Cloning of the replication origin from Drosophilavirilis mitochondrial DNA

Vol. 91, No. 4, 1979 December

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

28, 1979

Pages

1321-1329

CLONING OF THE REPLICATION ORIGIN FROM DROSOPHILA VIRILIS MITOCHONDRIAL DNA Akio

Sugino

Laboratory of Molecular Genetics Institute of Environmental Health Sciences National Institutes of Health P.O.Box 12233, Research Triangle Park, NC 27709

National

Received

November

1,1979

SUMMARY

Mitochondrial

DNA from Drosophila contains high "A+T"-rich region. starts in the 'A+T"-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the "A+T"-rich region of mitochondrial DNA from Drosophila virilis has been cloned in Escherichia coli. The chimeric plasmid DNA containing the "A+T"-rich region stimulates DNA replication --in vitro system from Drosophila virilis mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA.

Its DNA replication

INTRODUCTION Mitochondrial closed 9.9

circlar

to 12.4

CsCl

is

double X lo6

1.681

Electron

DNAs (mtDNA) stranded

(1,2).

of Drosophila

visualization

contour

length

is composed

of the

"A+T"-rich

region

given

species,

species

(1).

sequence

divergence,

possesses

complete

Differences species weight

varies it

whereas

from is

is

only

region remainder homology

weight explained

0.31

species

weights

of these

ranging

15 to 20% (1,3,4).

denatured

mtDNA molecules

25% of the

of A+T (l-3,5).

to 3.41 for

X lo6

for

of mtDNA molecules by differences

mtDNA molecules of a

extensive

of the molecule species

circular The size

mtDNA molecules

has undergone

in all

are

DNAs in neutral

approximately

constant

the

base sequence

can be completely

molecular

or entirely

The "A+T"-rich

"A+T"-rich

with

density

that

largely

while

of Drosophila

of partially

showed

in the molecular

of the

molecules

and the G+C content

melanogaster

of different

a variety

The buoyant

to 1.685,

microscopic

from

base

apparently tested

from

(3,6,7).

different

in the molecular

region. 0006-291X/79/241321-09$01.00/0 1321

Copyright All rights

@ I979

by Academic Press, Inc. in any form reserved.

of reproduction

Vol. 91, No. 4, 1979

BIOCHEMICAL

The origin

and direction

of Drosophila

have recently

studies

).

( 7-9

unidirectionally

location

of the origin the

nucleotide rich

contains

the

been tested( since

from

only

is

still

the molecule.

origin

I have cloned virilis

"AtT"-rich

1 ). This

a few restriction

However,

the precise

mtDNA fragments

to determine unique containing

Drosophila

DNA nucleotide cleave

the

more of its

the

virilis

sequence

and

feature

among many species

endonuleases

MATERIALS

microscopic region

In order

embryos.

some species

"A+T"-rich

and elucidate

region

simplifies

by electron

in the

unknown.

of the

Drosophila

smallest

starts

RESEARCH COMMUNICATIONS

of mtDNAs from

been determined

around

location

sequence,

region

of replication

DNA replication

proceeds

precisely

AND BIOPHYSICAL

which

"A+T"-

mtDNA have

determination,

"A+T"-rich

region.

AND METHODS

Drosophila Strain:Drosophila virilis strain D.V.15 established from single female captured in Southern Japan was used. Growth of flies, collection of larvae and preparation of mtDNA by use of CsCl density gradient centrifugation were as descrived( 6,lO ). Bacterial provided

Strains: E.coli by Dr. R.D&F

HBlOl(

recA,ri,ri

) and HBlOl(

pBR322

a

) were

Enzymes: T4 DNA ligase was a gift from Dr. K.L.Agarwal. All restriction endonucleases were from either Bethesda Research Laboratories or New England BioLabs, and were used as recommended. E.coli DNA polymerase I and pancreatic DNase I were provided from WorFhington Biochemical. Plasmid DNAs: E.coli HBlOl(pBR322) and plasmid DNA were grown at 37'C in 1 1 acids and 50 Pg/ml of ampicillin(Amp) further grown for 8 hours after addition The cells were collected by centrifuge descrived( 11 ).

