An N-acetylated peptide chain in tropomyosin

Vol.

23,

No.

4, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

AN N-ACETYLATED

RESEARCH

PEPIIDE

COMMUNICATIONS

CHAIN

IN TROPOMYOSIN.

R. E. Alving,

E. Moczar

and K. Laki

National Institute of Arthritis and Metabolic Diseases National Institutes of Health, Public Health Service U. S. Department of Health, Education, and Welfare Bethesda, Maryland 20014

ReceivedApril15, It

1966

has been reported

from

1961)

that

by the application

tamic

acid

could

be identified

tropomyosin

B.

rabbit's

Our experiments, tropomyosin residues

(Kominz

myosin

consisted

reported

that

p-mercapto weight also

half

suggested

tropomyosin tain

that

an N-terminal

rabbit pronase

lated

which

methionine

reduce

of

non-reactive

communication,

we report

contained in equal

acetic quantities.

containing

exhibited

a molecular

molecule.

This

finding

two chains.

But

chains

our findings

must

finding

which

peptide

a tripeptide

aspartic This

540

(Woods 1965)

if con-

to dinitrofluorobenzene.

tropomyosin, acid,

that

tropo-

solution

B has an N-acetylated of

that

one of the

residue

digestion

urea

contained

two chains,

of

as C-terminal

Recently,

native

glu-

residue

indicated

S-S bonds)

of the

and Kominz,

A suggested

chains.

tropomyosin

tropomyosin

After

This

B in 8 molar

of that

consists

In this that

(to

N-terminal

and serine

1957).

tropomyosin

(Saad

DNPB method,

carboxypeptidase

of two peptide

ethanol

about

as the

isoleucine

et al.,

laboratory

of Sanger's

using

B contained

this

acid, gives

show

chain. was iso

alanine further

and

Vol.

23,

No.

4, 1966

BIOCHEMICAL

support

to the

one starting lated

idea

with

AND

BIOPHYSICAL

that

tropomyosin

glutamic

acid,

RESEARCH

contains the

other

COMMUNICATIONS

two peptide one with

chains;

an acety-

residue. For

pared

these

experiments,

according

to Bailey

and stored

in the

was carried

out

justed

tropomyosin After

rechromatographed

peak 1.3

N.

Acid

(in

10% methionine). using acids

analyzer.

the

I) the

acetylated

yielded

aspartic

acid

column

determined of

the

of the from

and

by gas chroma1964).

peptide

The

amino

acid

yielded

I.

acetylated

tripeptide

tropomyosin. ~1 moles per u mole of myosin digested.

Residues acid

0.66

Alanine

0.61

Methionine

0.55

Acetic

alanine

sulfoxide

automatic

acetylated

roughly acid,

(Jackson,

by a Spinco

cycle,

collected.

peak

a sebacic

Components

were

of the

was determined

was

formate

concentration

acid

Hydrazinolysis

peptides

in the

Acetic

were

(by weight).

acid

acid,

1 mg

Dowex 50 col-

of 90% methionine

Table

Aspartic

through

column

ad-

contained

formic

of acetic form

mixture,

48 hours,

Two ml samples

hydrolysis

muscle by pronase

digestion

was passed

when the

(Table

and methionine

amino

for

was pre-

skeletal

in a 60 to 1 ratio

gradient.

came out

amounts

tography

the

25'C

containing

acid

rabbit

purity

The digestion

on Dowex 1 (X-8)

a formic

The main

equal

at

mixture

effluent,

from

way:

per ml and pronase

umn and the

reached

following

the

of high

state.

and kept

digestion,

using

(1948)

lyophilized

the

to pH 8.0

tropomyosin

acid

0.71

541

tropo-

Vol. 23, No. 4, 1966

BIOCHEMICAL

alanine,

showing

of this

tripeptide.

amino

acids With

that

teins,

actin

1966),

myosin

growing

class

finding (Alving (Offer, of

amino

The actual

remains the

this

AND BIOPHYSICAL

acid

RESEARCH COMMUNICATIONS

is the

sequence

C-terminal

of the

residue

other

two

muscle

pro-

to be established. reported

here,

all

and Laki,

1966,

Gaetjens

1965)

proteins

and tropomyosin that

have

three

and Barany, now belong

an acetylated

N-terminus.

