Mutation eliminating mitochondrial leader sequence of

Proc.

Nati.

Acad.

Sci.

USA

Vol.

87,

pp.

3147-3150,

April

1990

Biochemistry

Mutation

eliminating

mitochondrial

leader

sequence

of

methylmalonyl-CoA

mutase

causes

mut0

methylmalonic

acidemia

(molecular

cloning/enzyme

precursors/signal

peptides/inborn

error

of

metabolism/amino

acid

sequence)

FRED

D.

LEDLEY*t4§,

RUUD

JANSEN*,

SANG-UK

NHAM*,

WAYNE

A.

FENTON¶,

AND

LEON

E.

ROSENBERG¶

*Howard

Hughes

Medical

Institute,

and

Departments

of

tCell

Biology

and

§Pediatrics,

Baylor

College

of

Medicine,

Houston,

TX

77030;

and

IDepartment

of

Human

Genetics,

Yale

University

School

of

Medicine,

New

Haven,

CT

06510

Contributed

by

Leon

E.

Rosenberg,

January

8,

1990

ABSTRACT

Methylmalonyl-CoA

mutase

(EC

5.4.99.2)

is

a

mitochondrial

matrix

enzyme

whose

activity

is

deficient

in

the

inherited

disorder

methylmalonic

acidemia.

Previous

studies

on

primary

fibroblast

cell

lines

from

patients

with

methyl-

malonic

acidemia

have

delineated

a

variety

of

biochemical

phenotypes

underlying

this

disorder.

One

cell

line

with

primary

mutase

apoenzyme

deficiency

exhibited

a

particularly

unusual

phenotype;

it

expressed

an

abnormally

small

and

unstable

immunoreactive

protein,

which

was

not

imported

by

mitochon-

dria.

We

now

report

cloning

and

sequencing

of

the

cDNA

encoding

this

mutant

protein.

The

mutation

is

a

single

base

change,

a

cytosine

--

thymine

transition,

which

introduces

an

amber

termination

codon

at

position

17

within

the

mitochon-

drial

leader

sequence.

The

immunoreactive

protein

produced

by

these

cells

reflects

translation

from

AUG

codons

down-

stream

from

this

termination

codon

and,

hence,

lacks

a

mito-

chondrial

leader

peptide.

This

mutation

represents

a

complex

prototype

for

a

class

of

mutations

in

which

absence

of

the

mitochondrial

targeting

sequence

leads

to

absence

of

a

func-

tioning

gene

product.

Methylmalonyl-CoA

mutase

[MCM;

(R)-2-methyl-3-oxopro-

panoyl-CoA

CoA-carbonyl

mutase,

EC

5.4.99.2]

is

a

mito-

chondrial

matrix

enzyme

encoded

by

a

nuclear

gene

(1-3).

It

is

translated

on

cytoplasmic

ribosomes

as

a

742-amino

acid

(82,145

Da)

precursor

protein

comprising

a

32-amino

acid

mitochondrial

leader

sequence

and

a

710-amino

acid

(78,351

Da)

mature

apoenzyme

(4).

The

leader

sequence

directs

the

precursor

to

the

mitochondria

where

it

is

recognized,

trans-

located

across

both

mitochondrial

membranes,

and

cleaved

by

a

specific

matrix

endoprotease

to

form

the

mature

subunit

(3).

The

subunits

then

assemble

into

a

homodimer

and

bind

the

adenosylcobalamin

cofactor.

Inherited

deficiency

of

MCM

causes

methylmalonic

aci-

demia

(MMA),

an

inborn

error

of

organic

acid

metabolism

in

which

failure

of

methylmalonyl-CoA

degradation

leads

to

widespread

aberrations

in

organic

acid,

amino

acid,

and

carbohydrate

metabolism

(5).

MMA

can

result

from

two

general

classes

of

genetic

defects:

those

termed

cbl,

which

involve

genes

required

for

provision

of

the

adenosylcobal-

amin

cofactor

(McKusick

nos.

251100

and

151110)

(5,

6),

and

those

termed

mut,

which

involve

the

gene

encoding

the

MCM

apoenzyme

(McKusick

no.

