Restriction map of Chinese hamster mitochondrial DNA containing replication coordinates: comparison with Syrian hamster mitochondrial genome

Gene, 21 (1983) 249-255 Else&r

Biomedical

249

Press

Restriction map of Chinese hamster mitochondrial DNA comparison with Syrian hamster mitochondrial genome (Physical

maps;

pulse-chase

label analysis;

gel electrophoresis;

containing

hybridization;

replication

sequence

coordinates:

conservation

and

divergence)

Margit M.K. Nass Depa~f~ent of ~adjat~on Therapy, University of Pennsy/van;a School qf ~edicj~e. delphia. PA 19104 {U.S.A.) Tel. (215)898-576% (Received

September

13th. 1982)

(Accepted

December

9th. 1982)

Medicul ~bo~atori~s~~3~

Phila-

SUMMARY

A precise physical map, containing the structurally and operationally defined D-loop origin, terminal region, and direction of heavy-strand replication, has been constructed for mitochondrial DNA (mtDNA) from ovary (CHO-KI) and lung cells of Chinese hamster (Cricetulus griseus, 2 N = 22), and compared with our previously established 2 N = 44). All four HpaI

genome coordinates for mtDNA from Syrian hamster (~esocrjeer~ sites in Cricetulus are conserved in ~e~oc~~cef~~ (8 sites). Extensive

exists for hexanucleotides cleaved by EcoRI, between Chinese and Syrian hamster mtDNAs, for these six endonucleases, is estimated several other mammalian species contain initiation.

HindIII, PstI, KpnI and BantHI. Sequence divergence as reflected from analysis of the mapped recognition sites

as 559% base substitutions. mtDNAs from both hamster and a commonly conserved HpaI site in the region of light strand

et al., 1979, 1982; Avise et al., 1979; Brown and Simpson, 1981; Nass, 1981; Ferris et al., 1981a, b). In addition, a high degree of sequence stability has been observed in mtDNAs from different tissues and cell lines of mouse, Mus muscul~.~ (King et al., 198 l), and Syrian hamster, Mesocricetus auratus (Nass, 1981). The mechanism responsible for sequence homogeneity and variance within mtDNA molecule populations is unknown. Our preliminary data indicate that Chinese hamster (Crjcetulus griseus) mtDNA differs extensively from Syrian hamster mtDNA with respect

INTRODUCTION

mtDNA exhibits a high degree of sequence variability based on both interspecific and intraspecific comparisons, notably in certain rodent and primate

species (e.g., Potter et al., 1975; Brown

Abbreviations: kb, kilobase number strand

bp, base pairs; pairs;

mtDNA,

of chromosomes; origin;

ribosomal

p, base

CHO,

Chinese

mitochondrial O,,

heavy

substitutions

strand per

hamster

DNA;

ovary;

N. haploid

origin;

nucleotide;

0,.

light rRNA,

RNA.

0378-l 119/83/0000-0000/$03.00

aurutus, variation

0 1983 Elsevier Biomedical

Press

250

to

Hue111

(Nass.

and

1981).

HpaIl Chinese

+ Hind111

valuable

for the introduction

inherited

drug

resistance

mitochondrial and

alterations

Scheffler,

No data are available the

sequence

mtDNA mutational

changes

and

of

however, on

Chinese

the analysis

MATERIALS

sequence

AND

hamster

by Upholt

that

minimize

divergence

(Gotoh

(1977) and recommenda-

overestimation

_

F

+

p=l-

of sequence

et al., 1979; Nei and Li, 1979):

(F’

+ 8 F)

2

1’

I’~’ I’“,

in this DNA.

from which further

*=

deviation

[ P(l;P)]“‘,

where F is the fraction

of recognition

sites shared

between two DNAs; a and b. number of cleavage sites found in A and B DNA, respectively; c. cleavage sites common to A and B; u. cleavage sites found in either A or B but not both; N, number of independent nucleotide positions in cleavage sites [n(a + b - c)]: and n, number of base pairs recognized per cleavage site.

analysis.

