Am. J. Hum. Genet.

44:781-786, 1989

Chromosomal Localization of the Gene for the Human

Trifunctional Enzyme, Methylenetetrahydrofolate
Dehydrogenase-Methenyltetrahydrofolate
Cyclohydrolase-Formyltetrahydrofolate Synthetase
Rima Rozen,* David Barton,$ Jing Du,1 Dean W. Hum,T Robert E. MacKenzie,t and
Uta Francke4
*Departments of Pediatrics and Biology and tDepartment of Biochemistry, McGill University-Montreal Children's Hospital Research Institute,
Montreal; and WDepartment of Human Genetics, Yale University School of Medicine, New Haven, CT

Summary
A trifunctional protein in man, 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of tetrahydrofolate derivatives; these derivatives supply one-carbon units for intermediary metabolism. Somatic cell hybridization and in situ hybridization were used to localize the functional gene coding
for this protein-to human chromosome 14q24, near the c-fos and TGF-0i3 loci. A second hybridizing sequence, possibly a pseudogene, was identified near the centromere of the X chromosome, at Xpll.

Introduction

The de novo synthesis of purine nucleotides in eukaryotes involves a close physical association between various enzymes -either in the form of multifunctional proteins or as multienzyme complexes. A trifunctional
protein, designated MTHFD, consisting of the enzymes
5 ,10-methylenetetrahydrofolate dehydrogenase (E.C.
1.5.1.5), 5 ,10-methenyltetrahydrofolate cyclohydrolase
(E.C.3.5.4.9), and 10-formyltetrahydrofolate synthetase
(E.C.6.3..4.3) catalyzes the interconversion of tetrahydrofolate derivatives. These derivatives supply onecarbon units for formation of the purine ring, as well
as for synthesis of methionine and thymidylate. Although the eukaryotic trifunctional arrangement is present as a single polypeptide of 100 kD, the enzymes in
prokaryotes can be found as three separate polypeptides or as a bifunctional enzyme with dehydrogenasecyclohydrolase activities (MacKenzie 1984). The mulReceived November 29, 1988; revision received January 30, 1989.
Address for correspondence and reprints: Dr. Rima Rozen, McGill
University-Montreal Children's Hospital Research Institute, 2300
Tupper Street, Montreal, Quebec, Canada H3H, 1P3.
i 1989 by The American Society of Human Genetics. All rights reserved.

0002-9297/89/4406-0001$02.00

tifunctional arrangement is suggestive of a gene fusion

event, although the possibility of gene scission has not
been ruled out (Staben and Rabinowitz 1986).
10-Formyltetrahydrofolate, a product of the cyclohydrolase reaction, is the cofactor for glycinamide
ribonucleotide transformylase (GART), the third enzyme of de novo purine biosynthesis. The human gene
for GART was initially assigned to chromosome 14 on
the basis of complementation of the Chinese hamster
Ade - E mutant, presumed to lack transformylase activity (Jones et al. 1981). However, the mutation in the
Ade - E cell line was reevaluated and assigned to the
trifunctional protein which supplies the cofactor for
the transformylase rather than to the enzyme itself
(Hards et al. 1986). Consequently, the complementation of the Ade - E mutant by chromosome 14 indicated that the gene for the trifunctional enzyme resided
on that chromosome. The isolation of a cDNA, encoding the human trifunctional protein (Hum et al. 1988),
has enabled us to map the gene by Southern analysis
of Chinese hamster x human somatic cell hybrid DNA
and by in situ hybridization to human chromosomes.
Our results confirm the localization of the gene to chromosome 14, more precisely to 14q24. Our Southern
hybridization data, it is interesting to note, identify a
781

2Rozen et al.

782
second site of hybridization near the centromere of the
X chromosome.
Material and Methods
Somatic Cell Hybrids
Primary chromosomal assignment of MTHFD-related sequences was carried out with 14 hybrid clones
derived from six independent fusion experiments between Chinese hamster cell lines and human diploid
fibroblasts or lymphocytes. The origin and characterization of these hybrids have recently been summarized
(Yang-Feng et al. 1986). For regional mapping of
MTHFD sequences on human chromosome 14, Chinese hamster x human hybrids with defined regions
of chromosomes 14 and X were employed (Francke et
al. 1976; de Martinville et al. 1985).
Hybridization Probes