251000)

(5,

6).

mut

MMA

is

further

divided

into

phenotypic

subgroups

based

on

the

presence

(mut

-)

or

complete

absence

(muto)

of

detectable

enzyme

activity

in

primary

fibroblasts

(5,

7).

The

mut0

phenotype

itself

is

pleomorphic,

with

some

cells

containing

significant

amounts

of

cross-reactive

antigenic

material

(CRM),

some

expressing

severely

decreased

CRM,

some

expressing

unstable

proteins,

which

can

only

be

detected

by

pulse-chase

or

in

vitro

translation

methods

(8,

9),

and

some

expressing

no

detectable

CRM.

More

recent

studies

using

the

cloned

MCM

cDNA

have

shown

that

some

mut0

cells

have

extremely

low

levels

of

MCM

mRNA,

whereas

other

muto

and

all

mut

-

cells

have

mRNA

that

is

grossly

normal

in

quantity

and

size

(10,

11).

One

particularly

interesting

mut0

cell

line

(FB552)

has

been

described

in

which

the

mutant

MCM

gene

expresses

a

protein

that

is

smaller

than

the

normal

precursor

and

is

highly

unstable.

This

abnormal

protein

is

not

imported

or

cleaved

by

mitochondria

(9).

This

cell

line

exhibits

no

detectable

enzy-

matic

activity

and

no

stable

CRM

in

culture

(8,

9),

despite

having

grossly

normal

hybridizing

MCM

mRNA

(11).

These

data

suggest

that

the

mutant

gene

product

is

deficient

in

the

mitochondrial

targeting

signal

(9).

We

now

report

cloning

and

sequencing

of

the

MCM

cDNA

from

the

FB552

cell

line

and

identification

of

the

mutation

that

gives

rise

to

this

pheno-

type.

MATERIALS

AND

METHODS

Cloning

of

MCM

cDNA

by

the

Polymerase

Chain

Reaction

(PCR)

and

Determination

of

the

Nucleic

Acid

Sequence.

MCM

cDNA

was

cloned

by

reverse

transcription

and

PCR

ampli-

fication

from

total

RNA

by

using

the

methods

illustrated

in

Fig.

1A

(12).

Briefly,

RNA

was

prepared

by

the

hot

phenol

method

(13),

and

first

strand

cDNA

was

prepared

by

priming

reverse

transcription

with

an

oligonucleotide

complementary

to

sequences

in

the

3'

untranslated

region

of

MCM

(14).

Two

overlapping

portions

of

the

cDNA

were

amplified

by

using

oligonucleotides

corresponding

to

the

MCM

cDNA

se-

quence, subcloned

directionally

in

pGEM7zf(+),

and

se-

quenced

by

dideoxy

sequencing

by

using

oligonucleotides

complementary

to

vector

or

cDNA

sequences

as

illustrated

in

Fig.

1B.

PCR

mistakes

and

heterozygosity

were

evaluated

by

sequencing

single

isolates

of

cloned

material

and

pools

containing

15-20

independent

isolates.

Recombinant

DNA

procedures

were

carried

out

by

using

standard

methods

(14).

Oligonucleotide

sequences

will

be

given

elsewhere.

In

Vitro

Transcription

and

Translation

of

MCM

cDNA.

The

full-length

normal

and

mutant

cDNA

sequences

were

recon-

structed

by

three-part

ligation

of

the

5'

Cla

I-Acc

I

and

3'

Acc

I-Mlu

I

fragments

into

a

Cla

I-Mlu

I-digested

pGEM7zf

vector.

Subclones

of

the

full-length

MCM

cDNA

containing

5'

terminal

exonuclease

III

deletions

have

been

described

(4)

(see

Fig.

3A).

These

include

hMCM26

(bases

315-2770),

hMCM1.

1

(bases

424-2770),

hMCM5.1

(bases

721-2770),

and

hMCM8.3

(bases

928-2770).

Synthetic

RNA

was

produced

by

transcription

from

the

T7

promoter

by

T7

RNA

polymer-

ase

(Promega)

and

was

translated

into

protein

by

using

reticulocyte

lysate

(Amersham).