METHODS

Chinese hamster ovary cells (CHO-KI, prolinerequiring) and diploid lung cells (Don) (both from American Type Culture Collection) were grown in roller bottles in Ham’s F-12 medium with 10%’ fetal bovine serum. Baby hamster kidney cells (BHK,,/C,? and C,,/B,) from Syrian hamster were grown as described (Nass, 1980a; 1981). About twice as many CHO as C,,/B, cells were required for equivalent mtDNA yields. of mitochondrial

and standard

map posi-

(a) Cell culture

(b) Analysis

tions

per nucleo-

using the relationships de(1980), which are based on

of possible

tions can be derived and biologically important fragments selected for genome cloning and nucleotide

site variation

of base substitutions

et al.. 198la. b).

our previously established map for Syrian hamster mtDNA (Nass. 1980a. b). provides the necessary coordinates

The number

formulations

cell

This report presents a precise physical map for Chinese hamster mtDNA, which, in addition to

genome

of restriction

human

chloramphenicol

in the literature,

organization

(c) Evaluation

1978; Breen

to be

with single nucleo-

(Blanc

that would permit

sites been

tide (p) was estimated scribed by Cummings

likely

(e.g., Harris,

determined

in mtDNA

have

of cytoplasmically

mutations,

has been correlated

tide changes

cells

1980). In mouse

lines, cytoplasmically resistance

cleavage

hamster

DNA

The procedures for isolation of mitochondria. isolation of mtDNA in CsCl-propidium diiodide gradients, in vivo and in vitro radioactive labeling, isolation and hybridization of single-stranded Dloop sequences, electron microscopic localization of D-loops, and agarose gel electrophoresis of mtDNA were as described previously (Nass, 1980a, b; 1981 and in table and figure legends).

RESULTS

AND

(a) Physical Chinese

DISCUSSION

and functional

hamster

map of mtDNA

from

cell lines

Fig. 1 summarizes representative restriction fragment patterns of Chinese versus Syrian (Nass. 1980a) hamster mtDNA. The fragments have been ordered using standard double-enzyme digestion methods, coupled with alignment at both the single KpnI cleavage site and the D-loop origin of replication. The final map coordinates are given in Table I and Fig. 2A, C. [The key strategy for fragment order of Cricetulus mtDNA was (a) redigestion of fragment EcoRI-B with HpaI to yield three products (3.6, 0.45, 0.25 kb) inclusive HpaIB; (b) cleavage of fragment EcoRI-C with Hind111 to yield three fragments (1.2, 1.1, 0.5 kb) inclusive

251

106432 111111,

I

I

1

0.5

0.2

11111II

The

1 I

polarity

taking

of the map

advantage

dures (Nass,

I

of heavy strand

the D-loop

origin.

of isolated

D-loop

ment

HpaI-A,

marked

of two complementary

1980a) which operationally

5’ + 3’ direction

Hpa I

was established

strands

hybridization

fraction

to

used

from to frag-

the D-loop.

to fragment expanding

The less

HpaI-B

reflects

8-16 S strands, of the single-strand

for hybridization

sequences

replication

predominantly

which form a minor proportion These

procedefine the

Fig. 3A shows the hybridization

which carries

complementarity

by

correspond

(Fig. to newly

3A. inset). replicated

heavy strand, as they hybridize predominantly to light strand (Nass. 1980a, b). The operationally defined terminal region of heavy strand replication can be delineated on mtDNA restriction fragments by labeling with a short pulse of [ “Hlthymidine that is less than the generation time

I

Hind II

11,111,

10

1

I

,111,

1

I

0.5

6432

during

Fig. 1. Densitometric

I

0.2

restriction of each Short shared

fragments pair)

dashed

scan of the electrophoretic

of the IfpuI. and

of mtDNA Mesocrrretus

lines between

EcoRI.

