lY Characterization and Cloning of Mutant MCM. 
MCM can result from genetic defects in the apo- 
enzyme (termed mut) or genetic defects affecting 
synthesis of the adenosylcobalamin cofactor {cbt). A 
series of primary fibroblasts with mut MMA were 
identified, using an in vitro assay for the MCM apo- 
enzyme. Further differentiation of mut^ defects 
(which have no residual enzyme activity) from mut~ 
defects [which have residual enzyme activity often 
with abnormal (adenosylcobalamin)] was made 
using an in situ assay. Southern blotting and North- 
ern blotting were used to analyze mut cells. At least 
six distinct alleles are delineated by Hindlll and 
Taql haplotypes, the level of expression of MCM 
mRNA, and the biochemical phenotype. A statisti- 
cally significant association was identified between 
the //wdlll(-) polymorphism and alleles in mut~ 
cells. As described previously, several mut^ cell lines 
have decreased mRNA, suggesting a primary defect 
in mRNA transcription or processing. A mut^ cell 
line known to produce a small immunoreactive 
MCM protein that is not transported into mito- 
PUBLICATIONS 
chondria was found to have a termination mutation 
at codon 17. This mRNA produces antigenic mate- 
rial from an internal AUG distal to the mitochon- 
drial targeting sequence. This mutation represents 
a complex prototype for a class of mutations in 
which absence of a targeting sequence leads to ab- 
sence of a functioning gene product. Several dis- 
tinct mutations have been found in wMf° cell lines 
that alter amino acids preserved in alignments of 
human, mouse, and P. shermanii MCM sequences. 
Several sequence changes have also been identified 
in mut~ cells, although the eflfect of these sequence 
changes on enzyme activity has not yet been char- 
acterized. From these studies, Dr. Ledley and his 
colleagues hope to catalogue evolutionary se- 
quence variations that preserve apoenzyme func- 
tion and sequence changes that disrupt apoenzyme 
function, in order to highlight critical structural do- 
mains within the enzyme. 
Dr. Ledley is also Associate Professor of Cell Bi- 
ology and of Pediatrics at the Baylor College of 
Medicine. 
Books and Chapters of Books 
Armentano, D., Peng, H., MacKenzie-Graham, L., Seh, M., Shen, R.-F., Ledley, F.D., Darlington, G.J., and Woo, 
S.L.C. 1989. Retroviral-mediated gene transfer of human PAH into mouse primary hepatocytes. In Gene 
Transfer and Gene Therapy (Beaudet, A.L., Mulligan, R., and Verma, I.M., Eds.). New York: Liss, pp 355- 
363. 
Ledley, F.D. 1989- Human gene therapy. In Biotechnology, a Comprehensive Treatise (Jacobson, G.K, and 
Jolly S.O., Eds.). Weinhem: VCH Verlagsgesellschaft, vol 7b, pp 399-460. 
Ledley, F.D. 1989. Molecular genetic studies in methylmalonic acidemia. In Gene Transfer and Gene Therapy 
(Beaudet, A.L., Mulligan, R., and Verma, I.M., Eds.). New York: Liss, pp 335-344. 
Articles 
Cheng, S.V, Martin, G.R., Nadeau, J.H., Haines, J.L., Bucan, M., Kozak, C.A., MacDonald, M.E., Lockyer, J.L., 
Ledley, F.D., Woo, S.L.C, Lehrach, H., Gilliam, T.C., and Gusella, J.F. 1989. Synteny on mouse chromosome 
5 of homologs for human DNA loci linked to the Huntington disease gene. Genomics 4:419-426. 
Jansen, R. , Kalousek, F., Fenton, WA., Rosenberg, L.E., and Ledley, F.D. 1989. Cloning of full-length 
methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. Genomics 4:198- 
205. 
Jansen, R., and Ledley, F.D. 1989. Production of discrete high specific activity DNA probes using the polymer- 
ase chain reaction. Gene Anal Tech 6:79-83. 
Ledley, F.D. 1989. Genetic counselling and the outcome of phenylketonuria (letter). J Pediatr 114:684-685. 
Peng, H., Armentano, D., MacKenzie-Graham, L., Shen, R.-F., Darlington, G., Ledley, F.D., and Woo, S.L.C. 
1988. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary 
mouse hepatocytes. Proc Natl Acad Sci USA 85:8146-8150. 
Zoghbi, H.Y, O'Brien, WE., and Ledley, F.D. 1988. Linkage relationships of the human methylmalonyl CoA 
mutase to the HLA and D6S4 loci on chromosome 6. Genomics 3:396-398. 
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