MAMMALIAN DEVELOPMENTAL GENETICS 
Gregorys. Barsh, M.D., Ph.D., Assistant Investigator 
Genomic and Functional Characterization 
of the Mouse agouti (A) Locus 
A major focus in Dr. Barsh's laboratory has been 
the positional cloning and analysis of preexisting 
mouse developmental mutations. In the past year, 
candidate cDNAs for the mouse A locus have been 
isolated, and molecular defects associated with 
two recessive lethal A mutations have been 
characterized. 
Mice of agouti genotype A/ A have hairs with a 
subapical band of pheomelanic (yellow) pigment 
on an otherwise eumelanic (black) background. In 
general, recessive agouti mutations produce a com- 
pletely black hair and dominant agouti mutations 
produce a completely yellow hair. Some agouti mu- 
tations, such as {lethal non-agouti), result in 
embryonic lethality when homozygous; the labora- 
tory has previously shown that these recessive lethal 
genes represent three complementation groups. In 
addition, most dominant agouti mutations, such as 
A^ (lethal yellow) and A"^ {viable yellow) , are asso- 
ciated with premature infertility, adult-onset obe- 
sity, increased somatic growth, and increased sus- 
ceptibility to tumor formation. Transplantation 
studies suggest that the agouti gene acts in cells of 
dermal origin to control a signaling pathway that 
ultimately determines the type of pigment (pheo- 
melanin or eumelanin) synthesized by follicular 
melanocytes. 
To isolate the gene(s) affected by agouti muta- 
tions. Dr. Barsh has taken a systematic approach in 
which a physical map, based on the somatic cell 
genetic technique of radiation hybrid mapping, has 
been paired with the development of a genetic map, 
based on an interspecific backcross using the A*' and 
a'' alleles opposite a Mus castaneus chromosome 
(which carries the A allele) . The group found that 
the genetic maps surrounding A^ and a' are indistin- 
guishable and that two closely linked proviral inser- 
tions, Emv-15 and Xmv-10, are not responsible for 
the A^ and a mutations, respectively. The results 
also eliminated several candidate genes (phospholi- 
pase C, Src, phosphotyrosine phosphatase) from 
consideration as mediators of the tumor-promoting 
aspects of the A^ and A'^ alleles. (The work de- 
scribed above was supported, in part, by a grant 
from the National Center for Human Genome 
Research.) 
The occurrence of a radiation- induced mutation 
doubly heterozygous for limb deformity {Id) and 
extreme non-agouti has provided a fortuitous 
breakthrough in Dr. Barsh's efforts to understand the 
molecular basis of agouti mutations. As described 
by Dr. Rick Woychik and his colleagues, this muta- 
tion is associated with a chromosomal inversion, 
IslGsO, that allows the isolation of genomic se- 
quences closely linked to the A gene. Dr. Barsh's 
laboratory used these genomic fragments to isolate 
two cDNAs that span the IslGsO breakpoint and to 
investigate the structure of the A^ and a'' chromo- 
somes. One cDNA detects a 900-bp mRNA expressed 
in the skin and testes of A/ A mice and in many addi- 
tional organs (brain, kidney, liver, spleen) of A^/a 
mice. This cDNA has the potential to code for a pro- 
tein of ~ 1 7 kDa that contains two potential amino- 
linked glycosylation sites and a putative amino- 
terminal signal sequence. Similarity searches using 
the predicted peptide sequence are not revealing. 
However, the predicted 5'-untranslated region con- 
tains an ~180-bp region that is nearly identical to 
the nontranscribed strand of a retrotransposon ele- 
ment, VL-30. Expression of this candidate agouti 
cDNA in the same cell with one or more VL-30 ele- 
ments, which are developmentally regulated, is 
likely to result in the formation of double-stranded 
RNA. Thus it is possible that expression of the 
agouti gene is regulated, in part, via the production 
of natural antisense RNA. A second cDNA in- 
terrupted by the IslGsO breakpoint is 2.1 kb in 
length and detects a 6-kb mRNA expressed in the 
testes of A/ A and A^/a mice. Coding regions of the 
900-bp and the 2.1-kb cDNA are transcribed from 
opposite strands and are located within the same 
genomic fragment, but are spliced such that their 
exons are non-overlapping. 
Using genomic cloning and mapping, this labora- 
tory has determined that the A^ mutation is asso- 
ciated with a chromosomal rearrangement that 
affects the mobility of multiple high-molecular- 
weight restriction fragments centromere-proximal 
to the coding sequences of both the 900-bp and the 
2.1-kb cDNA they have isolated. By contrast, the a" 
mutation is associated with a deletion of at least 25 
kb of DNA centromere-distal to cDNA-coding se- 
quences. Dr. Barsh speculates that A^ and a" affect 
agouti coat color by altering regulatory sequences 
5' and 3', respectively, of the 900-bp cDNA, to pro- 
duce either a gain {A^) or a loss {a'') of function. 
To investigate the role of the cDNAs the laboratory 
has isolated in the increased tumor susceptibility 
observed in A'^/— and A^/— animals, they have, in 
collaboration with Drs. George Wolff and Wendy 
GENETICS 149 
