penditure and may reflect a genetic defect in the set 
point. Since the animals become profoundly dia- 
betic, their investigation also allows the dissection 
of the genes that play a role in the development of 
type II diabetes, which is often associated with obe- 
sity. More recently, efforts have begun to map and 
ultimately clone the fat and tubby mutations. 
Molecular Cloning of the Mouse ob 
and db Genes 
In 1950, during the course of experiments aimed 
at generating inbred mouse lines. Dr. George Snell 
(Jackson Laboratories) identified a recessive muta- 
tion, obese (ob), that resulted in massive obesity. 
Mutant animals often weigh three times as much as 
their lean litter mates at adulthood. Subsequent ge- 
netic studies localized this mutation to mouse chro- 
mosome 6. 
In 1964 Dr. Douglas Coleman identified a second 
mutation, diabetes (db). When bred on the same 
genetic background, this mutation, which was ge- 
netically localized to mouse chromosome 4, has a 
phenotype identical to ob. On the basis of studies 
employing parabiosis (crossed circulation studies) 
between obese and non-obese animals. Dr. Coleman 
concluded that animals carrying the ob mutations 
lacked a circulating appetite-suppressing factor and 
that animals with the db mutation lacked a receptor 
for this satiety factor. It has proved difficult, how- 
ever, to identify the ob and db gene products di- 
rectly, since the profound obesity of the animals 
results in numerous secondary endocrine and bio- 
chemical abnormalities. 
To isolate the o&and db genes, large crosses segre- 
gating the mutations between Mus castaneus mice 
and the two mutant strains, C57BL/6J ob/ob and 
db/db, have been completed and the progeny have 
been characterized. More than 1 ,000 meiotic events 
have been scored. These crosses have proved valu- 
able for the genetic mapping of both ob and db and 
have suggested an approach to the identification of 
genes that are responsible for the animals' type 11 
diabetes. 
In these genetic crosses the phenotype of the mu- 
tation is influenced by genes from the Mus casta- 
neus counterstrain to which the ob and db mice are 
bred. In general these variant alleles from the coun- 
terstrain tend to make ob/ob and db/db animals 
more diabetic. Careful measurement of plasma [glu- 
cose] and [insulin] among these progeny has not only 
ensured that correct assignment of genotype at the 
ob and db loci has been made but has also permitted 
identification of animals with severe diabetes, 
which is never seen among the progenitor C57BL/6J 
ob/ob or db/db animals. Thus the levels of plasma 
[glucose] and [insulin] have become important in 
their own right, since their distribution among the 
progeny of these crosses has suggested that ~80% of 
the variance in plasma [glucose] and [insulin] is 
genetic. 
The mathematical and analytical tools are 
currently available to dissect this genetic variance 
and localize the relevant genes. This strategy, which 
is currently being implemented, seeks to identify 
restriction fragment length polymorphisms (RFLPs) 
that are of the same haplotype as the counterstrain 
among the diabetic animals but are of the C57BL/6J 
haplotype in the nondiabetic animals. In such in- 
stances it can be deduced that the RFLP is linked to a 
gene that predisposes an animal to type II diabetes. 
The generation of crosses of the size referred to 
above also allows the compilation of very fine ge- 
netic maps that will facilitate the cloning of these 
mutant genes. Detailed genetic maps of the region 
around the mutations have previously been gener- 
ated. The gene order in the vicinity of ob is met- 
irp-Cf (cystic fibrosis) -0&-CPA (carboxypepti- 
dase A)-TcR/3 (T cell receptor-|8) . CF and CPA flank 
ob and are ~4 cM apart. RFLPs detected by each of 
these genes are designated met, irp, CF, and CPA. 
The gene order around db is ifa-c-jun-db-urod- 
D4Rpl-glutl. 
More recently several new RFLPs have been iden- 
tified from libraries made by chromosome microdis- 
section. The use of these libraries and a chromo- 
some-specific library for chromosomes 4 and 6 has 
also led to the compilation of a dense genetic map of 
these two chromosomes. (This work is funded by a 
grant from the National Institutes of Health.) 
These libraries have also yielded probes in close 
proximity to the ob and db mutations. In the case of 
ob, a new RFLP, D6Rckl 3, has been isolated that has 
not recombined with ob mice in 900 meioses. This 
suggests that ob has been localized to a region of 
~500 kb. In the case of db, two distal probes are 
available that are both <0.4 cM from the mutant 
gene. A proximal marker has also been identified 
that is 0.5 cM from db. These probes can be used as 
starting points with which to initiate chromosome 
walking experiments using yeast artificial chromo- 
somes (YACs) . YAC clones have been isolated for all 
of the new probes as well as for CPA ( 1 cM from ob) . 
Five independent YACs have been identified for 
the D6Rckl3 microclone that is nonrecombinant 
with ob. 
In separate experiments with Dr. Rudolph Leibel, 
another mutation, the fatty rat mutation (fa), was 
segregated in crosses with +/+ Brown Norway rats. 
Genetic linkage analysis of the progeny revealed 
that the fatty mutation is flanked by the same RFLPs 
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