HBlOl containing the chimeric of Mg medium containing 1% Casamino to 5 ~108 cells/ml. The cells were of 150 ug/ml of chloramphenicol(Cam). and plasmid DNA was isolated as

Cloning of Drosophila virilis mtDNA: Each 1 pg of the purified mtDNA and plasmid pBR322 DNA were digested completely with Hind III and in the reaction mixture( 20 ~1 ) containing 1 unit of theenzyme at 37'C for 90 min. After inactivation the enzyme by heating at 65°C for 10 min, the DNA were mixed, annealed at 37°C for 10 min and ligated with 2 unitsof T4 DNA ligase at 10°C for 24 hours in the reaction mixture( 200 ~1 ), containing 50 mM tris-HCl,pH8.0, 60 uM ATP, 1 mM dithiothreitol, and 5 mM MgC12. The ligated DNA was dialized against 50 mM CaC12 and used to transform E.coli HBlOl. Transformatiom and selection of Amp-resistant, tetracycli$Tc)sensitive transformants were the same as published( 11 ). The cloning experiments described here were done under NIH Guidelines for Research Involving Recombinant DNA molecules utilizing P2 + EK-1 conditions.

1322

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Nick-translation of the cloned DNA: The cloned mtDNA T. Hind III C fragment was cut out from the plasmid DNA with -Hind III and purified by agarose gel electrophoresis as published (21). One ug of the cloned DNA was first nicked in the reaction mixture (100 ~1) containing 50 mM tris-HCl, pH7.5, 10 mM MgCl , 5 mM dithiothreitol, 50 ug/ml BSA, and 0.1 pg of Then, 3 mumoles each pancreatic DNase Q for 1 m@ at room temperature. three dNTPs, 1 mvmole [ ct- P]dTTP (sp.act. 100 C/mole, ICN) and 4 units of r. coli DNA polymerase I were added the reaction was continued at 15°C for 3 hours. The reaction was stopped by addition of 10 mM EDTA and the DNA was purified by Sepharose 48 (Pharmacia) column chromatographs . In vitro DNA replication system from Drosophila virilis mitochondria: Mitochondria from Drosophila virilis larvae (50 g) was prepared as published (6), excepting that a sucrose density gradient centrifugation was omitted. The isolated mitochondria was suspended into 2 ml of Buffer A (50 mM Hepes buffer, pH7.8, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol).After addition of 0.1% Triton X100, the suspension was incubated at 30°C for 10 min, chilled at 0°C for 5 min, and centrifuged at 40,OOOrpm for 30 min in a Spinco SW50.1 rotor (at 2'C). Supernatant was precipitated with 50% saturation of ammonium sulfate, the precipitates were suspended into 1 ml of Buffer A, and dialyzed against Buffer A for 4 hours at 0°C. DNA synthesis reaction mixture (50 ~1) contained 30 mM Hepes buffer, pH7.8, 10 mM MgCl 1 ~lj dithiothreitol, 5 mM ATP, 1 mM DPN, 100 UM each three rNTPs, 20 $'[aP]dTTP (sp. act. l,OOOcpm/pmole), 33 pM each three dNTPs, 100 pg/ml BSA, 250 pmoles DNA (total nucleotide concentration),and 10 pl crude extract from mitochondria. DNA synthesis was measured at 20°C for 15 min. RESULTS AND DISCUSSION Figure Hind lb,

III

lb shows

digestion

inside

produces

of circle)

nucleotide

a physical

long.

map of Drosophila

four

contains

DNA fragments the

The DNA origin

mtDNA (6,9).

and H&d

"A+T"-rich

(7,9)

virilis

region

III

C (Figure

and is about

and direction

(7)

was cloned

in r.

