to hypoxia is determined by both increased tran- 
scription and a novel mechanism for increasing 
RNA stabiUty. 
III. Measurement of mRNA by Competitive Polymer- 
ase Chain Reaction. 
Understanding the regulation of hematopoiesis 
depends in part on the ability to measure mRNA 
^ecies of hematopoietic growth factors in defined 
cell populations accurately. Conventional methods 
of mRNA analyses are not sensitive enough to de- 
tect mRNA in samples limited by either low cell 
number or low copy number per cell and permit 
only crude quantitation of mRNA. Because of ex- 
traordinarily high sensitivity, PCR is being used for 
amplifying cDNA copies of low-abundance mRNA. 
However, quantitation has been unreliable, be- 
cause the amount of PCR product increases expo- 
nentially with each cycle of amplification, and 
therefore minute differences in any of the variables 
that affect the rate of amplification can alter prod- 
uct yield dramatically. Drs. Gary Gilliland and Kerry 
Blanchard and Dr. Bunn have devised a simple and 
inexpensive method utilizing competitive PCR for 
highly accurate quantitation of mRNA from a small 
number of cells. Serial dilutions of a competitor 
DNA fragment that differs from the cDNA of interest 
only by having either a small intron or a mutated 
internal restriction site are added to aliquots of the 
PCR reaction mix. Therefore the same primers are 
used to coamplify the unknown and the competi- 
tor. The ratio of products remains precisely con- 
stant through the amplification. The relative 
amount of each provides a precise measure of 
cDNA concentration. This technique has been used 
to quantitate mRNA levels of three inducible 
cytokines: interleukin-3 (IL-3) and granulocyte mac- 
rophage colony-stimulating factor (GM-CSF) in 
MLA-144 cells before and after induction with 
phorbol myristate acetate (PMA) and Epo in Hep3B 
cells before and after induction with hypoxia. Mes- 
senger RNA was undetectable in unstimulated cells 
by PCR. However, with appropriate induction, 
mRNA for each of these cytokines was detected and 
quantitated from as few as 100 cells. This strategy 
PUBLICATIONS 
can be applied to a variety of studies on gene ex- 
pression requiring accurate measurement of mRNA 
species in low abundance or in small numbers of 
cells isolated by fluorescent sorting or plucked 
from colonies grown in semisolid medium. More- 
over, competitive PCR should also be useful in anal- 
yses of DNA, such as measurement of gene dosage 
(both amplification and deletion) or quantitation of 
low-abundance DNA species, e.g., somatic cell mu- 
tations and integration of viral DNA, both of which 
may involve only a small minority of a cell popula- 
tion. 
lY Identification of a Gene Encoding a Retrovirus 
Receptor. 
Murine type C ecotropic retrovirus infection is in- 
itiated by virus envelope binding to a membrane 
protein expressed on mouse cells. Dr. Cunningham 
and his colleagues have identified a cDNA clone 
that encodes for this protein. Human EJ cells that 
express the cDNA acquire a millionfold increase in 
virus infectivity. The predicted 62 2 -amino acid se- 
quence of this putative receptor is extremely hydro- 
phobic; 14 potential membrane-spanning domains 
have been identified. A model of the structure of 
the protein in the membrane is different from other 
virus receptors, and a computer-based search of se- 
quence data banks has not identified another pro- 
tein with significant similarity. Therefore this pro- 
tein may function in a novel way to permit virus 
entry into the cell. The structural basis for virus in- 
teraction with the receptor is currently being inves- 
tigated by examination of chimeric receptors con- 
structed by exchanging small regions of the 
receptor cDNA with its human homologue (which 
is nonpermissive for infection). These experiments 
have identified a single domain that is responsible 
for the virus receptor function. Site-directed muta- 
tions within this domain are being prepared to 
identify the specific amino acid residues required 
for virus infectivity. 
Dr. Bunn is Professor of Medicine at Harvard 
Medical School and Director of Hematology Re- 
search at Brigham and Women's Hospital. 
Articles 
Albritton, L.M., Tseng, L., Scadden, D., and Cunningham, J. M. 1989. A putative murine ecotropic retrovirus 
receptor gene encodes a multiple membrane-spanning protein and confers susceptibility of virus infec- 
tion. Ce// 57:659-666. 
Continued 
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