Genetic Defects in the Metabolic Pathways 
Interconnecting the Urea 
and Tricarboxylic Acid Cycles 
David L. Valle, M.D. — Investigator 
Dr. Valle is also Professor of Pediatrics, Medicine, and Molecular Biology and Genetics at the Johns 
Hopkins University School of Medicine. He received both his undergraduate degree in zoology and his 
medical degree from Duke University. His internship and residency in pediatrics were completed at the Johns 
Hopkins Hospital. His postdoctoral research in metabolism was done at the NIH. 
TTTTUMAN biochemical genetics has been a 
JLXfruitful area of study since its beginning 
with the work of Sir Archibald Garrod early in this 
century. Inherited defects in our body's chemis- 
try or, as Garrod called them, inborn errors of 
metabolism, are intrinsically interesting and 
serve as important models for all genetic diseases. 
My colleagues and I have been involved in the 
study of several aspects of these disorders, in- 
cluding clinical diagnosis, biochemical charac- 
terization, delineation of pathophysiologic mech- 
anisms, development of new therapeutic 
approaches, and molecular studies of the caus- 
ative mutations. 
We have focused on disorders of amino acid 
metabolism, particularly those involving two 
fundamentally important areas of metabolism: 
the urea cycle, which is involved in the conver- 
sion of excess nitrogen from a toxic to a nontoxic, 
readily excreted form, and the tricarboxylic acid 
cycle, an essential component of energy metabo- 
lism. Recently we have extended these interests 
to include inborn errors in the biogenesis of the 
peroxisome, a ubiquitous, subcellular organelle 
that contains about 40 enzymes important in a 
variety of anabolic and catabolic processes. 
One of the amino acid disorders that we are 
studying is an inborn error of ornithine metabo- 
lism known as gyrate atrophy of the choroid and 
retina (GA) . This progressive, blinding chorioret- 
inal degeneration with associated cataract forma- 
tion is inherited as an autosomal recessive trait. 
The primary biochemical defect is deficiency of 
the enzyme ornithine-5-aminotransferase (OAT), 
which results in an approximate 1 0-fold accumu- 
lation of ornithine in all bodily fluids. 
Despite the systemic nature of the metabolic 
abnormality in GA, the clinical phenotype is lim- 
ited to the eye. Thus GA is one of a very few iso- 
lated, inherited retinal degenerations for which a 
primary biochemical defect is known. As such, 
GA serves as a model for more common inherited 
retinal degenerations whose biochemical basis 
remains enigmatic. We have been involved in a 
variety of GA studies, including the development 
and testing of potential therapies, investigation of 
possible explanations for the sensitivity of the ret- 
ina to the systemic biochemical abnormalities, 
elucidation of regulatory mechanisms control- 
ling the expression of the OAT gene, and determi- 
nation of the molecular defects causing GA. The 
last depends on the results of earlier studies in 
our laboratory, including isolation and sequenc- 
ing of a full-length cDNA clone of human liver 
OAT and determination of the structure and orga- 
nization of the human OAT gene. 
The OAT-catalyzed reaction is an essential step 
in the pathway that interconnects the urea cycle 
with the tricarboxylic acid cycle. Thus it is not 
surprising that the regulation of OAT is complex 
and, in liver, coordinated with other urea cycle- 
related enzymes. We recognized a sequence mo- 
tif in the 5'-flanking region of OAT that is similar 
to sequences in the 5'-flanking region of several 
other urea cycle-related genes, including orni- 
thine transcarbamylase, argininosuccinate synthe- 
tase, arginase, and glutamine synthetase. We des- 
ignated this candidate cis-acting regulatory 
element a urea cycle element (UCE) . Expression 
studies with hybrid constructs of various portions 
of the 5'-flanking region of the OAT gene linked 
to a reporter gene indicate that the UCE motifs 
have functional roles. DNase I footprint assays 
and gel retardation experiments with a series of 
synthetic oligonucleotides corresponding to the 
normal and mutant variations of the UCE se- 
quence indicate that at least three proteins, each 
with distinct but overlapping recognition sites, 
bind to the UCE. 
Using a concatamer of the UCE sequence as a 
radiolabeled probe, we have cloned cDNAs for 
two specific DNA-binding proteins that are candi- 
dates for trans-acting regulators of the homeo- 
static mechanisms that adjust ureagenic capacity 
to nitrogen intake. Transient transfection experi- 
ments in which we co-transfect one of these 
cDNAs with a second construct containing two or 
four UCE in the promoter of a reporter gene indi- 
cate that the protein product of our cDNA clone 
does activate the expression of genes with UCE in 
their promoters. 
To better understand the coordinated regula- 
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