Incorporation

of

[35S]methi-

Abbreviations:

MCM,

methylmalonyl-CoA

mutase;

MMA,

methyl-

malonic

acidemia;

PCR,

polymerase

chain

reaction;

CRM,

cross-

reactive

antigenic

material.

*To

whom

reprint

requests

should

be

addressed.

3147

The

publication

costs

of

this

article

were

defrayed

in

part

by

page

charge

payment.

This

article

must

therefore

be

hereby

marked

"advertisement"

in

accordance

with

18

U.S.C.

§1734

solely

to

indicate

this

fact.

Proc.

Natl.

Acad.

Sci.

USA

87

(1990)

A

Clal

Accl

Kpnl

BcIl

AAAAA

*-

33

53

3

28

2

C

a

ll

pna

pGEM7zf(+)

pGEM7zf

(+)

B

5P6-.

SP

...

...

2

48

-

T7

NORMAL

cDNA

MUTANT

cDNA

SiNGLE

POOLED

IA

T

C

G

A

T

C

G

A

T

C

Gc

=

S.

t~

i~~~

GLN

...

.ACCTGAGGCAG...

...ACCTGAGGTAG

...

TOP

READING

FRAME

t

t

C(128)

HindlIl(-)

BASES

AAAAA

cDNA

SEQUENCE

1000

2000

3000

T7

-

34

-

35--

16

-

-*28

-*37

-5SP6

-*

15

-

17

BASES

1000

2UUU

FIG.

1.

Strategy

for

cloning

and

sequencing

cDNA

from

FB552.

(A)

Schematic

showing

oligonucleotides

used

for

reverse

transcrip-

tion

and

PCR

amplification

of

FB552

cDNA.

The

restriction

sites

used

for

directional

cloning

of

PCR

products

are

shown.

(B)

Strategy

for

sequencing

FB552

cDNA

showing

the

positions

of

oligonucleo-

tide

primers

internal

to

the

cDNA

sequence

and

the

direction

of

sequencing.

The

full-length

cDNA

is

described

in

ref.

4.

The

se-

quences

of

the

numbered

oligonucleotides

will

be

given

elsewhere.

onine

(Amersham)

into

protein

was

assayed

on

SDS/7-15%

polyacrylamide

gradient

gels.

Demonstration

of

Mutation

in

Genomic

DNA

by

PCR.

Genomic

sequences

spanning

the

putative

mutation

site

in

exon

II

and

a

previously

described

HindIII

polymorphism

in

exon

III

were

amplified

by

PCR

using

oligonucleotides

com-

plementary

to

exon

and

intron

sequences

(S.-U.N.,

R.J.,

and

F.D.L.,

unpublished

data)

(see

Fig.

4A).

The

presence

of

the

mutation

was

confirmed

by

direct

sequencing

of

PCR

prod-

ucts

using

methods

described

(16,

17).

The

HindIII

polymor-

phism

was

identified

by

digestion

of

PCR

products,

separa-

tion

of

fragments

on

a

15%

acrylamide

gel,

and

staining

with

ethidium

bromide

(1

gg/ml)

(S.-U.N.,

R.J.,

and

F.D.L.,

unpublished

data).

In

the

direct

analysis

of

PCR

products

by

sequencing

or

restriction

digestion,

PCR

mistakes

represent

minor

species,

which

are

not

detectable.

RESULTS

Sequence

of

the

FB552

cDNA.

Several

differences

from

the

published

reference

MCM

sequence

(hMCM7.1)

(4)

were

observed

in

the

cDNA

cloned

from

FB552.

The

mutation

identified

in

the

FB552

cDNA

is

a

transition

of

C128

-T128

which

changes

the

CAG

codon

encoding

Gln-17

to

a

TAG

(amber)

termination

codon.

There

was

no

evidence

of

the

C128

base

in

the

pooled

sample,

indicating

that

all

of

the

mRNA

contained

the

T128

variant

(Fig.

2).

We

also

observed

a

previously

described

HindIII

polymorphism

at

base

712.

