GENETIC DEFECTS IN THE METABOLIC PATHWAYS INTERCONNECTING 
THE UREA AND TRICARBOXYLIC ACID CYCLES 
David Valle, M.D., Investigator 
Dr. Valle is interested in human biochemical ge- 
netics, with the objective of understanding the mo- 
lecular basis, heterogeneity, pathophysiology, and 
treatment of a variety of inborn errors. During the 
past year he has continued his studies of the molec- 
ular biology, structure -function relationships, and 
inherited defects of ornithine-8 -aminotransferase. 
Deficiency of this mitochondrial matrix enzyme 
causes gyrate atrophy of the choroid and retina, a 
rare, autosomal recessive, blinding condition asso- 
ciated with accumulation of ornithine in all body 
fluids. He has also analyzed the phenylalanine 
hydroxylase gene in black Americans with phenyl- 
ketonuria, with the goal of determining the molec- 
ular basis and origin of the phenylalanine hydrox- 
ylase mutant allele (s) in this population. Finally, Dr. 
Valle has collaborated in a study of the molecular 
basis of pseudohypoparathyroidism, a dominantly 
inherited inborn error in calcium homeostasis. 
I. Gyrate Atrophy of the Choroid and Retina. 
A. Molecular and cell biology of ornithine-h-ami- 
notransferase (OAT). To understand better the reg- 
ulation of OAT transcription, John Engelhardt, a 
student in Dr. Valle's laboratory, has constructed a 
set of hybrid OAT promoter chloramphenicol 
acetyltransferase (CAT) constructs containing vari- 
able regions of the immediate 5 '-flanking region of 
the OAT gene. Transfection of these hybrid con- 
structs into a variety of mammalian cells has shown 
that the 5'-flanking region to -134 (where +1 is 
the transcriptional start site) is sufficient for maxi- 
mal promoter activity. Furthermore, constructs con- 
taining this same region transfected into human 
retinoblastoma cells (Y79) but not human liver or 
kidney lines exhibit a 3-5 times increased expres- 
sion in response to estrogen. The same construct 
exhibits cAMP-dependent expression (3-5 times) in 
human and rodent liver cell lines and in human 
kidney cells. These observations are consistent with 
the prediction from sequence analysis of possible 
estrogen and/or cAMP cis-acting elements in a 32 
bp region overlapping the transcriptional start site 
(-25 to +7) that exhibits incomplete dyad symme- 
try and has homology with both estrogen and cAMP 
responsive elements. DNase I footprint analysis of 
this same region with nuclear extracts from various 
cell lines shows clear footprints over three Spl- 
binding sites and to the CAAT box at -73. However, 
a clear footprint over the suspected estrogen and/ 
or cAMP responsive elements has not been detect- 
ed. This may indicate that the candidate sequence 
is not a region of protein binding or, more likely, 
that the binding is more complex (perhaps involv- 
ing interaction of several proteins) and the correct 
conditions have not yet been produced. Additional 
footprinting, gel-shift, and mutagenesis experi- 
ments are in progress to clarify this point. 
Dr. Valle and his colleagues have also found that 
alternative splicing of OAT mRNA occurs: most ma- 
ture transcripts lack exon 2, an 87 bp exon in the 
5'-untranslated region of the OAT mRNA, even 
though it is flanked by acceptable splice consensus 
sequences. Dr. Valle and his colleagues have now 
found that <5% of the OAT mRNA in a variety of 
human cell lines and tissues contains exon 2, and 
this proportion does not change when OAT expres- 
sion is stimulated by cAMP in HepG2 cells. By con- 
trast, >50% of OAT transcripts in mRNA isolated 
from a variety of monkey cell lines and tissues con- 
tain exon 2. Thus, as has been shown for the meta- 
bolically related enzyme argininosuccinate syn- 
thetase, there is a species-dependent, quantitative 
difference in the splicing pattern of OAT mRNA. 
Whether this is related to cis-acting, sequence dif- 
ferences in the relevant introns between monkey 
and human OAT or due to trans-acting differences 
in the splicing apparatus is being investigated. 
B. Analysis of the mutations causing gyrate atro- 
phy (GA) of the choroid and retina. Dr. Valle and 
his colleagues have characterized the OAT mRNA 
and OAT antigen phenotype in fibroblast samples 
from 72 GA pedigrees (27 Finnish, 45 non-Finnish), 
92% of which express a normal or only moderately 
reduced amounts of normally sized OAT mRNA. Of 
the mRNA^ probands, 67% are CRM"; all of the 
mRNA" lines are also CRM". Using a combination of 
RNase A protection assays and polymerase chain re- 
action (PGR) amplification and sequencing of OAT 
exons in genomic DNA or fibroblast OAT cDNA, Dr. 
Valle and his co-workers have delineated 15 differ- 
ent mutant alleles that account for 75% or 52% of 
the 144 possible abnormal alleles from probands in 
the 72 pedigrees. Two alleles recently identified in- 
clude one with two missense mutations in close 
proximity (20 codons apart) and a second with a 
Continued 
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