For Southern analysis, two probes were used that gave

consistent results. One was a 230-bp cDNA, cloned
into the EcoRI site of pUC 13, which was isolated by
screening a Xgt 11 human liver cDNA library with a
polyclonal antibody against purified porcine trifunctional protein (Hum et al. 1988). The otherprobe, used
also for in situ hybridization, was a 2-kb cDNA, cloned
into the EcoRI site of a BluescriptTm vector (Stratagene
Cloning Systems, La Jolla), which contained the middle portion of the coding sequence for the human
trifunctional protein. The full-length cDNA (3.1 kb),
from which the 2-kb fragment was derived, had been
isolated by screening a Xgt 10 cDNA library, made from
the human colon carcinoma cell line LS-180, with the
230-bp cDNA (Hum et al. 1988).

donor, in situ hybridization of tritium-labeled probe,

and chromosome banding were carried out according
to the method of Harper and Saunders (1981) with
modifications (Yang-Feng et al. 1985).
Results

On Southern blot analysis of BamHI-digested DNA,

two human bands were detected, with molecular weights
of approximately 15 kb and 10 kb, in addition to a single 5-kb band in Chinese hamster DNA. In somatic
cell hybrids between a Chinese hamster cell line and
human diploid cells with normal chromosomes, the two
human bands did not cosegregate (fig. 1). The 15-kb
fragment was concordant with chromosome 14, and
the 10-kb fragment was concordant with the X chromosome (table 1). Furthermore, in BamHI-digested
DNA from a patient with a 48,XXXX karyotype, the
10-kb fragment was much more prominent than the
15-kb fragment.
For regional mapping of the sequence on chromosome 14, we used somatic cell hybrids derived from a
patient with a translocation t(X;14) (p22;q21) (Francke
et al. 1976) and found that the MTHFD signal was
concordant with the region 14q21--qter. When Chinese hamster x human somatic cell hybrids retaining

kb
15
10

Southern Analysis

DNA of Chinese hamster x human somatic cell

hybrids and control cell lines was extracted, digested
with BamHI, size-fractionated by agarose gel electrophoresis, blotted to nitrocellulose filters, and hybridized to the 230-bp or 2-kb cDNA insert, labeled by
the random primer method (Feinberg and Vogelstein
1983) to a specific activity of 108-109 cpm/ pg. Hybridization and washing conditions were similar to those
described elsewhere (Rozen et al. 1985; Barton et al.
1987).
In Situ Hybridization

Chromosome preparations from synchronized blood

lymphocytes of a normal male and a normal female

5-

BamHl
Southern filter of 230-bp cDNA probe hybridized
Figure I
with BamHI-digested DNA of Chinese hamster V79/380-6 (lane
1), human 46, XY (lane 2) and 48,XXXX (lane 10), and Chinese
hamster x human hybrid cell origin (lanes 3-9). Photos of lanes
3 and 10 are derived from a shorter exposure of the filter. The human
chromosomal content of the hybrids, as established by karyotyping,
is as follows: hybrids in lanes 3, 5, 6, 8, and 9 contain human chromosome 14, and those in lanes 4 and 7 do not. The regions of the
X chromosome present were Xp22-cen (lane 3), Xpll.3-qter (lane
4), Xpter-21 (lanes 5, 6), Xp2l-qter.(lanes 7, 9), and complete X
(lane 8). The 10-kb fragment shows X-specific dosage in lane 10 and
can be assigned to region Xpll.3-cen.

783

Trifunctional Folate-dependent Enzyme

Table I
Correlation of Trifunctional Enzyme with Human Chromosomes
in Chinese Hamster x Human Somatic Cell Hybrids

HUMAN
FRAGMENTS

HUMAN CHROMOSOMES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

15 kb

10 kb

XII-12B aza ..........

XV-1 8A-8b-Gl ........

+
+

XV-18A-lOb-D4 ......
XV-18B-7a-N4 ........
XVII-18B-lOa .........
XVIII-23H-a HAT

+
+
+

+
+
+

+
XVIII-54A aza ........
+
XVIII-54A-3a HAT ....
XXI-22A-g-la .........+
XXI-23A-2c ..........

+
+

P + P + + P - + - - + + P + + + + P - + P - + + +
+
+
P +PP+
- + P - - + - - - - + + - - - + - + - P ----+
- + + + + + - - - - + + +
-- + + + -

HYBRID CLONE

.....

XXI-51B .............

+
-

+ + + - - + + + +

+
-

+
+

+
+

+
+

+ ++
+ +

+
+ + +
++
+ +

+ + + + + +
+ - - - L - - + - + + - + - - + - + + ++ - + - + + + - - L .+ + - - +
+ - ----+ + -+ -- + - + -+- + + + + + + - +
P +
P - - + - - + - + - P + + +
+++
+++++ - - + + + ++ + + - + P +

Fragment Distribution

15 kb:
2 3
No. of discordant hybrids ....55....
9 11 9 10
.......
Total no. of hybrids .....
10 kb:
4 5 6
No. of discordant hybrids ....5....
9 11 9 10
.......
Total no. of hybrids .....