2,200

are shown

by an

arrow. The mtDNA digested for

a vector

colonies to Tc.

as Materials

which These

emerged,

because

-Hind

site

assay(

endonuclease

8,000

of pBR322

gel

were

than

recutting

III

Of about

tested

(11).

is All

plasmid parental experiments

lost

10,000

colonies

when

inserts

76 colonies

were

DNA with

a lower

revealed

that

using

pBR322

Amp-resistant to be sensitive

may contain are

cloned

chimeric at the

shown by the toothpick

electrophoretic

pBR322 DNA. However,

1323

coli

and 76 found

and Tc-sensitive

Tc-resistance

12 ) to contain

in an agarose

Hind

and Methods.

Amp-resistant

plasmids III

with

only

Hind

mobility III

restriction

13 of those

colonies

Vol. 91, No. 4, 1979

A

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A+ T -rich

4

l

abc

c

940 w

660,4

320 +

385-j

A t3

380

t----520

2301

kl45

195-j

i C

D

01

02 Figure-l: Physical map of mtDNA from Drosophila virilis embryos, showing recognition sites for restriction endonucleases, the "A+T"-rich region and the origin and direction of DNA replication. (a) Detail map of mtDNA Hind III C fragment cloned in E.coli. The numbers indicate the size in b.p, of the DNA fragments created by restriction endonucleases. (b) Simplified physical map of whole mtDNA (9). The contour length is 15,000 b.p.; Hind III fragments are 6,140, 5,600, 2,090, and 1,200 b.p.; u II fragmez are 10,150, and 5,300 b.p.; and &R I fragments are 9,500, 5,200, and 830 b.p. (9). of the cloned Hind III C fragment to mtDNA from 0.2 ug of mtDNA f= Drosophila virilis was digested in a 20-~1 reaction mixture containingunit of Hind 50 mM and 50 pg/ml of BSA,xd pH 7.5, 10 mM MgCl through a 1% agaro Z' e gel for 20 hours at 2.5 volts/cm. with 0.5 pg/ml ehtidium bromide and transferred to a hybridization was carried out as described P-labelled cloned Hind III C fragment (sp.act. brcmid e stainingpattern of Hind III of DNA marker. (b) Ethidium bromide staining pattes? of Hind III P-labelled of Droso hila virilis mtDNA. (c) Hybridization of DNA --fe--to b . A, B, C, and D indicate Hind III fragments of Drosophila mtDNA.

Fi ure 2: Hybridization &+liTTa virilis. at 37 C for 1 hour III, 50 mM tris-HCl, then electrophoresed The DNA was stained

digest digest cloned virilis

1324

BIOCHEMICAL

Vol. 91, No. 4, 1979

contain

mtDNA-Hind

may contain

nuclear

extensively

purified

Four,

III

restriction

III

dization

This

using

Figure

2, the

of native

32 P-cloned

base pairsf

4,362

b.p.(

17 ).

with

sequences,

of Southern's

an agarose over

III

gel(

A&

-EcoR I, -EcoR I*,

Mbo I and II, -

--Pst

cleaved

the Hind

means(

Figure

37°C but also HindIII

the

la

).

various

pH ( 18 ),or

the preliminary

contains

many :!$;I results

pNS213 and the To test

whether

I, I.

stabilize only that

nucleotide sequences(

).

this

A. Sugino,

is

for

its

the preferred

Clpa I and II, and III,

region

H. Kojo,

orientation,

only

at

of the

EcoR I* was detected

of the Hind

pattern )'I( III

[low

Figure

la )