Again,

there

was

no

evidence

for

heterogeneity

in

the

mRNA;

all

of

the

cDNAs

exhibited

the

HindIII(-)

sequence.

FIG.

2.

Mutation

in

FB552

cDNA.

The

sequencing

gel

shows

the

sequence

of

a

normal

cDNA

clone,

an

isolated

single

mutant

cDNA

clone,

and

a

pool

of

15

mutant

cDNA

clones.

The

C128

-T128

transition

is

apparent

when

comparing

the

normal

and

isolated

clone

sequences.

The

pooled

sequence

shows

only

T128,

indicating

that

all

of

the

mRNA

contains

this

mutation.

This

mutation

introduces

an

amber

termination

codon

at

position

17

within

the

mitochondrial

leader

sequence.

The

position

of

the

mitochondrial

leader

sequence

(open

box),

mature

apoenzyme

sequence

(solid

box),

and

cDNA

3000

sequence

are

shown

schematically

along

with

the

position

of

the

mutation

and

HindI11

polymorphic

site.

In

Vitro

Transcription

and

Translation

of

cDNA

Bearing

the

FB552

Mutation.

The

5'

segment

of

the

FB552

cDNA,

comprising

bases

52-375,

was

recombined

with

a

normal

3'

cDNA

segment

in

the

vector

pGEM7zf(+).

RNA

was

tran-

scribed

from

this

clone,

from

a

control

plasmid

containing

the

normal

52-2774

sequence,

and

from

a

series

of

clones

con-

taining

5'

terminal

deletions

(Fig.

3A).

These

clones

were

selected

such

that

the

first

AUG

of

each

clone

represents

a

different

internal

AUG

(methionine)

codon

and

potential

initiation

site

(Fig.

3A).

In

vitro

translation

of

the

normal

sequence

produces

the

expected

precursor

protein

of

-82

kDa

as

well

as

a

series

of

smaller

translation

products

(Fig.

3B,

lane

3).

The

recon-

structed

mutant

clone

does

not

exhibit

the

82-kDa

peptide,

as

expected,

although

all

of

the

smaller

translation

products

are

present

(Fig.

3B,

lane

2).

The

terminally

deleted

clones

exhibit

a

sequential

loss

of

the

larger

molecular

mass

trans-

lation

products

(Fig.

3B,

lanes

5-8)

corresponding

to

initia-

tion

at

AUG

codons

within

the

cDNA,

which

can

be

indi-

vidually

identified

by

reference

to

the

terminally

deleted

subclones

(Fig.

3A).

Specifically,

clone

hMCM26

lacks

the

first

internal

methionine

codon

at

position

311

and

does

not

make

either

the

82-kDa

protein

or

the

70-kDa

protein.

Clone

hMCM1.1

(Fig.

3B,

lane

5)

lacks

the

second

internal

methio-

nine

codon

at

position

371

and

lacks

the

63.9-kDa

translation

product.

The

dominant

band

of

material

translated

from

FB552

corresponds

to

the

product

of

-45

kDa

translated

from

clone

hMCM8.3

(Fig.

3B,

lane

7).

It

is

not

possible

to

correlate

all

of

the

bands

observed

by

in

vitro

translation

of

the

recombinant

RNA

with

those

observed

from

purified

cellular

mRNA,

because

plasmid

and

polylinker

sequences

at

the

5'

end

of

the

recombinant

RNA

transcript

may

affect

translational

efficiency

(18).

There

are

no

other

reading

frames

in

the

MCM

cDNA

that

would

encode

proteins

>10

kDa.

Identification

of

Mutation

and

Polymorphisms

in

FB552

Genomic

DNA.

To

confirm

the

presence

of

the

C128

->

T128

mutation

in

the

FB552

genome,

DNA

from

the

FB552

cell

line

was

amplified

by

PCR

and

subjected

to

direct

sequencing

by

3148

Biochemistry:

Ledley

et

al.

Proc.

Natl.

Acad.

Sci.

