Genetic Defects in the Metabolic Pathways Interconnecting the Urea 
and Tricarboxylic Acid Cycles 
chemistry reveals an additional complication: 
OAT expression is limited to one zone of the he- 
patic lobule, namely a small population of hepa- 
tocytes surrounding the central vein, whereas 
most other urea cycle-related enzymes are in the 
periportal region. This zonal expression persists 
even when OAT activity is induced 40 -fold by 
alterations in dietary protein. We hope to identify 
cis- and trans-acting elements that mediate this 
aspect of OAT expression. Furthermore, to un- 
derstand coordinated aspects of OAT regulation 
better, we are cloning the genes for other en- 
zymes that are metabolically related to OAT. We 
used complementation in Saccharomyces cere- 
visiae mutants to clone the human cDNA for 
pyrroline-5-carboxylate reductase, the enzyme 
that catalyzes the conversion of the product of 
the OAT reaction to proline. We have now cloned 
and mapped the structural gene for this enzyme 
and are beginning a comparison of the promoter 
regions of the reductase to that of OAT. 
Our interest in GA has stimulated us to identify 
other genes that may be involved in inherited reti- 
nal degenerations. We have proceeded along two 
lines of investigation: 1) cloning genes important 
for photoreceptor function and 2) using posi- 
tional cloning to identify candidate genes from a 
region of the genome known to harbor genes for 
several retinal degenerations (Xpl 1 .2-Xpll.3). 
As part of the former strategy, we have cloned the 
cDNA and cloned and mapped the structural gene 
for recoverin. Recoverin is a calcium-binding 
protein whose expression is limited to the pho- 
toreceptor. When intraphotoreceptor calcium 
falls, recoverin stimulates retinal guanylate cy- 
clase, so that photoreceptor cGMP concentra- 
tions return to high, dark-adapted levels. We are 
beginning to examine the possible role of re- 
coverin in a variety of retinal degenerations. In 
our positional cloning studies of the Xpl 1.2 re- 
gion of the human genome, we have assembled 
yeast artificial chromosome (YAC) contigs cover- 
ing most of this region and have utilized one of 
the YACs as a probe to screen a human retinal 
cDNA library. We have cloned at least five cDNAs, 
all of which map back to the correct Xpl 1.2 re- 
gion. These are being sequenced and will be used 
as probes in Northern blots of patient samples to 
test for their possible involvement in these 
disorders. 
We have also begun an investigation of inborn 
errors of peroxisome biogenesis and function. 
Zellweger syndrome, a neurodevelopmental dis- 
order fatal in infancy, is the disease paradigm. 
Cells and tissues from these patients exhibit defi- 
ciency of virtually all peroxisomal enzymes and 
lack normal-appearing peroxisomes. We have 
cloned the genes for two peroxisomal membrane 
proteins, the 70-kDa peroxisomal membrane 
protein (PMP70) and the 35-kDa protein 
(PMP35). PMP70 isamemberof theATP-binding 
cassette (ABC) transporter protein family that 
also includes the mammalian multiple-drug resis- 
tance protein (MDR) and the CFTR protein in- 
volved in cystic fibrosis. We have cloned the en- 
tire PMP70 cDNA, determined its sequence, and 
used it to clone, map, and characterize the 
PMP70 gene. In collaboration with Hugo Moser, 
we are analyzing the possible role of PMP70 in 
Zellweger syndrome. We have identified three 
PMP70 mutant alleles, and our results suggest 
that PMP70 mutations account for one of the 
Zellweger complementation groups. We also 
have determined the complete sequence of hu- 
man PMP35 and identified one mutant allele in 
another Zellweger complementation group. We 
now are focusing on expression systems to test 
directly the functional consequences of these 
mutations on peroxisomal biogenesis. 
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