REFERENCES Alving, R. E. and Iaki, K., Fed. Proc., 3 223 (1966). Bailey, K., Biochem. J., 43, 271 (1948). Gaetjens, E. and Barany, IL, Fed. Proc. 3 223 (1966). Jackson, R. B., J. Chromatography, & 306 (1964). Kominz, D. R., Saad, F., Gladner, J. A. and Iaki, K., Arch. Biochem. Biophys., 2 16 (1957). Offer, G. W., Biochim. Biophys. Acta, 111, 191 (1965). Saad, F. M. and Kominz, D. R., Arch. Biochem. Bio92 541 (1961). Phys., 207, 82 (1965). Woods, E. E, Nature,

542

to the

Vol.

23,

Nlo. 4, 1966

BIOCHEMICAL

INTERSPECIES

AND

TRANSFORMATION

BETWEEN

BACILLUS

Ivan

D.

S.

Army

1.

Received

Goldberg,

Dubnau --subtilis

and

g.

(1965)

system have

low

that

been

able

di.fference

in

efficiency

as

specific

transformants

the

transformants specific

Maryland

with

only

-1

this

the

the

resiswith

a

and

we

least

a

of

When number

times

the

on

number

number

the

A loo-fold the

recipient

the

DNA.

lo-'

work

at

DNA,

times

to

transformation.

as

subtilis to

able

depending

homologous B.

10

-4

were

transformation

heterologous 10

a strep-

streptomycin

indicates

licheniformis

of

to

reciprocal

B.

from they

confirmed

transformation,

B.

isolated

subtilis

of

between

direcand

of

2.

inter-

intraspecific B.

of

licheniformis interspecific

number

of

intra-

transformants.

3.

were

AND

Thorne

Frederick,

DNA

crosses

transform

was

-B.

the

MATERIALS

g-

B.

Detrick,

percent

perform

was

obtained to

higher

With source

transformants used

Curtis

licheniformis,

reciprocal

cross.

--subtilis

was

SUBTILIS

transformation

We have

to

the

DNA

B. of

a much

of

the

of

frequency.

comparison

of

and

Using

strain

gives

also

ti.on

reported

strain

a

Gwinn Fort

licheniformis.

a sensitive at

D.

Center,

al.

tc'mycin-resistant

tance

BACILLUS

COMMUNICATIONS

15, 1966 et

transform

RESEARCH

LICHENIFORMIS

Darrel

Biological

April

BIOPHYSICAL

subtilis

subtilis obtained

W168 168

-ind-. from

was g.

C.

G.

AND

isolated

METHODS as

licheniformis Leonard.

a spontaneous 9945A

g.

543

subtilis

revertant

Ml8 SB-1

s-

and his-,

of Ml7 -ind-

-adwas

Vol. 23, No. 4, 1966

BIOCHEMICAL

obtained

from

tomycin

sulfate

DNA

was

E.

of

by

subtilis

168

tially (1961).

10

DNA

treatment

(Millipore

-ind-

HA

plates

then biotic

and

marker

were

the

samples

0.45

cl).

to

83

agar.

With

agar on

2 hours

exposure

amino tomycin plemented

grow to

minimal

10 acid

1 mg/ml

the agar for

marker. with

on

the

prior

supplemented

with

40

expression

both

of g.

-B.

subtilis

tryptophan

SB-1, and

544

histidine

at

37

C.

not

be

allowed

but

used.

transfer

to

pen-

attained

normal it

was

that

killed

after by

of

transformed minimal

difficult

colonies

overcome

at

on

express

were

was

When

to

Thus

the

Anti-

sulfate.

could

micrograms/ml

subtilis

(Difco

agar

to

the

were

done

and

problem

allow

to

was

transformants

This

time

expression

never

plates

to agar.

agar

plates.

expression

After

membranes

were

antibiotic.

With

the

the

for

+ 0.75%

incubation

colonies

true

on

filters

streptomycin

hours'

medium the

between

to

suitable

penassay

this

scored

transferred

media

and

streptomycin-containing

differentiate

a

recipient

Spizizen

membrane

penassay

48

essen-

way.

then

transformants on

were

following

were

subtilis,

+ streptomycin, the

started

g.

as

streptomycin

than

+

after

used

the

expressed

agar)

by and

onto

containing

scored was

subtilis

size

plates

were g.

transformed

growth

become

+ 0.75%

were

licheniformis

assay

in

C for

&-

Transformants

filtered

37

U.V. Com-

(1965).