USA

87

(1990)

3149

A

LANE

V

w

WWv

wV

w

3

v

4

t

v

5

v

6

v

7

v

CLONE

LARGEST

PRODUCT

MCM7.1

82,145

MCM26

70,801

MCM

1.1

63,942

MCM5.1

56,751

MCM8.3

47,046

A2/

HlndIII(+/-)

t

EXON

II

,'~~N

II

#64

-0

'O

#47

88bp1168bp

#23

4

*

#59

B

A

T

C

G

1000

2000

3000

BASES

B

1

2

3

4

5

6

7

8

..

rs

A

CT

1

2

8

-=

-

-

z

_

t.

RDo

w~~~9

7

_

w

66.3

FIG.

3.

In

vitro

transcription

and

translation

of

cDNA

with

the

FB552

mutation

and

of

controls.

(A)

Schematic

of

potential

initiation

sites

within

the

long

open

reading

frame

of

the

MCM

mRNA

and

the

terminally

deleted

subclones

used

for

in

vitro

transcription

and

translation.

v,

Position

of

"in-frame"

methionine

(AUG)

codons;

A,

position

of

the

normal

termination

codons;

I

,

position

of

the

amber

mutation

(position

128).

No

alternative

reading

frames

that

could

code

for

translation

products

>10

kDa

are

present.

(B)

In

vitro

translation

of

clones

containing

normal

MCM

cDNA,

of

cDNA

carrying

the

FB552

mutation,

and

of

deletion

subclones.

Lane

1,

no

RNA;

lane

2,

translation

of

mRNA

carrying

FB552

mutation;

lane

3,

translation

of

normal

MCM

mRNA;

lane

4,

translation

of

mRNA

from

clone

hMCM26;

lane

5,

translation

of

mRNA

from

clone

hMCM1.1;

lane

6,

translation

of

mRNA

from

clone

hMCM5.1;

lane

7,

translation

of

mRNA

from

clone

hMCM8.3;

lane

8,

molecular

size

markers

(sizes

in

kDa

are

indicated).

using

the

scheme

illustrated

in

Fig.

4A.

Direct

sequencing

revealed

the

presence

of

the

C128

-*

T128

change

within

exon

II

but

also

uncovered

heterozygosity

at

this

position;

the

normal

cytosine

base

was

also

detected

(Fig.

4B).

The

HindIII

polymorphic

site

within

exon

III

was

assayed

after

PCR

amplification

(Fig.

4A)

and

was

similarly

found

to

be

heterozygous

(Fig.

4C).

DISCUSSION

The

phenotype

of

the

FB552

cell

line

has

been

characterized

in

several

previous

publications.

This

cell

line

exhibits

no

detectable

MCM

enzymatic

activity

either

by

in

vitro

enzy-

matic

assays

or

by

propionate

uptake

in

cultured

cells

and

is

hence

designated

mut0

(8,

9,

11).

Steady-state

CRM

was

not

detected

in

this

cell

line

in

an

initial

survey

(8),

although

detailed

analysis

of

CRM

production

by

using

pulse-chase

methods

revealed

the

presence

of

an

abnormally

sized

and

unstable

translation

product,

which

was

neither

translocated

into

mitochondria

nor

proteolytically

processed,

either

in

cells

or

in

vitro

(9).

Subsequent

studies

demonstrated

that

the

FB552

cell

line

contained

a

qualitatively

normal

MCM

mRNA

(11).

C

k

.

56

--1

68

88

FIG.

4.

Demonstration

of

heterozygosity

in

FB552

genomic

DNA.

(A)

Scheme

for

amplification

of

exon

II

sequences

containing

the

FB552

mutation

and

exon

III

sequences

containing

the

Hind1II

polymorphism.

The

position

of

oligonucleotides

and

regions

ampli-

fied

by

PCR

are

indicated.

Oligonucleotide

sequences

are

as

follows:

no.

23,

CCGCTCGAG1TTCCATGACTATGA;

no.

47,

CATACTG-

GCGGATGGTCCAG;

no.

59,

AATTCCTACATTCAAGGAAC-

TATAG;

no.

64,

TACTCTATGTLTCTITTCTAGGTCAG.

(B)

Se-

quence

analysis

of

exon

II

PCR

products.

Heterozygosity

is

apparent

by

the

presence

of

both

the

cytosine

and

thymine

residues

at

position

128

as

indicated.

