Conserved gene structure and genomic linkage for D-dopachrome
tautomerase (DDT) and MIF
* Marcia Budarf,
Christine A. Kozak,
Section on Molecular Structure and Function, National Eye Institute, Building 6 Room 331, National Institutes of Health,
Bethesda, Maryland 20892-2740, USA
Dept. of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, 19104-4399, USA
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases National Institutes of Health,
Bethesda, Maryland 20892 USA
Received: 12 February 1998 / Accepted: 8 May 1998
Abstract. Macrophage migration inhibitory factor (MIF) and D-
dopachrome tautomerase (DDT) are small proteins, which are re-
lated both by sequence and by in vitro enzyme activity. Here we
show that the gene for DDT in human and mouse is identical in
exon structure to MIF. Both genes have two introns that are located
at equivalent positions, relative to a twofold repeat in protein struc-
ture. Although in similar positions, the introns are in different
phases relative to the open reading frame. Other members of this
superfamily exist in nematodes and a plant, and a related gene in
C. elegans shares an intron position with MIF and DDT. In addi-
tion to similarities in structure, the genes for DDT and MIF are
closely linked on human Chromosome (Chr) 22 and mouse Chr 10.
Macrophage migration inhibitory factor (MIF) is a small protein
originally identified with a classic lymphokine activity of the same
name (Bloom and Bennett 1966; David 1966; Weiser et al. 1989).
There is a single functional MIF gene in the human genome (Par-
alkar and Wistow 1994), located on Chr 22q11.2 (Budarf et al.
1997). Mouse also has a single functional MIF gene located on Chr
10 but, in contrast to human, the mouse genome also contains
several processed pseudogenes (Bozza et al. 1995; Kozak et al.
1995; Mitchell et al. 1995).
A protein involved in an unusual pathway of melanin synthe-
sis, rat D-dopachrome tautomerase (DDT; Zhang et al. 1995)
shows low but significant similarity to MIF in protein sequence.
Surprisingly, although the two proteins share less than 30% amino
acid identity, MIF too can catalyze an isomerization of D-
dopachrome (Rosengren et al. 1996), even though this is unlikely
to be the physiologically relevant activity of either protein.
Here we compare the gene structures of MIF and DDT and
show that both genes have equivalent introns in positions that
delineate protein structural elements related by a twofold repeat.
Furthermore, MIF and DDT genes are closely linked in both hu-
man and mouse genomes. Database searches show that DDT and
MIF belong to a wider superfamily with members in nematodes
and plants. One nematode gene shares an intron position with the
Materials and methods
cDNA and gene sequence analysis.
Primers were designed from the
rat DDT sequence (Zhang et al. 1995) and expressed sequence tag (EST)
databases and were used for 5Ј and 3Ј RACE (Frohman 1990) of both
human and mouse DDT cDNA. For RACE, 1g of total human or mouse
liver RNA was transcribed with the appropriate primer by use of Super-
script RT (Life Technologies, Gaithersburg, Md.) following manufactur-
er’s protocols. Of the resulting cDNA template, 10% was then used for
PCR amplification with Taq polymerase (Boehringer Mannheim, India-
napolis, IN) with 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C
for 1 min, and a final 10 min extension at 72°C. 5Ј and 3Ј RACE was
performed with reagents from Life Technologies according to manufactur-
er’s instructions. Products were subcloned with the pCRII system (Invit-
rogen, Carlsbad, CA). Genomic clones were obtained by PCR from human
and mouse genomic DNA. Primer sequences are available on request.
Chromosomal localization in human.
Regional localization on human
Chr 22 used 11 somatic cell hybrids as described before (Budarf et al.
1996). For interphase fluorescence in situ hybridization (FISH), human
fibroblast nuclei (GM5565) were prepared as described (Yokota et al.
1995) except that sodium borate was omitted from the hypotonic solution.
Biotin labeling of cosmid probes and FISH were performed as previously
described (Holmes et al. 1997). DAPI was used instead of propidium
iodide to visualize the DNA. Labeling with digoxigenin was performed
according to Lichter and associates (1988).
Chromosomal localization in mouse.
A 460-bp fragment of the mouse
Ddt gene corresponding to exon 1, intron 1, and exon 2 was amplified from
genomic DNA, with primers 3345: CCCGCTAACATGCCATTCGTT and
3512: ATCAACAGGGTCATGCCAGGT. DDT was mapped by following
inheritance of RFLPs in two sets of genetic crosses: (NFS/N or C58/J ×
M.m.musculus) (Kozak et al. 1990) and (NFS/N × M.spretus)×M.spretus
or C58/J as described previously (Adamson et al. 1991).
Total RNA was extracted from 2-day and 10-month-
old mouse tissues and from NIH 3T3 cells, using RNA STAT-60 (Tel-Test
Inc., Friendswood, TX). Northern blotting followed standard methods
(Davis et al. 1986), with 20g total RNA loaded per lane on a 1.5%
agarose gel using formaldehyde buffer. A 375-bp mouse DDT cDNA
fragment was labeled with
P by random priming (Life Technologies).
Hybridization in aqueous buffer was carried out at 65°C. Blots were
stripped and reprobed with a cDNA probe for 18S RNA.
Results and discussion
Cloning mouse and human DDT. The cDNA sequences of human
and mouse DDT were obtained by RT-PCR and by 5Ј and 3Ј
*Present address: The Wilmer Ophthalmological Institute, Johns Hopkins
Hospital, Baltimore, MD 21287-9289, USA.
Correspondence to: G. Wistow
Mammalian Genome 9, 753–757 (1998).
© Springer-Verlag New York Inc. 1998