6 4 8 4 7 9 3 3 8 0 6 3 7 6 2 5 4 4 1 5
11 8 10 10 10 11 9 11 11 11 11 11 11 10 11 11 10 11 8 11
7 3 7 7 7 8 4 4 7 4 9 6 6 7 5 6 5 7 0 7
11 8 10 10 10 11 9 11 11 11 11 11 11 10 11 11 10 11 8 11

NOTE.-P = partial chromosome; L = low frequency (P and L data were excluded from primary assignment).

different regions of the human X chromosome were

tested for presence or absence of the 10-kb fragment,
the sequence could be localized to the proximal short
arm near the centromere (region pll.3--cen).
In situ hybridization, initially to normal human male
cells, was carried out with the 2-kb cDNA. When no
significant label above background was obtained on the
X chromosome, the experiment was repeated using female cells. The results from both experiments were combined. In a total number of 105 cells, 356 labeled sites
were identified. A specific site of hybridization was detected at band 14q24; 38 (36%) of all cells had label
at this site, and 38 (11%) of all grains were at this site.
The distribution of label over all human chromosomes
is shown in figure 2. On the X chromosome a small
peak was seen at the centromere and band p1l. This
peak may reach borderline significance if the fact is considered that 48 of the 105 cells scored were derived from
a male with a single X chromosome.
Figure 3 summarizes the localization of MTHFD on
chromosome 14. The bracket on the left indicates localization of the sequence on the basis of the transloca-

tion hybrids with a breakpoint in band q21, as indicated by the arrow. The autoradiographic silver-grain
distribution shown on the right indicates the peak of
grains in band q24.
Discussion
Our assignment of the gene for the human trifunc-

tional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, to chromosome 14q24,

by somatic cell hybridization and by in situ hybridization, is in agreement with previous mapping data obtained by complementation analysis (Hards et al. 1986).
Both the detection of a second hybridizing sequence
with two cDNA probes and its localization on the X
chromosome are novel findings. The X chromosomal
sequence was assigned close to the centromere by Southern blot analysis of hybrid cell lines but in in situ hybridization experiments did not hybridize as well as the
one on chromosome 14.
The localization of MTHFD in band 14q24 places

784 Rozen et al.

784

IL0I

I,1

Ip

IP

.11.I

qI p

q IP

**

p
8

20E

0.

.1. .

10

11

12

13

14

15

16

17

18

19

20 21 22

HIgure 2
In situ hybridization of tritium-labeled 2-kb cDNA probe yielded tnis autoradiographic silver-grain distribution over chromosomes in 48 male and 57 female metaphase spreads.

it close to the loci for c-fos (Ekstrand and Zech 1987)

and TGF-D3 (Barton et al. 1988) but distant from the
genes for al-antitrypsin and for the immunoglobulin
heavy-chain gene cluster (Cox et al. 1982; Ropers et
al. 1987).
Since human chromosome 14 complemented the mutation in the Chinese hamster Ade - E cell line, deficient
in trifunctional enzyme activity, the gene on chromosome 14 must be expressed. Whether the gene on the
X chromosome is a functional one or a pseudogene
remains to be determined. We have recently isolated
genomic sequences, coding for this folate-dependent
protein, from an X chromosome library (data not
shown). Characterization of these sequences should
prove useful in this regard. If the gene on the X chromosome proves to be a pseudogene, it would represent
another addition to the lengthy list of pseudogenes on
the X chromosome gene map (Willard et al. 1985).
The fourth enzyme of purine synthesis, phosphor-

MTHFD

p
_
1
11.2

I*

11.2

12
0@

13

_4
q

21
22

0*

23

24
*

31
32
[.