In

contrast,

normal

DNA

shows

only

the

cytosine

at

this

position.

(C)

Ethidium

bromide-stained

PCR

products

from

exon

III

showing

heterozygosity

for

HindII1

polymorphism.

The

HindIII(-)

allele

is

identified

in

genomic

DNA

by

the

presence

of

a

256-bp

PCR

fragment

("-").

The

HindIII(+)

allele

is

identified

by

cleavage

of

the

PCR

products

into

168-

and

88-bp

fragments

("+

").

Heterozygosity

is

established

in

FB552

genomic

DNA

("552")

by

the

presence

of

the

256-,

168-,

and

88-bp

bands.

Cloning

and

sequencing

cDNA

from

the

FB552

cell

line

revealed

a

nonsense

(amber)

mutation

at

position

17,

which

terminated

translation

from

the

proper

AUG

in

the

midst

of

the

leader

sequence.

The

previously

described

immunoreac-

tive

proteins

are

apparently

produced

by

translation

from

AUG

codons

internal

to

the

normal

reading

frame

3'

of

the

mutation.

These

truncated

peptides,

lacking

the

mitochon-

drial

leader

sequence

and

a

portion

of

the

amino

terminus

of

the

mature

apoenzyme,

are

not

transported

into

the

mito-

chondria

and

do

not

express

MCM

activity

in

vivo.

These

studies

describe

the

genotype

of

a

complex

mutation

in

a

nuclear-encoded

mitochondrial

matrix

protein.

It

has

been

suggested

that

this

mutation

represents

a

prototype

for

a

class

of

mutations

in

which

the

absence

of

a

mitochondrial

targeting

sequence

leads

to

the

absence

of

a

functioning

gene

product

(9).

The

failure

of

mitochondrial

transport,

however,

is

only

one

of

several

consequences

of

this

mutation.

First,

normal

translation

of

the

long

open

reading

frame

terminates

after

amino

acid

17.

This

premature

termination

of

translation

is

the

ultimate

cause

of

the

deficiency

of

steady-

state

immunoreactive

protein

and

assayable

enzyme

activity

in

this

cell

line.

It

is

interesting

that

sensitive

methods

for

assaying

translation

indicate

that

immunoreactive

protein

is

produced

in

cultured

cells

and

can

be

produced

by

in

vitro

-

WN

.Z

-"W

--*W

Biochemistry:

Ledley

et

al.

Proc.

Natl.

Acad.

Sci.

USA

87

(1990)

methods.

When

the

FB552

cDNA

is

translated

in

vitro,

the

truncated

proteins

appear

to

be

produced

by

coincident

translation

from

internal

AUG

sequences

rather

than

by

reinitiation

of

translation

following

premature

termination,

because

the

smaller

translation

products

are

generated

in

roughly

equivalent

amounts

from

the

normal

and

FB552

cell

lines.

In

contrast,

when

terminally

deleted

subclones

are

used

as

controls,

greater

translational

efficiency

is

seen

from

the

internal

initiation

sites.

These

observations

are

consistent

with

previous

data

indicating

that

the

translational

efficiency

of

mRNA

purified

from

the

FB552

cell

line

is

significantly

lower

than

that

of

other

mRNAs

(9).

Second,

the

translated

products

are

unstable

and

can

only

be

detected

in

cells

by

pulse-chase

or

in

vitro

translation

(9).

There

are

many

potential

explanations

for

this

instability,

including

the

primary

amino-terminal

sequence

(19)

or

ter-

tiary

configuration

of

the

truncated

peptide

or

a

failure

of

proper

compartmentalization

(9,

20).

The

present

work

does

not

address

the

mechanism

underlying

this

instability.

The

impaired

translation

of

the

long

open

reading

frame

distal

to

the

amber

mutation

and

the

relative

instability

of

this

abnor-

mal

protein

product

constitute

the

proximal

cause

of

the

absence

of

steady-state

protein

and

assayable

enzyme

activ-

ity.

Third,

in

the

absence

of

a

leader

sequence,

the

truncated

translation

products

are

not

transported

into

mitochondria.

