and reduce the steady-state level of the mutant 
mRNA. Expression studies to investigate these ef- 
fects directly are in progress. 
Part of the project described above was supported 
by a grant from the National Eye Institute, National 
Institutes of Health. 
Cloning of genes for enzymes metabolically re- 
lated to OAT. Seeking a better understanding of co- 
ordinate regulation of OAT with other, metaboli- 
cally related enzymes, Kristianne Dougherty, a 
human genetics graduate student in Dr. Valle's 
laboratory, cloned a cDNA for A'-pyrroline-5- 
carboxylate (P5C) reductase. This enzyme catalyzes 
reduction of P5C, the product of the OAT reaction, 
to proline. This human cDNA was cloned by func- 
tional complementation of the corresponding mu- 
tant in Saccharomyces cerevisiae. Utilizing the P5C 
reductase cDNA, Dougherty cloned and character- 
ized its structural gene (P5CR. 1) and a second gene 
that appears to encode a closely related P5C reduc- 
tase (P5CR.2) with 86% amino acid identity with 
P5CR. 1 . Studies are under way to investigate the pos- 
sibility that the qualitative biochemical differences 
in P5C reductase activity of various tissues may re- 
sult from tissue-specific differences in the expres- 
sion of P5CR. 1 and P5CR.2. 
Regional expression of OAT in liver. In collabora- 
tion with Dr. James Darnell (Rockefeller Univer- 
sity), Dr. Valle and his co-workers discovered that 
expression of OAT in human and rat liver is local- 
ized to hepatocytes surrounding the central vein of 
the hepatic lobule in a pattern identical to that of 
glutamine synthetase. In contrast, expression of or- 
nithine transcarbamylase and the other enzymes of 
the urea cycle is limited to periportal hepatocytes. 
Exclusion of OAT from periportal hepatocytes is hy- 
pothesized to allow for accumulation of ornithine 
necessary for ureagenesis. Transgenic experiments 
with an OAT minigene driven by a periportal- 
specific promoter are in progress to test this 
hypothesis. 
Animal models for GA. Why is the retina 
uniquely sensitive to the metabolic disturbances in 
GA? To answer this question, Tao Wang, a graduate 
student in human genetics working with Dr. Valle, is 
attempting to produce a mouse model for GA by 
gene knockout. Initial studies utilizing a targeting 
construct containing nonisogenic murine OAT gene 
sequences did not yield homologous recombination 
events. Therefore, Wang has cloned OAT gene se- 
quences from the AB- 1 embryonic stem cell line and 
is producing targeting constructs containing these 
isogenic sequences. 
Cloning and Analysis of Genes Whose 
Expression Is Limited to Photoreceptors 
Carol Freund, another human genetics graduate 
student, has cloned the human homologue of re- 
coverin, a protein that mediates the Ca^"^-dependent 
stimulation of photoreceptor guanylate cyclase and 
plays a role in recovery of the photoreceptor to the 
dark-adapted state. Freund has cloned and se- 
quenced the human recoverin cDNA; cloned, 
mapped, and characterized the recoverin structural 
gene; and identified several intragenic polymor- 
phisms useful for linkage studies. Currently she is 
investigating the possible involvement of recoverin 
in a variety of retinal degenerations. 
Reverse Genetic Approach to Cloning 
Retinal Degeneration Genes Mapping 
in or Near Xpll.2 
Dr. Michael Geraghty, an HHMI-supported postdoc- 
toral fellow in the laboratory, has developed yeast arti- 
ficial chromosome (YAC) contigs covering the OATLl 
and OATL2 loci in the Xpl 1.2 region. These loci each 
contain several OAT-related pseudogenes and are sepa- 
rated by 1 -2 mb. Dr. Geraghty used a YAC from OATLl 
to screen a human retinal cDNA library and cloned 
seven cDNAs whose genes map back to this region. 
Four of these originate from single-copy genes in 
Xpll.2; the others have considerable repetitive se- 
quences and appear to represent false positives. The 
possible role of these genes in various X-linked retinal 
degenerations that map to this region of the X chromo- 
some is under investigation. 
Molecular Studies of Inborn Errors 
of Peroxisome Biogenesis 
Drs. Jutta Gartner and Emily Germaine-Lee, post- 
doctoral fellows in the laboratory, are testing the 
hypothesis that genes encoding peroxisome- 
specific integral membrane proteins may be in- 
volved in disorders of peroxisomal biogenesis such 
as ZS. Study of these genes also should enhance un- 
derstanding of how normal peroxisomes are assem- 
bled and function. Initial studies have focused on 
PMP70, which is a member of the ATP-binding cas- 
sette (ABC) transponer protein family. In 35 pro- 
bands with ZS, representing five complementation 
groups (provided by Dr. Hugo Moser), Dr. Gartner 
found two complementation group 1 patients with 
mutations in PMP70. One mutation is a simple mis- 
sense allele, G17D; the other results in aberrant 
splicing. The splicing mutation results in replace- 
ment of the normal carboxyl-terminal 25 amino 
acids of PMP70 with an unrelated sequence of 23 
GENETICS 279 