HuzdIII.

patterns and

from Cncerulus

grwus

aurana

of each

(lower

HpaI and HpoII pairs

the pulse are labeled

in the region

that is

synthesized last, which is near the terminus (cf., Nass, 1980b). Fig. 3B. C demonstrates pulse-chase radioactivity accumulated in fragments HpuI-C

kb

autoradiographs)

of a mtDNA molecule, followed by brief chase periods, so that molecules already in the process of replication and completed to closed circular forms

(jzPIfpuII (upper

and EcoRI-D, E, respectively; based on these data (Fig. 3) the map polarity was obtained for Fig. 2. The application of these rapid procedures (Fig. 3) to the unambiguous determination of the direction of heavy strand replication is of particular advantage in cell types where electron microscopic analysis of replicating mtDNA reveals only a low frequency fragments.

of stable D-loops

on isolated

restriction

pair).

designate

fragments.

HindIII-C; (c) bisection of fragment HindIII-D by EcoRI (0.52, 0.45 kb); and (d) digestion of fragments PstI-C, HindIII-A and HpaI-C with EcoRI to yield four products inclusive EcoRI-D and E in each case; the order D, E was consistent with partial fragment analysis of HpaI-C.] This map is identical for mtDNA from CHO-KI and Don cells. The single KpnI cleavage site, which occurs at different genome locations in Chinese and Syrian hamster species, is particularly useful for cloning full-length mtDNA molecules.

(b) Conserved and divergent quences in hamster mtDNA

hexanucleotide

se-

Both conserved and variable cleavage sites occur in all quadrants of the two hamster mtDNA genomes (Fig. 2B, C). Six closely clustered variant sites are located between map positions 40 and 50 [in human, cow and mouse mtDNA the analogous region is in or near the gene for ATPase subunit 6 (Anderson et al., 1981; 1982; Bibb et al.. 1981)]. Three variant sites occur between positions 80 and 100; this region contains the two rRNA cistrons in a number of mammalian mtDNAs, and their conserved and divergent features may differ in closely

252

TABLE

I

Size estimates

(kb) ’ and map positions

of Crrcetulus

(Chinese

hamster)

mtDNA

fragments

(A) .Fragments

A

B

c

D

D-loop location

E

h

Hpa I

6.21, a

3.59,

3.32.

2.58

A.

2.0990.50-3.75

Hind111

8.15,

5.50,

1.13.

0.97

A.

5.X0-0.50-2.05

Ec0RI

7.83.

4.32,

2.80.

0.51,

A.

3.92-0.51-3.56

Psr I

7.19,

6.21,

2.41

A.

3.6220.50~3.08

KpnI

15.64

0.27

A. 14.44-0.51-0.82

(B) Cleavage

Distance

site

from 0,

1 KpnI

’

Cleavage

Distance

Cleavage

Distance

site

from 0 n

site

from Ott

8.2

7 HlndIlI

50.5

13 EcoRI

70.4

2 H/ndIII

15.5

8 EcoRI

53.1

14 EcoRI

73.2

3 PsrI

22.5

4 EcoRI

25.0

5 HpaI 6 HpaI a Fragment DNA

comment

15 EcoRI

75.5

61.7

16 PstI

76.5

26.9

II Hind111

64.2

17 HpaI

86.6

50.0

12 Hpuf

66.0

sizes (kb) by electrophoretic on genome

deviation

and additional

and T7 HpuI

electron

restriction

microscopic

fragments

(above 4 kb) analysis

as size markers

(Nass.

(6 to 8 determinations):

1980a). Standard

deviation

+X174 + 3%. See

size in legend to Fig. 2.

microscopic

analysis

side of the D-loop

’ Distance

and

9 Hind111

(5386 bp), @X174 Hind11

’ Electron each

56.6

10 PstI

of glyoxal-fixed

(underlined)

mtDNA

to the borders

restriction

fragments

of fragments

HpaI-A.

as described HtndIII-A.

(Nass.