14
Figure 3

Right, Grain distribution on chromosome 14 reveals

peak at band 14q24. Left, Brackets indicate region of chromosome
14 retained in series XII hybrids with translocation breakpoint at
band 14q21 (arrow). Hybrids with region 14q21-qter were positive
for the 15-kb human BamHI fragment.

ibosylformylglycinamide amidotransferase (FGART),

has also been assigned to human chromosome 14 (Jones
et al. 1981). FGART cannot be part of the 100-kD polypeptide with dehydrogenase, cyclohydrolase, and synthetase activities, since all domains have been accounted
for; a more complex association at the level of the gene
cannot be ruled out.
GART, the third enzyme of de novo purine synthesis, requires 10-formyltetrahydrofolate as a cofactor. The
transformylase has been purified from several sources,
including a murine lymphoma cell line (Caperelli 1985),
chicken liver (Daubner et al. 1985), and HeLa cells
(Daubner et al. 1986). GART was first shown to be
part of a trifunctional protein in Drosophila (Henikoff
et al. 1986); the other two activities on the polypeptide
are phosphoribosylglycinamide synthetase (GARS) and
phosphoribosylaminoimidazole synthetase (AIRS), the
second and fifth enzymes of purine biosynthesis, respectively. A similar arrangement has been documented in
avian liver (Daubner et al. 1985) and HeLa cells (Daubner et al. 1986). Using the somatic cell genetics approach, Patterson et al. (1981) demonstrated coordinated
regulation of GARS and AIRS and assigned these two
activities to human chromosome 21. Subsequently,
GART was also mapped to chromosome 21 (Hards et
al. 1986). The combined data from these reports are
highly indicative of a single locus, coding for a trifunctional polypeptide with GART, GARS, and AIRS activities, situated on chromosome 21 in man.
Several enzymes in purine and pyrimidine synthesis
have been shown to be part of multifunctional proteins
or multienzyme complexes (Caperelli et al. 1980). Presumably this type of arrangement contributes to moreefficient regulation of these critical pathways. The definitive mapping of one trifunctional protein and of FGART
to chromosome 14 and the assignment of three other
purine biosynthetic enzymes to chromosome 21 may

785

Trifunctional Folate-dependent Enzyme

have interesting implications for disorders of nucleotidemetabolism that map to those regions of the genome.
It has been known for some time that patients with
trisomy 21 have elevated purine levels (Fuller et al. 1962);
however, the contribution of GART, GARS, and AIRS
to the pathogenesis of Down syndrome remains to be
determined. By contrast, there have been no reports on
the clinical significance of the trifunctional folatedependent protein on chromosome 14. It is possible
that a mutation in this enzyme, central to folic acid metabolism, is incompatible with life. Further investigation of the Ade - E mutant, with reduced cyclohydrolase activity, should yield some information on the
metabolic consequences of a deficiency of this protein.

Acknowledgments
This paper is dedicated to the memory of Maria Belen
Escobar-Pelletier, whose tragic death occurred during her assistance with this project. We thank M. Ranger for preparation of the manuscript. This work was supported by the Medical Research Council of Canada and by NIH research grant
GM26105. R.R. and D.W.H. are recipients of a ChercheurBoursier Award and a Pre-doctoral Fellowship, respectively,
from the Fonds de la Recherche en Sante du Quebec. J.D.
was supported by a fellowship from China Medical University in Shen-yang.

References
Barton, D. E., M. Arquint, J. Roder, R. Dunn, and U. Francke.
1987. The myelin-associated glycoprotein gene: mapping
to human chromosome 19 and mouse chromosome 7 and
expression in quivering mice. Genomics 1:107-112.
Barton, D. E., B. E. Foellmer, J. Du, J. Tamm, R. Derynck,
and U. Francke. 1988. Chromosomal mapping for transforming growth factors I2 and 3 in man and mouse: dispersion of TGF-1 gene family. Oncogene Res. 3:323-331.
Caperelli, C. 1985. Mammalian glycinamide ribonucleotide
transformylase: purification and some properties. Biochemistry 24:1316-1320.
Caperelli, C. A., P. A. Benkovic, G. Chettur, and S. J. Benkovic.
1980. Purification of a complex catalyzing folate cofactor
synthesis and transformylation in de novo purine biosynthesis. J. Biol. Chem. 255:1885-1890.
Cox, D. W., V. D. Markovic, and I. E. Teshima. 1982. Genes
for immunoglobulin heavy chains and for a-1-antitrypsin
are localized to specific regions of chromosome 14q. Nature 397:428-430.
Daubner, S. C., J. L. Schrimsher, E J. Schendel, M. Young,
S. Henikoff, D. Patterson, J. Stubbe, and S. J. Benkovic.
1985. A multifunctional protein possessing glycinamide

ribonucleotide synthetase, glycinamide ribonucleotide

transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis. Biochemistry 24:7059-7062.
Daubner, S. C., M. Young, R. D. Sammons, L. F. Courtney,
and S. J. Benkovic. 1986. Structural and mechanistic studies
on the HeLa and chicken liver proteins that catalyze glycinamide ribonucleotide synthesis and formylation and aminoimidazole ribonucleotide synthesis. Biochemistry 25:
2951-2957.

de Martinville, B., L. M. Kunkel, G. Bruns, F. Morle, M.