Even

if

these

proteins

retained

determinants

for

necessary

catalytic

activity,

it

is

doubtful

that

this

gene

product

would

be

biologically

active

in

the

cytoplasmic

space.

The

adeno-

sylcobalamin

cofactor,

required

for

holoenzyme

formation

and

activity,

is

synthesized

within

the

mitochondria

and

is

not

transported

into

the

cytoplasm.

In

addition,

the

methyl-

malonyl-CoA

substrate

is

also

synthesized

within

the

mito-

chondria

(5).

In

addition,

protein

factors

responsible

for

proper

assembly

of

mitochondrial

proteins

are

not

present

in

the

cytosol

(15).

Finally,

although

only

one

cDNA

species

was

cloned

from

this

cell

line,

analysis

of

genomic

DNA

indicates

that

this

cell

line

is

heterozygous

for

both

the

C128

mutation

and

a

poly-

morphic

HindIII

site.

It

was

previously

suggested

that

the

multiple

bands

of

immunoreactive

protein

observed

in

the

FB552

cell

line

represented

translation

products

of

mRNA

species

arising

from

heterozygous

alleles.

Analysis

of

poly-

morphisms

within

the

MUT

genomic

locus

indicates

that

this

cell

line

is

indeed

a

compound

heterozygote.

One

allele

contains

the

C128

-*

T128

mutation.

The

second

allele,

how-

ever,

apparently

does

not

express

a

clonable

mRNA

because

pools

of

amplified

cDNA

contain

only

T'28/HindIII(-)

tran-

scripts.

Thus,

it

is

likely

that

the

multiple

translation

products

observed

previously

arose

from

coincident

usage

of

alterna-

tive

AUG

codons

within

the

mutant

mRNA,

as

seen

in

the

in

vitro

translation

experiment,

rather

than

from

translation

from

heterologous

alleles.

Previous

studies

have

shown

that

up

to

one-third

of

mut°

cell

lines

express

a

"low

message"

phenotype

in

which

the

levels

of

hybridizable

mRNA

are

significantly

lower

than

in

normal

cell

lines

(11).

This

observation

is

consistent

with

protein

data,

which

predicted

the

presence

of

"null"

alleles

among

the

CRM+

cell

lines

(9).

It

is

likely

that

these

low

message

alleles

are

common

contributors

to

compound

het-

erozygosity

in

both

mut-

and

mut°

forms

of

MMA.

The

nature

of

this

mutation

(or

mutations)

in

genomic

DNA

has

yet

to

be

determined.

This

work

represents

an

application

of

molecular

cloning

to

the

characterization

of

mutations

in

mut

MMA.

These

studies

confirm

the

predictions

made

by

somatic

cell

complementa-

tion

and

analysis

of

the

biochemical

phenotype

of

MMA

cells

that

the

mut

complementation

group

represents

cells

with

allelic

mutations

within

the

MUT

locus.

A

variety

of

muta-

tions

have

now

been

described,

which

underlie

mut

MMA

(R.J.,

S.-U.N.,

and

F.D.L.,

unpublished

results).

There

is

no

evidence

from

population

genetic

data

or

molecular

cloning

that

MMA

represents

a

discrete

set

of

common

mutations.

Rather,

this

relatively

rare

disorder

appears

to

result

from

a

large

variety

of

mutations

as

would

be

expected

if

a

genetic

equilibrium

exists

between

new

mutations

and

the

generally

lethal

nature

of

MCM

deficiency.

The

authors

gratefully

acknowledge

the

contributions

of

Ana

Maria

Crane

and

Adelle

Hack

in

characterizing

the

FB552

cell

line

and

Tammy

Reid

in

preparing

this

manuscript.

This

work

was

supported

by

National

Institutes

of

Health

Grants

HD-24186

(F.D.L.)

and

DK-12579

(L.E.R.)

and

the

Mental

Retardation

Re-

search

Center

at

the

Baylor

College

of

Medicine

(P30-HD-24064

to

Dr.

Ed

McCabe).

F.D.L.

is

an

Assistant

Investigator

of

the

Howard

Hughes

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