MOLECULAR GENETIC APPROACHES TO METABOLISM AND METABOLIC DISEASES 
Fred D. Ledley, M.D., Assistant Investigator 
This laboratory is involved in molecular genetic 
studies of methylmalonyl CoA mutase (MCM) and 
its deficiency state in humans, methylmalonic aci- 
demia (MMA). This enzyme provides a system in 
which to investigate several areas of biological, evo- 
lutionary, and medical significance. Previous work 
in this laboratory identified a full-length human 
MCM cDNA clone that provides information about 
the primary sequence, chromosomal location, and 
expression of this mRNA in mutant cells. In the past 
year this work has been extended to include clon- 
ing and characterization of murine MCM, character- 
ization of the MCM gene locus (MUT), and molecu- 
lar cloning and analysis of mutant MCM cDNAs 
from MCM-deficient cells. 
I. Characterization and Cloning of Murine MCM. 
Murine MCM enzyme activity was assayed in pri- 
mary murine fibroblasts and crude liver extracts 
and found to exhibit activity and reaction kinetics 
similar to human MCM. A full-length mouse MCM 
cDNA was cloned from a murine liver cDNA library, 
using the human MCM cDNA as a probe. The murine 
cDNA is 3,217 bases in length and encodes a pro- 
tein of 748 amino acids (82,965 Da). There is 82% 
nucleic acid identity and 92% predicted amino acid 
sequence identity with human MCM. Extensive se- 
quence divergence is apparent in the mitochondrial 
uptake sequence, with only 20/32 base identities. 
The murine Mut locus was identified at chromo- 
some 17C-D by in situ hybridization in cell lines 
containing a 2:17 Robertsonian translocation. This 
locus is syntenic with other loci on human chromo- 
some 6 and murine chromosome 17, including 
the histocompatibility (HLA/H2) and glyoxalase 
(GLOl/Glo-1) loci. Linkage analysis among these 
loci performed in collaboration with Dr. Huda 
Zoghbi (human locus) and Dr. Jan Klein (mouse 
locus) indicates that although these loci are syn- 
tenic, they are not coUinear, as a result of a series of 
rearrangements within these chromosomes subse- 
quent to species divergence. 
MCM expression has been studied by Northern 
blots of mRNA from various mouse tissues, includ- 
ing embryonic stem (ES) cells. MCM mRNA was 
found to be expressed ubiquitously (including ES 
cells) with levels highest in the liver and kidney. 
The level of expression roughly parallels the levels 
of assayable MCM and is consistent with the pre- 
viously described "housekeeping" distribution of 
this enzyme. 
II. Homology of Higher Eukaryote MCM with Pro- 
karyote MCM. 
A prokaryote MCM from Propionibacterium 
shermanii was cloned by Dr. Peter Leadlay. This 
MCM is a heterodimer of MUTA and MUTB sub- 
units (human and mouse MCM are homodimers), 
and its function in vivo is to catalyze the reaction 
succinyl CoA-»methylmalonyl CoA, which is the re- 
verse of the reaction catalyzed by the human and 
mouse enzymes in vivo. Despite these structural 
and functional differences, homology is apparent 
among these sequences, with human MCM exhibit- 
ing 22% identity with MUTA and 65% identity with 
MUTB. No homology is apparent between the pro- 
karyotic sequences and the mitochondrial targeting 
region of the eukaryotic protein. 
III. Structure and Polymorphism Within the MUT 
Locus. 
The human MUT locus has been cloned as a 
series of overlapping phage clones, restriction- 
mapped, and the exons and intron boundaries have 
been sequenced. There is a single gene encoding 
MCM that comprises 13 exons, spanning >50 kb. 
Consensus splice donor and acceptor sites were 
identified adjacent to each exon, and a GC-rich re- 
gion is identified 5' to the first exon consistent with 
the structure of a housekeeping promoter. A pre- 
viously described Hindlll polymorphism was local- 
ized within the coding sequence, where it does not 
affect the protein sequence. A method for detection 
of this polymorphism using the polymerase chain 
reaction has been developed and distributed to in- 
vestigators interested in linkage or MMA pedigrees. 
Another polymorphism was identified, manifest as a 
24 bp insertion/deletion in the open reading frame 
of the cDNA adjacent to the exon 6 boundary. This 
appears to be a rare but benign polymorphism. The 
genomic basis for this variant mRNA has not been 
elucidated. Additional sequence polymorphisms 
were identified at sites recognized by Taql, Xhol, 
and Ndel. No discordance has been found between 
the segregation of mutant alleles and HindWl or 
Taql polymorphic markers in a small number of 
families. 
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