1980a). Relative

etc.. oriented

distances

(kb) on

as in Fig. 2A. Standard

i 5% (N = SO). (map units in %) from D-loop

more distantly

related

origin of replication

mtDNAs

(position

(cf., Ander-

son et al., 1981; 1982; Bibb et al., 1981; Ferris et al.. 198 la, b; Brown and Simpson, 198 1). Several restriction site changes found outside rRNA genes represent single-base substitutions (cf. refs. in Brown and Simpson, 1981; Nass, 198 1); the majority of point-mutational changes analyzed in protein-coding sequences of mtDNA in closely related species are transitions and silent substitutions (Brown et al., 1982; Brown and Simpson, 1982). It is remarkable that a conserved tipuI site, which occurs in both hamster species at map position 66 to 67 in the region of the light strand origin of replication (Nass, 1980a, b; Fig. 2), is also found in the same map region in mouse (Martens and Clayton, 1979; Bibb et al., 198 1; Parker and Watson, 1977) bovine (Laipis et al., 1979; Anderson et al., 1982), several ape (Ferris et

0). Accuracy

of map positions

assessed

as + 1%.

al., 19Slaj and human (Anderson et al., 1981) mtDNAs, and at least 4 of the 6 bp of this site are still present (as a Hind11 site) in rat mtDNA (Brown and Simpson, 1981; Parker and Watson. 1977). This genome region (65 to 67) has shown extensive homology with origin-related sequences of SV40 and (pX 174 DNA (Martens and Clayton, 1979). From the mapped recognition sites for HpaI, EcoRI, HindIII, PsfI, BumHI, and KpnI on Chinese and Syrian hamster mtDNA, an estimate of 5-9s sequence divergence (p = 0.069 + 0.019) was obtained. It should be noted that the relatively high number of conserved EfpaI sites biases towards lower p. The mapped site method employed here is considered more accurate than the fragment size method; the former also can detect small genome rearrangements, if present (cf., Upholt, 1977; Brown and Simpson, 198 1; Ferris et al.,

253

IElF Ci II DE i

I

A

C I”

i

A

,ili

A

A

“1’

D

C;

B

1 1

11

11

Eco RI Hind Ill Pst I

11

1

11

11

1

I

60

40

20

loo/O

Hpa I >

C;

B

11

Bo

j

D

B

A

I

60

*I

D

B

Map units 0

Mesocricetus

Fig. 2. Restriction

cleavage

A, linearized

cleavage

heavy strand

origin (0,)

indicate restriction detected mtDNAs

estimates

current

measured).

Upper

(0) HpaI, (A) EcoRI, genome

mtDNA.

gnseus.

Short arrows

(Mesocricerus)

are shown as corresponding

known;

and Syrian hamster

from Cricetulur

digest analyses.

in C. grrseus mtDNA. of absolute

C Cricetulus

is to the right (arrows).

sites on Syrian

valid regardless molecules

maps of Chinese

map of mtDNA

PsrI sites in double cleavage

auratus

hatched

Fragment

reflecting

give position

portion

and Chinese (A) HindIII.

variant

sizes estimated

(0) BarnHI,

hamster

are 15.7kO.3

is subject

kb for both Syrian

hamster

mtDNA,

and

(Brown et al.. 1980)]. Asterisks

designate

cleavage

to adjustment

(Nass.

sites shared between

1981a; Cummings, 1980; Upholt and Dawid, 1977). Differences in mtDNAs of 0.2-1.8% and 0.2-9.6% were found within two species of rats, respectively (Brown and Simpson, 1981) 4-10% between species of Paramecium (Cummings, 1980) 6- 11% between sheep and goat (Upholt and

mtDNA

(Nass.

mtDNA.

respectively.

when the entire

microscopy,

(Table

sequences.

of replication

sequence

lines of

No Barn HI sites are

sites. The genomes of each hamster (Syrian)

of both

from 0,.

are

mtDNA

I and by electron microscopy,

N = 21) for Golden

from

dashed

1980a). B, C. distribution

given in Table IB as % distance

1980a) and Chinese

16.3 kO.5 kb (by electron

hexanucleotide

site; short vertical

(m) PsrI, (X) KpnI cleavage

to 100 map units. [The precise map coordinates. size; the latter

and conserved

in Table IA. Direction

of single KpnI cleavage

shows Mesocricetus (Cricetulus)

griseus

hamster

is

N = 42 mtDNA

the two mtDNAs.