(1966)).

membranes

to

(1961). Ml7

Mattheis

markers

scored

at

and

Anagnostopoulos

Stull

The

incubated

to

Transformants

more

by

strep-

spores

Marmur

arg-

were

streptomycin-free

Medium

g.

and

his-

were

of

Ml8

auxotrophic

(Thorne

marker

penassay

z-,

described

transferred

for

SB-1

1 rag/ml of

procedure

Leonard

for

were

streptomycin

2.

of

and

containing plates

the

to

exposure

99458

procedure

agar

streptomycin

following to

RESEARCH COMMUNICATIONS

resistant

licheniformis

procedures

the

The

isolated

Transformants

minimal

to

g.

the

the

Mutants

according

cells

prepared

Nester.

were

extracted

petent

If

W.

AND BIOPHYSICAL

the for 10 was

a concentration

had

using required the

strepsup-

Vol. 23, No. 4, 1966

of

40

agar

BIOCHEMICAL

micrograms/ml. was

due

medium,

We

to

the

feel

extremely

resulting

in

inhibited

that

AND BIOPHYSICAL

the

a heavy growth

that

our

fast

growth

difficulty

the

with

of

background of

RESEARCH COMMUNICATIONS

of

g.

penassay

subtilis

on

this

non-transformed

cells

transformants.

RESULTS In of

order

to

obtain

transformants

sary

to

for

determine

marker.

Under

--subtilis

became

sence

of

sion.

Table

tion

between

for

used,

expressed

done

subsequent

under

conditions results

-B.

and

g.

the

intraspecific

transformation.

This

the

interspecific

transformation

systems

the

difference

2.

subtilis->

2.

the

intraspecific

10

thousand-fold.

number 10-l

of to

could

be

with among various

times It

in

all

Dubnau

et

number

should

cases.

transferred

them

other

hand, -B.

the

None between

al.

(1965),

streptomycin

species

of

in

noted of the

who

was

significantly

is

a

resistance,

could

Bacillus.

545

by

Dubnau

the

date.

However,

obtained

in

cross

was

cross,

transformants

was

only be

the only

subtilis->

saturating

II.

levels markers is certain

transferred

all

interspecific

reciprocal

This

than to

number

B.

al.

common

licheniformis

the

et

lower

to

auxotrophic

that

expres-

intraspecific

feature

the

2 species. found

ab-

transforma-

is

the

in that

the

maximum

shown

subtilis

obtained be

gave

in

2.

the

B.

transformation

and

licheniformis-> the

in

reported

cross

this and

growth

transformants

licheniformis->

cross. used

of

licheniformis

On

B.

--subtilis

number

2.

162

were

the

necesof

and

As

DNA.

first

interspecific

transformation

in

of

that of

was

numbers

licheniformis

licheniformis

homologous

interspecific

-B.

All

1).

the

expression

6 hr

(fig.

subtilis

the

both

of it

after

the

using the

required

1 indicates

transformation (1965)

marker,

conditions

fully

were

streptomycin

time

streptomycin

experiments

measurement

the

the the

a meaningful

of tested

in

agreement markers, between

DNA

Vol.

23,

No.

4, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISCUSSION We have

demonstrated

directions

between

calculated

from

of

3 to

1.

and

a difference

Goldberg

Ravin

(1965)

in

reciprocal

obtained

Interspecific

transformation

with

T m (Gwinn,

transformation TABLE

2 species

Chen

4%.

interspecific

and

in

Thorne,

compared

the

crosses

transformation

in

between

GC content

unpublished

results)

efficiencies

of

between

2.

both

pneumo-

subtilis

and

g.

licheniformis.

Recipients 2. licheniformis 99458 M-18 DNA

source

(1.7

x

lo"",

(2.6

Transformants

per

g. subtilis w-168 sr

1.1

x

lo2

g. licheniformis 99458sr

1.2

x

lo6

subtilis

source

2.0

x

Transformants

10

x

x lo6

J!j. licheniformis 99458s=

1.7

x

105

x

lo1

6.5

x

LO5

subtilis

x

<

3.2

+

10

*

10

(his-,

lo6

Jo&

per

ml

his+ -

2.4

x

lo6

2.3

x lo4

)

x log)

Sr

lo6

+

x

Transformants

ml

ml

<

SB-1 (2.0

2.3

per -ad

4.8

log)

-ind

2.9

lo6

g.

per

(ad-) -

log)

Transformants

(&-)

Sr g. subtilis W-168 sr

x

Sr

<

168

(2.9 DNA

ml 3'

Sr

2.

g, licheniformis 994512 M-17

(arg-)

