hrs,

i.e.

If the

B

this

period

of oxygen than

From

DNA.

during

rate

was

growth.

of about

of nuclear

but more slowly

the possibility

to

of this

B component

amount

enzymes

culture's

enzymes,

mutants

its

observations DNA is the

at a

consumption

mass,

with

the

precisely

what

which would accontrols synthesis

therefore cytoplasmic

are entirely genetic

"p".

A further of

function variation

Q. would decrease. This in fact is et al2 The simplest hypothesis 1956). - _- ) findings would be that the B DNA directly

these

segregational

mitochondrion.

the

of certain determinant

in the

functional

with

the

Certainly 100 pro-

at least

of cytoplasmic

rate

exponentially,

(Ephrussi for

of the

detection

T 1965) for a fully

the

and enzymes

possibility.

-et --al ) 1956; shows that the presence

of its

during

cytoplasmic

(Ephrussi

a controlling

exponentially

component should

that

its

constituents.

the

code for

ability

1964)]

cytochromes

this

could

structures

for

one third

constant

certain with

and the

observation

appears

at about

from

-et --al requirement

support by our

2 it

was absent

consistent

of a

in mitochondria

G component

Avers

sole

presence

mitochondrial

Nevertheless,

Yotsuyanagi,

Fig.

by the

(Sherman,

of certain

mutant,

mitochondrial

Further

to as "p"

are known to lack

size.

deficient

provided

for

in yeast

in terms

synthesis

clearly

form partial DNA is

[referred

fi DNA in the mitochondrion

of average

respiratory

function

h as been interpreted

be accounted

directs

"petite"

of mitochondrial

1953)

determinant

of a DNA which

(Slonimski,

inheritance

Slonimski,

feature Although

is

of our observations the

evident in the

of the

exact

from final

nuclear

its

stages DNA must

course enhanced

the

increased.

60

the

synthesis

proportion

of growth, have

concerns

of its

control after

in the rate

stationary

of its Thus,

of synthesis 8 hrs

synthesis

during

this

is

not phase relative period

Vol. 23, No. 1, 1966

as well

BIOCHEMICAL

as during

the

of the mitochondrial pendent explained

therefore

respiration

rate

observed

than

that

its

cally

amount

variable

sidered

it

is

component

as a possible

such

not

of enzyme synthesis. Its

varied

nucleolus

indebted

to Dr.

during

to Dr.

B. D. Hall

and M. Steinhardt

for

During

final

of this

(McLeish,

preparation

their

help

in the

cell.

the

in the

cyto-

The fact growth of the

and a metaboli-

1964) might

be con-

about

the possible

nuclear

his

hospitality

and

and advice.

manuscript,

the presence of the y component -151:687, 1966) reported preparations and its absence from isolated mitochondria, our views

unlikely.

constituent,

B. L. Roman for

g&.

supporting

presence

location

a chromosomal

as the

of the mitochondrial

candidate. We are

the

clear.

inde-

depression

(1956) may be

repression

not

synthesis

seemed to be under

--et al. of synthesis

to the CYcomponent

that

Acknowledgements.

is

the

glucose-induced

by Ephrussi

makes a mitochondrial

relative

suggested

growth,

the

of direct

y component

mutants

that

inhibition

as a consequence

"petite"

culture

of a specific of the

culture's

DNA components

appears

The function plasmic

of the

It

in terms

DNA, rather

stages

and nuclear

control.

of relative

early

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

location

Corneo

et -- al.(Science in whole yeast DNA thus

indirectly

of this

material

REFERENCES

Avers, C., Pfeffer, C., Rancmourt, M., J. Bact. 90, 481 (1965) Burton, K., Biochem. J., 62, 315 (1956) Chun, E., Vaughan, M., Rich, A., J. Mol. Biol. 1, 130 (1963) Chun, E., Littlefield, J., J. Mol. Biol. 1, 245 (1963) Eddy, A.A., and Williamson, D. H., Nature, l&, 1101 (1959) Ephrussi, B., Hottinguer, H., Chimenes, A. N., Ann. Inst. Past. 76, 351 (1949) Ephrussi, B., Nucleo-cytopla,smic relations in microorganisms. Clarendon Press Oxford, England. (1953) Ephrussi, B., Slonimski, P., Yotsuyanagi, Y., Tavlitski, J., Compt. Rend. Lab. Carlsberg 26, 87 (1956) M., Epstein, H. T., Schiff, J. A., Bioch. Bioph. Res. Comm. Leff, J., Mandel, 12, 126 (1963) Luck, D. J., and Reich, E., .Proc. Natl. Acad. SC. U.S. z, 931 (1964) McLeish, J., Nature, 2& 36 (1964) Meselson, M., Stahl, F. W., Vinograd, J., Proc. Natl. Acad. SC. U.S., 43,

581 (1957)

floustacchi, E., and Marcovich, H., Ann. Inst. Past. z, 841 (1962) Nass, M. M., Nass, S., Afzelius, B., Exptl. Cell. Res. 37, 516 (1965) 3gur, M., Minckler, S., Lindegren, G., Lindegren, C. C., Arch. Bioch. Biophys., 40, 175 (1952) jchildkraut, C. L., Marmur-, J., Doty, P., J. Mol. Biol. 2, 430 (1962) Schmidt, G., and Thannhauser, S. J., J. Biol. Chem., 161, 83 (1945) Sherman, F., Genetics, 49, 3'3 (1964) Slonimski, P., Formation des enzymes respiratoires chez la levure, Masson Paris (1953) Suyama, Y., and Preer, J. R., Genetics 52, .LO51 (1965) I'ewari, K. K., Jayaraman, J., Mahler, M. R., Bioch. Bioph. Res. Comm. I,

141 (1965)

Vinograd, J., and Hearst, J. E. in Progress in the Chemistry of Organic Natural Products (Ed. L. Zeichmeister) Vol.xx,P.372, Springer-Verlag, Vienna (1962) llotsuyanagi, Y., J. Ultrastruct. Res. 1, 12.1 (1962) and 1, 141 (1962) 61