Dawid, 1977) 4-23% between primate species (Ferris et al., 1981a), and 30-35% between primates and rodents, primates and cow, and cow and mouse (Anderson et al., 1982; Bibb et al., 198 1; Brown et al., 1979, 1982). The interesting possibility has been raised

Preliminary

A

c-

analysis

mtDNA

from

hamster

revealed

Syrian

and

differences

quence I

I

I

B

A

studies

evolution

from both

mtDNAs

between

(data

not

further

these

and

of

of Armenian

to determine

of type

base substitution

The present

I

hamster

in terms

patterns

cells

differences

It will be of interest

specific

C

extensive

shown).

the sequence

D

restriction lung

Chinese

mtDNAs

ml

of

cultured

hamster

frequency

of a

(cf., Brown et al., 1982).

corroborate

the extensive

of the mitochondrial

se-

genome,

which has been assessed

to evolve at a higher rate

than single-copy

DNA (Brown et al., 1979).

nuclear

Hpa I ACKNOWLEDGEMENTS

_L D

This work was supported by research grants CA 13814 from the National Institutes of Health and PCM 79-22654 from the National Science Foundation. I wish to thank Hazel Williams and Gregg

C

Eco RI

C

iL C

of the direction

tion from the D-loop

origin

hybridization equimolar

of D-loop D-loop

tured-renatured tation

strands

CHO

B

of heavy strand

tion of 27 and 37 S mtDNA

B. distribution of terminally

mtDNA

in viva

with

distribution terminally

of pulse-chase replicating

with [‘Hhhymidine,

min)

radioactivity

mtDNA

labeled

aedimen-

indicate

in HpuI fragments (10

pattern

from dena-

label radioactivity labeled

A.

denatured.

velocity

1980a); arrows

species,

assistance.

poai-

of pulsereplicating

[ ‘Hlthymidine:

in EcoRI

fragments

C. of

in viva (IO min pulse

30 min chase with unlabeled

thymidine).

Anderson.

genome.

DNA:

mitochondrial

genome.

D.P.. Roe. Staden,

conserved

A.R.. Eperon. sequence

features

1.C..

of bovme

of the mammalian

J. Mol. Biol. 156 (1982) 683-717. C., Laerm.

Lansman,

R.A.: Mitochondrial

phylogeny

within and among

J., Patton,

J.C. and

DNA clones and matriarchal geographic

populations

Geomys prnerrs. Proc. Nat]. Acad.

gopher,

R.

of the human

290 (1981) 457-465. Coulson.

I.G.: Complete

Avise, J.C.. Giblin-Davidson.

pocket

A.J.H..

and organization

Nature

F. and Young,

I.C., Nierlich.

P.H.. Smith,

M.H.L..

mitochondrial

B.C., de BrutJn, M.H.L.,

J., Eperon.

I.G.: Sequence

S.. de Bruijn.

Sanger.

of the

Set. USA

76 (1979) 6694-6698. Bibb. M.J.. Van Etten. R.A., Wrrght. Clayton.

D.A.:

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D.A.:

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H.. Wright.

ton. sistant

mouse

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Cell 26 (1981) 167-180.

cells contains

H., Adams,

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M.W. and

C.T., Bibb. M.J.. Wallace.

Proc. Nat]. Acad. Blanc,

C.T.. Walberg.

and gene organization

Mrtochondrial

the region encoding

(Margulis, 1970) that Mesocricetus (nuclear karyotype 2 N = 44) was derived relatively recently by centromeric fissioning from a Chinese or Armenian (Cricetulus migratorius) hamster (both 2 N = 22).

A.T., Barrel.

F.. Schreier.

mitochondrial

replica-

(electrophoreais

by sucrose

(Nass.

and Young.

~rwzw.s mtDNA.

to Immobilized

Bank&,

A.R.. Drouin.

B.A.. Sanger.

order

( - 7s) were isolated

mtDNA

(inset) as described

S.,

Coulson,

of Criwtulus

strands

of Hpo I fragments

amounts

Fig. I. top);

Anderson.

fragment

Fig. 3. Determination

for expert technical

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Michaels.

Mapping

DNA of individual

Communicated

by A.-M. Skalka

of mitochondrial

rapid evolution

in the