The Molecular Biology of Feeding Behavior 
larly the mouse db gene has been mapped rela- 
tive to RFLPs for interferon-a and a complement 
gene. 
To identify other RFLPs that are more tightly 
linked than these probes, we have used the chro- 
mosomal microdissection technique: small slices 
of individual chromosomes are dissected and 
tKen cloned. Separate libraries have been made 
from proximal chromosome 6, where ob maps, 
and from mid chromosome 4, where db maps. 
Two probes from the chromosome 4 library that 
flank db are ~ 1 cM apart, and two probes that 
flank ob are ~1.5 cM apart. The proximity of 
these markers and their density will enable us to 
use evolving techniques in an attempt to clone 
the ob and db genes. Techniques such as pulsed- 
field gel electrophoresis and the cloning of large 
fragments in yeast artificial chromosomes are 
currently being used. The cloning of these genes 
should further our understanding of the mecha- 
nisms that control food intake and body weight. 
Cholecystokinin Regulation, Function, and 
Expression in Human Tumors 
The hormone cholecystokinin (CCK) was origi- 
nally found in the small intestine by virtue of its 
ability to stimulate gallbladder contraction and 
pancreatic secretion in response to feeding. High 
levels of CCK have also been found in neurons of 
the mammalian brain, where it functions as a neu- 
rotransmitter. The first demonstration that CCK 
could affect behavior was reported by Gerry 
Smith and Dick Gibbs, who showed that peripher- 
ally administered CCK had an appetite-suppress- 
ing effect on rats. It has also been demonstrated 
that CCK antagonists increase feeding behavior in 
rodents. These observations suggest that the regu- 
lation and function of this gene are important in 
the control of appetite. 
We have been conducting experiments to elu- 
cidate the molecular mechanisms controlling the 
expression of the CCK gene in both brain and 
intestine. In addition, we are using a variety of 
techniques to explore the function (s) of this hor- 
mone, with particular reference to the role of the 
CCK gene in the control of feeding behavior. 
To define DNA elements involved in the regula- 
tion of the CCK gene, it was necessary to identify 
cultured cell lines that express this gene. Tumor 
cell lines derived from peripheral neuroepithe- 
lioma (a rare pediatric nerve tumor that usually 
develops in the chest wall) were found to synthe- 
size CCK. Cell lines derived from another pediat- 
ric tumor, Ewing's sarcoma of bone, also express 
CCK mRNA, and subsets of other pediatric tu- 
mors, including rhabdomyosarcoma (a malignant 
muscle tumor) , also appear to make CCK. 
Characterization of these tumor cell lines also 
suggested that there is a class of pediatric tumors 
that express this hormone and that synthesis of 
this peptide may be of diagnostic and prognostic 
value in pediatric solid tumors. Of note, how- 
ever, is that biologically active CCK is generated 
only after enzymatic cleavage of a precursor mole- 
cule (CCK prohormone) to an active form. The 
tumors that we have analyzed appear to synthe- 
size the precursor but lack the ability to cleave it. 
The failure to note an association between these 
tumors and the expression of the CCK gene previ- 
ously was likely a consequence of the failure of 
these tumors to synthesize processed CCK. Since 
the tumors do synthesize CCK mRNA and also se- 
crete the CCK precursor, we have, together with 
Bruce Schneider, developed a novel radioimmu- 
noassay that specifically detects the CCK precur- 
sor. In preliminary experiments it appears that 
the blood levels of the CCK precursor are ele- 
vated in patients with these tumors. 
The identification of human tumor cell lines 
that express the CCK gene will make it possible 
to identify the DNA sequences required for cell- 
specific expression of this gene. These observa- 
tions also suggest that CCK overproduction could 
in some cases have pathophysiologic effects in 
humans. 
To describe the possible effects of ectopic CCK 
production on the control of feeding behavior, 
we have, in collaboration with Richard Palmiter 
(HHMI, University of Washington) and Ralph 
Brinster (University of Pennsylvania), artificially 
expressed high levels of this hormone in rodents 
by fusing the metallothionein (MT) promoter to 
the CCK-coding sequence and introducing the 
MT-CCK transgene into mice. Transgenic mice 
that express high levels of the CCK precursor in 
liver, plasma, and elsewhere are now available 
and have been characterized. Most of the tissues 
that expressed the CCK gene were similar to the 
human tumor cell lines, in that none of the ex- 
pressing tissues processed the CCK precursor. 
Experiments to target CCK expression to tissues 
(such as stomach and pituitary) that can process 
the precursor to active forms are under way. 
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