C fragment

and K. Nakayama,

inserted are

II,

by conventional

by Mntt(*19

determination

Hint

endonucleases

out not

for

and was

recognition

EcoR I* cutting

of the

1325

II

"A+T"-rich

of Mgtt

III

different

carried

sequence

The orientation

rationa'le

the

Hind

were mapped

one site

favor

replacement

with

7 restriction

the

DNA is

pNS213 DNA was

and III,

were

as

pBR322 DNA is

with

sites

C fragment

plasmid

of plasmid

Only

III

in

DNA, designated

-BamH I, -Hind

and these

Surprisingly, conditions

the Hind

digestion

The enzyme digestion

although

published

-Hinf

B and D

13 ). As shown

parental

&II

III

-Hind

a mtDNA-Hind -

This

endonucleases

C fragment

at 15°C to help

and high

20 ) after

both

Hind

plasmid

whereas

Sau 3A, and Sal -

III

C fragment.

under salt

I,

This

I, Ava I and II,

-Hha I and II,

with

C fragment

20 restriction

including

only

III

by DNA-DNA hybri

characterization.

) long,

MtDNA-Hind

both

method(

virilis.

further b.p.

contained

DNA containing

DNA hybridized

for

6,600

treated

from

mtDNA-Hind

colonies

was confirmed

Drosophila

about

from

had a plasmid

a modification

was selected

isolated

contained

Three

identification

mtDNA from

pNS213,

plasmids

mtDNA was extracted

of mtDNA and one contained

one colony

C fragment.

though

of the 13 colonies respectively.

A and D fragments Only

, even

the other

mitochondria.

one and three

fragments.

enzyme fragments;

DNA inserts

A, 6, and Cl fragments, III

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

un-

DNA in plasmid shown

in Figure

pNS213 DNA was cut

3.

BIOCHEMICAL

Vol. 91, No. 4, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 3: Orientation of the inserted mtDNA-Hind III C fragment in the pTasmid Orientation was determined using pBR322 and pNS213 DNAs P %iyed with j2 P as in Figure 2 and restriction endonuclease Taq I. MtDNA-md III C fragment has one l& I site (Figure la) and pBR322 DNA has several, including one site 338 nucleotides away from the EcoR I site. If the orientation of inserted DNA were as shown in FiguK3, then the third-smallest Tag I fragment of pBR322 DNA (315 b.p.) (16) should be unchanged in the chimeric DNA. If the orientation were oPPosite, the fragment should be lost and a new one about 700 nucleotide ibng would be found. Actual experiment was the former case. Thick and thin lines represent the inserted DNA and the vector pBR322 DNA, respectively. Only relevant sites for Taq I are shown. with

III,

Hind

HBlOl

treated

contained for

religated with

CaC12.

plasmid

of the

contained

plasmid

III

Hind

pNS213.

It

is

of the mtDNA insert one colony

DNA was quite

growth.

One possible

begines

not

only

mtDNA replication opposite,

both

finish

replication

tested

using

Tomizawa

tha

the

and his

same as in the in the opposite

plasmid If

of the entire

colleagues(

ColEl

plasmid.

This

some support

1326

genome.

days of

is

that

but

compete

replication

also

and could

possibility system for

this

Only

this

of each replication

DNA replication

14 ).

several

of

as in

however,

origin

would

Nine

of replication

plasmid

preference

the unidirection systems

the direction

after

replication

selected

C fragment.

orientation;

this

which

same direction

original

and was lost

replication

the --in vitro

in the that

randomly

III

Hind

E.coli

colonies

as pNS213 were of the

for

into

Tc-sensitive

to note

explanation

origin.

then

orientation

unstable

from

and transformed

C mtDNA oriented

insert

plasmid

same size

interesting

is

had the

T4 DNA ligase

Ten Amp-resistant,

DNA of the

determination

these

with

from

the were

not

is now being developed idea

by was obtained

BIOCHEMICAL

Vol. 91, No. 4, 1979

Table

1. Copy

number segments

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of plasmid DNAs containing from Drosophila --virilis

mtDNA embryos.