1.2

x

<

-ind lo6

10

8.0

+

x lo5

<

10

All the other Numbers in parentheses are numbers of recipient cells/ml. numbers in the table refer to numbers of transformants per ml. The concentration DNA was 22 pg/ml; that of B. licheniformis DNA was 22.5 @g/ml. of B. subtilis Botk levels were saturating for intraspecific and interspecific transformations. Cells transformed to streptomycin resistance (Sr) were scored on penassay agar Cells transformed to prototrophy were containing 1 mg/ml streptomycin sulfate. scored on minimal 10 agar. With 2. subtilis SB-1, transformants for the histidine marker were scored on minimal 10 + 40 pg/ml L-tryptophan and transformants for the indole marker were scored on minimal 10 + 40 Kg/ml L-histidine. Untreated cells and cells treated with DNA, that had been preincubated with 100 pg/ml deoxyribonuclease (Worthington) were run as controls in all experiments.

546

Vol. 23, No. 4, 1966

BIOCHEMICAL

FIGURE 1.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Time required for expression of streptomycin resistance by JJ. subtilis and B. licheniformis interspecific transformants.

with

lis cells transformed DNA of 2. licheniformis

g. licheniformis cells formed with DNA of g.

60

120

180 240 300 Time (min)

360 420

transsubtilis

480

Millipore filters were incubated on streptomycin-free media for the times indicated and they were then transferred to plates of penassay agar containing 1 mg of streptomycin sulfate per ml. Complete procedure as in text. coccus

and the

resistance

Challis

marker.

strain They

found

transformation

to be much more

They

suggested

that

grated

into

DNA into likely

the

the

the

Streptococcus

the

genome of

efficient

in our

system

the

->

than

the

streptomycin

Streptococcus reciprocal

DNA was more easily

Streptococcus genome.

for

pneumococcus

pneumococcal

pneumococcal

possibility

of

than

We have that

was the not

-B. subtilis

cross. inte-

streptococcal

eliminated can take

the

un-

up -B.

Vol. 23, No. 4, 1966

BIOCHEMICAL

licheniformis up

DNA

subtilis

2.

DNA.

a more

efficient

Boyer,

1964)

than

basis

of

on

the

a small the

ported

core

is

of

of

that

is

This

streptomycin

intraspecific

transformation the

core

would

hypothesis.

nucleotides

are

nucleotides

adjacent

to

those

frequency

of

transformation

the

interspecific

affect

the

1958),

thus

lowering

We wish T.

Bryan

and

involved

in

to

acknowledge

R.

Gregoire.

both

that

the

lower

than

the

seem,

at

the

first

These

to that efficiency

efficiency glance,

same

recombinations.

of to

that

argue

sequences

of

However, a given

of

inter-

resistant

fact

determining

re-

a conserved

marker

the

as

Bacillus. -~

the

Presumably

(1965)

well

relatively

The

that

al.

Bacillus

include

resistance. is

of

genus is

suggested

conserved

et as

species the

has

studies are

take

1962;

(1965)

hybridization

would

transformation

Dussoix,

Dubnau

that core

and

sequences

in

material

licheniformis

hybridization

several

there

change.

interspecific

against

acid

can

-B.

Igarashi

Bacillus.

among

genetic

evolutionary

and

nucleotide genus

licheniformis that

DNA-RNA

the

RESEARCH COMMUNICATIONS

(Arber

Doi

nucleic

stable

possible

subtilis.

identical

suggested

determines

also

II.

mechanism

transformation

of

than

interspecies

interspecific

authors

of

It

g.

members

specific

efficiently

restriction

number

among

more

AND BIOPHYSICAL

marker marker

the might

(Shaeffer,

efficiency. able

technical

assistance

of

REFEERENCES Anagnostopoulos, Arber, W. and Boyer, H., J. Chen, Kuo-Chun, Doi, R. H. and Dubnau, D., I. Sci. U.S. Leonard, C. G. Marmur, J., J. Shaeffer, P., Thorne, C. B.

C. and J. Spizizen, J. Bacterial. s, 741 (1961). D. Dussoix, J. Mol. Biol. 1, 18 (1962). Bacterial. E, 1652 (1964). and A. W. Ravin, Genetics 52, 434 (1965). R. T. Igarashi, J. Bacterial. 90, 384 (1965). Smith, P. Morel1 and J. Marmur, Proc. Natl. Acad. Ift, 491 (1965). and M. J. Mattheis, J. Bacterial. 90, 558 (1965). Mol. Biol. 2, 208 (1961). Symp. Sot. Exptl. Biol. 12, 60 (1958). and H. B. Stull, J. Bacterial. 3, 1012 (1966).

548