Gene dosage effect for glycinamide ribonucleotide synthetase in human fibroblasts trisomic for chromosome 21

Vol. 93, No. 4, 1980 April 29, 1980

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1286-1289

GENE DOSAGE EFFECT FOR GLYCINAMIDE RIBONUCLEOTIDE

TRISOMICFOR CHROMOSOME 21

HUMAN FIBROBLASTS

JamesA. Bartley Departments University

Received

March

J. EpsteinI

and Charles

of Pediatrics of California,

and of Biochemistry and Biophysics San Francisco, California 94143

L2,1980

Summary: The mean activity of glycinamide ribonucleotide fibroblasts from fetuses with trisomy 21 is 1.64 - times This gene dosage effect is confirmatory of the assignment this enzyme to human chromosome 21. Glycinamide

ribonucleotide

third

enzyme in the purine

coded

for

cular

interest

syndrome, ment.

since,

by that

demonstrated

for

known to be carried exists

of an extra

pathway

for

soluble

21 and normal

retardation

with (3-5),

superoxide

by chromosome GARS, and thereby

fetal

state,

it

synthetase of that of normal cells. of the gene for

(GARS; E.C.6.3.4.13), (l),

has been

This

21 (2).

chromosome

effect,

chromosome

gene to chromosome

MATERIALS

of mental

gene dosage

determined

its

biosynthetic

in the trisomic

a common cause

The presence

effect

(GAR) synthetase

by a gene on human chromosome

commensurate

SYNTHETASE IN

is

is generally

21 (4). to test

21, we have compared

with

Down of develop-

accompanied in the activity

and such a gene dosage

the validity the activities

another

whether

by a of enzymes

effect

(SODS, SOD-l), -To determine

to be

is of parti-

and abnormalities

50% increases

dismutase

reported

chromosome

associated

the

has been gene

such a dosage

of the assignment

of

of GARS in trisomy

fibroblasts.

AND METHODS

Fibroblast Strains Fibroblast strains were developed from the lung, skin, and/or diaphragm of 3 trisomic and 4 normal fetuses between 19 and 40 gestational weeks of age. Aside from a pair of dirygotic twins, one trisomic and the other normal, all donors were unrelated. Preparation of Cellular Extracts Celis were grown in Dulbecco modified Eagle medium (Gibco, Grand Island, N.Y.) containing 10% fetal calf serum, 100 u/ml penicillin and 100 ug/ml streptomycin in 5% CO2 at 370C. The cells were harvested when they reached 50 to 75% confluence, washed in saline G and in a buffer of 10 n+l Tris-HCl, 0006-291X/80/081286-04$01.00/0 Copyright All rights

0 1980 by Academic Press, of reproduction in any form

Inc. reserved.

1286

Vol.

93, No.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

pH 7.3, 0.14 M NaCl, 5 mM MgC12, and 0.29 M sucrose, and lysed in 10 mM Tris-HCl, pH 7.3 with 0.29 M sucrose, as described by Oates (6). Protein concentrations were determined by the method of Lowry using bovine serum albumin as the standard. Assays

of Enzyme Activity The assay for GARS was performed as described by Oates (6). [14C]glycine (113 in Ci/mmole, New England Nuclear) was used as the radioactive substrate and was converted to the labile irrmediate substrate of the reaction, [I4Cl phosphoribosylamine, by the activity of phosphoribosylpyrophosphate (PRPP) amidotransferase in the extract. The assay for PpgP amidotransferase was performed as described by Martin (7). Oowex l-X2 cleaned [ CJL-glutamate (1.6 Ci/mmole, New England Nuclear) was used as the radioactive substrate. For both assays, the reaction products were spotted on OEAE paper discs, washed in de-ionized water, dried, and counted in a Beckman LS 8100 liquid scintillation spectrometer. The concentration of the original cell extracts was 0.8 ml protein/ml and the final concentration in the assay 0.4 mg/ml. RESULTS Three

separate

performed, activity with

experiments,

and the results in the

p = 0.025,

order

test.

1.66,

and 1.53,

each with

are

shown

freshly

in Figure

trisomic

cells

was significantly

0.005,

and 0.05,

respectively,

The individual

ratios

respectively,

I

0’

1.72

T

N EXP. 1

1.

cell

extracts,

In each experiment, greater

than

by the one-tailed

of trisomic/normal

with

T - = N

prepared

activities

were the GARS

in normal

cells,

Wilcoxon were

1.72,

a mean of 1.64.