Molecules/cell Plasmid

DNA size (b.p.1

Inserted mtDNA

no drug

none

10

+Cam

+Amp

+Amp and

15

l,OOO-2,000

pBR322

4,362

pNS213

6,600

Hind

III

C

3-5*

15

5

15

pNSOl1

5,400

Hind

III

D

15

450

15

400

pNS451

10,500

Hind

III

A

10

350

20

450

pNS225

9,950

Hind

III

B

15

350

30

500

ColEl**

6,400

15

1 ,ooo-2,000

none

Copy number of various plasmid medium containing 1% Casamino Acids and and 50 pg/ml of Amp as published (15,16). medium contained 5 pg/ml of thymine. * Some

loss

** --E. coli

from

of

plasmid

could

JC411 (ColEl)

a measurement

and absence

vector

cloning

Drosophila

virilis

mtDNA-Hind 15,16

).

replication

in the Hind

vitro

virus

system

We have developed and can complement

15,16

)( Table

1 ).

DNA and the chimeric A, B and D fragments

could

was not of this

be because

C fragment

were

the

origin

too many copies

are

present.

of mechanism

of DNA replication

is

has been greatly

systems(

22 ).

essential

for

an --in vitro exogenous

DNA replication protein

factors

1327

such --in vitro which system from

in

replication

and

by developping

Particularly,

by

of mtDNA

in procaryotes

advanced

eucaryotes

amplified

DNA,was reduced

if

systems

containing

significantly

the

the cell

presence

Parental

plasmids

for

DNA replication

replication

Amp.

competes

and kills

III

of

HBlOl.

pNS213 DNA in the

the copy number

of Cam and Amp. This

some animal

absence

pNS213 DNA, however,

by Cam and in fact

Understanding

the

of plasmid

III

the absence

apparatus

in

of --E. coli

( Cam )(

pBR322 DNA, ColEl

Cam as reported( amplified

be detected

of copy number

-

DNAs were measured in M9-glucose with or without 150 pg/ml of Cam In the case of ColEl, the

was used instead

of chloramphenicol

l,OOO-2,000

Cam

-in DNA

are missing

genetics.

which

--in vivo

Drosophila

mimic virilis

Vol. 91, No. 4, 1979

Table

BIOCHEMICAL

2.

Template

AND BIOPHYSICAL

specificity from Drosophila

of

RESEARCH COMMUNICATIONS

in vitro DNA replication virilisitochondria

DNA added

DNA synthesizing (pmoles/50

no DNA

system

activity ~1/15 min)

0.2

Drosophila

virilis

mtDNA

Drosophila

melanogaster

Drosophila

simulans

3.1

0.8

mtDNA

0.9

mtDNA

pNS213

DNA

2.9

pNSOl1

DNA

0.4

pNS451

DNA

0.3

pNS225

DNA

0.4

pBR322

DNA

0.3

DNA synthesis was measured as Materials and Methods in --In vitro the presence or absence of 250 pmoles DNA templates(sophir melano aster and Drosophila simulans mtDNAs were the gifts from Dr. D. lTiT&+ After 15 min at 2O"C, the reaction was stopped with 1 ml of 5% trichloroacetic acid containing 5 mM sodium pyrophosphate. After 20 min at O"C, DNA was collected on a GF/C filter disc and the filter was washed twice with 10 ml of 5%trichloroacetic acid and 5 mMsodium pyrophosphate, and once with 5 ml of ethanol, and dried. Radioactivity was measured in a toluene-base scintillation fluid.

mitochondria(

K.

As shown

in Table

of

stimulated

mtDNA

the

parental

but not merit

--in vitro

other

plasmid

and

Sugino,

pBR322, species

support

as did

native

other Hind

III

of DNA replication.

system

provides

possibilities

origin

and purification

In order

C fragment

to compare

mtDNA from

another

Drosophila

melanogaster

strain,

than

not

virilis, the chi-

stimulate

DNA

virilis

DNA replication

of exact

location

of

proteins.

the structure

Drosophila

hand,

--in vitro

of determination

of DNA replication

higher

of Drosophila

And this

region

Drosophila

other

of mtDNA did

).

