GENETIC ANALYSIS OF GROWTH CONTROL 
Donna L. George, Ph.D., Assistant Investigator 
Cellular proliferation and differentiation are in- 
tricately regulated; both are controlled by the coor- 
dinated interactions of a large number of genes. 
Genetic events that lead to the altered or inappro- 
priate expression of any of a broadly based class of 
genes, included among the proto-oncogenes, can 
result in the perturbation of normal cell growth 
and development; such uncontrolled proliferation 
and concomitant loss of differentiative ability are 
hallmarks of transformation and tumorigenesis. Re- 
search in this laboratory has focused on the identi- 
fication and characterization of such growth-con- 
trol genes, as well as the molecular mechanisms 
governing their expression. 
L Amplified DNA Sequences in a Transformed 
Mouse Cell Line. 
A number of studies have demonstrated that the 
amplification and consequent overexpression of 
particular cellular genes play a direct role in the eti- 
ology of some tumors. This laboratory is currently 
characterizing the function and organization of am- 
plified DNA sequences located in stably maintained 
extrachromosomal double minute nuclear ele- 
ments (DMs) in a spontaneously transformed deriv- 
ative of mouse 3T3 cells (3T3-DM). 
No known oncogene is amplified in these cells. 
However, because of the stable presence of DMs in 
these cells in the absence of selective pressure, the 
amplified domain is expected to contain a pre- 
viously unrecognized gene with a role in cellular 
proliferation/growth control. A multifaceted ap- 
proach has been used for the analysis of the ampli- 
fied DNA, including differential screening of cDNA 
libraries, chromosome walking, cDNA and genomic 
cloning, and pulsed-field gel mapping of CpG is- 
land-associated transcription units. It would appear 
that three genes are amplified and overexpressed in 
these cells. To date there is no evidence for the 
presence or overexpression of other amplified 
genes. 
For each of these three amplified genes, a full- 
length genomic equivalent has been introduced 
into nontransformed recipient cells and amplified 
appropriately to mimic the state in 3T3-DM cells. 
Results obtained in repeat studies indicate that the 
overexpression of one of these genes (mdm-1) in 
Rat-2 cells is associated with a distinct morphologi- 
cal alteration that is characteristic of some trans- 
formed derivatives. Additional experiments, includ- 
ing cotransfection studies with c-myc and nude 
mouse injections, are in progress to confirm and 
extend these findings. Because of the potential role 
of this gene in a pathway of cellular grov^T:h control, 
it is of interest that immunofluorescence studies 
have provided evidence that at least one of the 
mdm-l -encoded proteins is nuclear localized, in a 
pattern representative of a nuclear matrix compo- 
nent; in addition, this gene has enhanced expres- 
sion developmentally during spermatogenesis. 
An intriguing observation has been made con- 
cerning another of the amplified genes in the 3T3- 
DM cells. A cDNA probe from this gene detects an 
mRNA species that is present in several normal tis- 
sues and nontransformed cell lines but is conspicu- 
ously absent in a number of tumorigenic cell lines. 
If these initial studies are substantiated, Dr. George 
and her colleagues will proceed with studies to 
address how this differential expression might be 
related to the transformed properties of these 
tumors. 
II. Promoter Region of the cKi-ras Proto-oncogene. 
Another area of investigation is aimed at elucidat- 
ing sequence elements mediating transcription of 
the cKi-ras proto-oncogene. Alterations in the 
structure or level of expression of this gene have 
been implicated in the etiology of a number of 
mammalian tumors. Enhanced expression of an al- 
tered cKi-ras gene has been shown to augment the 
metastatic potential of some primary tumor cells. 
Thus it is important to clarify the various factors 
that regulate the expression of this gene. 
Results obtained in transient expression assays 
allowed the identification of a 160 bp DNA frag- 
ment critical for promoter activity. Subsequently, 
gel mobility shift and DNase protection assays indi- 
cated that this region is composed of at least two 
sequence domains that bind nuclear proteins and 
most likely represent transcriptional regulatory ele- 
ments. One domain includes a binding site for the 
transcription factor Spl. Another domain, highly 
conserved among human and mouse cKi-ras genes, 
shows no sequence homology to binding sites for 
previously identified transcription factors and may 
represent recognition elements for as yet un- 
characterized DNA-binding proteins. Included in 
this latter domain is a homopurine-homopyrimi- 
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