T - = N

1.66

T EXP. 2

N

T - = N

1.53

T

N EXP. 3

The activity of glycinamide ribonucleotide synthetase in Figure 1 extracts of trisomy 21 and normal human fetal fibroblasts. The results of three independent experiments are shown, and the mean ratio of trisomic to normal (T/N) activities was 1.64.

1287

rank

Vol.

93, No.

BIOCHEMICAL

4, 1980

The generation requires

of the

the action

of this

immediate

that

the

a difference

differences

fibroblast

in the

two types

Therefore,

extracts

However,

of extracts

were

COMMUNICATIONS

phosphoribosylamine,

in GARS activity

in PRPP amidotransferase.

transferase

RESEARCH

amidotransferase,

and normal

observed

BIOPHYSICAL

GARS substrate,

of PRPP glutamine

enzyme in trisomic

insure

AND

the

activity

was determined

did

not

actually

the activities

not found

to represent

of PRPP amido-

to be sign i ficantly

different. DISCUSSION The 1.64-fold individuals

with

of 1.5.

which

which

adds yet

the expected

the mapping

the

and 0.47,

of the

do in trisomic

another

mammalian

gene dosage

consistent

such functional

in Down syndrome

its

deleterious

of the

possibility

that

the de novo purine

here,

trisomic

(9).

serves

in this for

to confirm

and coworkers dosage

if

uric this

acid

chromocells

concentration

would

explain

believe

that anueploidy

(10).

However,

in view

earlier

in the pathway

pathway

(111,

may be rate

the hypothesis

in trisomy

that

21 remains

limiting increased

to be critically

tested.

1288

one

by which

enzymes

ACKNOWLEDGEMENTS This work was supported by Grant GM-24309 of Health. J.A.B. was supported by NIH training is an investigator of the Howard Hughes Medical

of

the mechanisms

to be understood

significant

for

in trisomic

21, and we strongly

important

(8) effects

and monosomic

If true,

are

is functionally

to occur

of enzymes

with

of the serum

effects

biosynthetic

list

GARS activity

of trisomy are

dosage effect

Therefore,

also

Scoggin

reported

increased

to the elevation

explanations

produces

is demonstrable,

those

the

consequences

appear

from

21.

with

that

reported

functional

GARS activity

effect

gene

do not

enzyme to the

in fi broblasts

contributing

expected

obtained

erythrocytes.

of th s manuscript,

respectively,

has been

to the

as they

They suggested

is a factor

close

in fibroblasts

of enzyme activities

preparation

results

some 21.

21 is quite

of GARS to chromosome

During published

in mean GARS activity

elevations

21 fibroblasts,

finding,

which

trisomy

Spurious

trisomy

1.54

increase

from the National grant GM-07085, Institute.

Institutes and C.J.E.

in

Vol.

93, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES

3. 4.

Buchanan, J.M. (1959) Harvey Lect. 64, 104-130. Moore, E.A., Jones, C., Kao, R., and Oates, D.C. (1977) Amer. J. Hum. Genet. 29, 389-396. Krone, W. and Wolf, U. (1977) Hereditas 86, 31-36. Feaster, W., Kwok, L.W., and Epstein, C.J.71977) Amer. J. Hum. Genet.

5.

29, 563-570. Epstein, C.J.,

::

6. 7. a. 9. 10.

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Tucker, G., Travis, B., and Gropp, A. (1977) Nature 267, 615-616. Oates, D.C. (1976) Ph.D. Dissertation, University of Colorado, Boulder. Martin, D.W. (1972) Anal. Biochem. 46, 234-243. Scoggin, C.H., Bleskan, J., Davidson, J.N., and Patterson, D. (1980) Clin. Res. 28, 31A. Pant, S.S., Moser, H.W., and Krane, S.M. (1968) J. Clin. Endo. Metab. 3, 472-478. Epstein, C.J., Epstein, L.B., Cox, D.R., and Weil, J. Hum. Genet. in press Seegmiller, J.E. (1980) in Metabolic Control and Disease, 8th edit., pp. 777-937, Bondy, P.K. and Rosenberg, L.E., eds., Saunders, Philadelphia.

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