"A+T"-rich

10 fold

mtDNA from On the

part

preparation

the

about

of Drosophila.

that

in

DNA containing

mtDNA has an origin the

manuscript

DNA replication

DNAs containing

This

A.

2, pNS213 plasmid

plasmid

from

synthesis.

Nakayama,

of the

I tried

mtDNA digested

1328

with

"A+T"-rich several

either

region times

Hind

III

in the

to clone

or EcoR I

Vol. 91, No. 4, 1979

into

--E.coli

BIOCHEMICAL

using

nick-translated detected groups

both

pBR322 DNA and agt-xB

native

neither

Drosophila

colonies

D. Clayton,

Why no one has succeeded is

nor

have been unsuccessful

D. Walstenholm,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DNA cloning

melanogaster plaques

in cloning

mtDNA.

Drosophila

and M. Meselson, this

Using

mtDNA as a probe,

containing

in cloning

vectors. I

Also,

other

melanogaster

mtDNA(

personal

comnunications

mtDNA segments

in --E.coli

).

unknown. ACKNOWLEDGMENTS

I am indebted for

providing

mtDNA and for versity

to Drs D. M. Shah and C. H. Langley

me with

an unpublished

discussion.

of Chicago,

for

I also

physical thank

criticizing

Dr.

in this

Institute

map of Drosophila N. R. Cozzarelli,

virilis the

Uni-

the manuscript.

REFERENCES 1.

Fauron, C. -R., and Wolstenholme, 73, 3623-3627.

2.

Bultman, H., Zakour, R. A., and Sosland, M. A. (1976) Biochim. Biophys. Acta 454, 21-44. Peacock, W. J., Brutlag, D., Goldring, E., Appels, R., Hinton, C. and Lindsley, D. C. (1974) Cold Spring Harbor Symp. Quant. Biol. 38, 405416. Bultman, H., and Laird, C. D. (1973) Biochim. Biophys. Acta 299, 196209. Klukas, C. K., and Dawid, I. B. (1976) Cell 9, 615-625. Shah, D. M., and Langley, C. H. (1979) PlasmTd 2, 69-78. Wolstenholme, D. R., Goddard, J. M., and Fauron, C. M. -R.(1979) J. Supramol. Structure, Supplement 3 136. Goddard, J. M., and Wolestenholme, D. R. (1979) Proc. Natl. Acad. Sci. 75, 3886-3890. Shah, D. M., and Langley, C. H. submitted. Shah, D. M., and Langley, C. H. (1977) Nucleic Acids Res. 4, 2949-2960. Bolivar, F., Rodriguez, Z. L., Greene, P. J., Betlach, M. C., Heyneker, Gene 2, 95-113.

3. 4. ii* 7: a. 9. 10 1, 1,:

D. R. (1976)

Proc.

Natl.

Acad.

Sci.

M., and Helinski, 16. Sutchiffe, J. G. (1978) Nucleic Acids Res. 2, 2721-2728, and Cold Spring Harbor Symp. Quant. Biol 43, 77-90. 17. Polisky, B., Greene, P., garfin, D. E., McCarthy, 6. J., Goodman, H. M., and Boyer, H. W. (1975) Proc. Natl. Acad. Sci. 72, 3310-3314. 18. Hsu, M., and Berg, P. (1978) Biochemistry lJ, 131-138. 19. Frank, B., and Ray, D. S. (1970) Virology fi, 168-187. 20. Tabak, H. F., and Flavell, R. A. (1978) Nucleic Acids Res. 5, 2321-2332. 21. Cedar, H., Solage, A., Glaser, G., and Rozin, A. (1979) Nucleic Acids Res. 5, 2125-2132. 22. Cold Spring Harbor Symp. Quant. Biol. 43 (1978).

1329