Koenig, J. L. Mandel, A. Horwich, S. A. Latt, G. F. Gusella,
D. Housman, and U. Francke. 1985. Localization of DNA
sequences in region Xp2l of the human X chromosome;
search for molecular markers close to the Duchenne muscular dystrophy locus. Am. J. Hum. Genet. 37:235-249.
Ekstrand, A., and L. Zech. 1987. Human c-fos protooncogene mapped to chromosome 14, band q24.3-q31:
possibilities for oncogene activation by chromosomal rearrangements in human neoplasms. Exp. Cell Res. 169:
262-266.
Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high
specific activity. Anal. Biochem. 132:6-13.
Francke, U., N. Busby, D. Shaw, S. Hansen, and M. G. Brown.
1976. Intrachromosomal gene mapping in man: assignment
of nucleoside phosphorylase to region 14cen- 14q21 by interspecific hybridization of cells with a t(X;14)(p22;q21)
translocation. Somatic Cell Genet. 2:27-40.
Fuller, R. W., M. W. Luce, and E. T. Mertz. 1962. Serum
uric acid in Mongolism. Science 137:868-869.
Hards, R. G., S. J. Benkovic, M. L. Van Keuren, S. L. Graw,
H. A. Drabkin, and D. Patterson. 1986. Assignment of a
third purine biosynthetic gene (glycinamide ribonucleotide
transformylase) to human chromosome 21. Am. J. Hum.
Genet., 39:179-185.
Harper, M. E., and G. F. Saunders. 1981. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83:431-439.
Hienkoff, S., M. A. Keene, J. S. Sloan, J. Bleskan, R. Hards,
and D. Patterson. 1986. Multiple purine pathway enzyme
activities are encoded at a single genetic locus in Drosophila. Proc. Natl. Acad. Sci. USA 83:720-724.
Hum, D. W., A. W. Bell, R. Rozen, and R. E. MacKenzie.
1988. Primary structure of a human trifunctional enzyme:
isolation of a cDNA encoding the human trifunctional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. J. Biol. Chem. 263:15946-15950.
Jones, C., D. Patterson, and F. T. Kao. 1981. Assignment of
the gene coding for phosphoribosylglycineamide formyltransferase to human chromosome. 14. Somatic Cell Genet. 7:399-409.
MacKenzie, R. E. 1984. Biogenesis and interconversion of
substituted tetrahydrofolates. Pp. 255-306 in R. L. Blakley and S. J. Benkovic, eds. Folate and pterins: chemistry

786
and biochemistry of folates. Vol. 1. John Wiley, Philadelphia.
Patterson, S., S. Graw, and C. Jones. 1981. Demonstration,
by somatic cell genetics, of coordinate regulation of genes
for two enzymes of purine synthesis assigned to human
chromosome 21. Proc. Natl. Acad. Sci. USA 78:405-409.
Ropers, H. H., T. Gedde-Dahl, Jr., and D. W. Cox. 1987.
Report of the Committee on the Genetic Constitution of
Chromosome 13, 14, 15 and 16. Cytogenet. Cell Genet.
46:213-242.
Rozen, R., J. Fox, W. A. Fenton, A. L. Horwich, and L. E.
Rosenberg. 1985. Gene deletion and restriction fragment
length polymorphisms at the human ornithine transcarbamylase locus. Nature 313:815-817.
Staben, C., and J. C. Rabinowitz. 1986. Nucleotide sequence

Rozen et al.
of the Saccharomyces cerevisiae ADE3 gene encoding Cltetrahydrofolate synthase. J. Biol. Chem. 261:4629-4637.
Willard, H. F., M. H. Skolnick, P. L. Pearson, and J.-L. Mandel. 1985. Report of the Committee on Human Gene Mapping by Recombinant DNA Techniques. Cytogenet. Cell.
Genet. 40:360-489.
Yang-Feng, T. L., L. J. DeGennaro, and U. Francke. 1986.
Genes for synapsin I, a neuronal phosphoprotein, map to
conserved regions of human and murine X chromosomes.
Proc. Natl. Acad. Sci. USA 83:8629-8633.
Yang-Feng, T. L., G. Floyd-Smith, M. Nemer, J. Drouin, and
U. Francke. 1985. The pronatriodilatin gene is located on
the distal short arm of human chromosome 1 and on mouse
chromosome 4. Am. J. Hum. Genet. 37:1117-1128.