Project: Research project

Project Details


We will continue investigations into the role of the mouse mammary
tumor virus (MMTV) long terminal repeat region (LTR) and LTR open
reading frame (ORF) products in tumorigenesis and in the life cycle
of MMTV. The phenomenon of tissue tropism by MMTV will be
addressed by studying the interaction of specific nuclear DNA
binding proteins with MMTV LTR DNA. This interaction will be
studied with the full length as well as the truncated forms of the
LTR found in amplified proviruses of certain leukemia cells. DNA-
protein binding will be tested with nuclear extracts from various
cell types, before and after treatment of the cells with
glucocorticoids and phorbol esters. Cloned MMTV LTR cDNAs will be
ligated into an inducible mammalian expression vector to study the
post-translational processing of the LTR proteins as well as the
effects of LTR ORF gene expression in various cell types. The
interaction of the LTR protein with the MMTV genome will be studied
using ORF translation product purified from a bacteriophage lambda
gt11 expression vector. The altered LTR of the amplified provirus
found in DBA/2 leukemia cells will be molecularly cloned and
sequenced in order to define the alteration in the LTR region. The
cloned LTR DNA will then be used for studying DNA binding protein
interactions. Enhancer activity of the altered LTR will be
measured by fusion into a plasmid containing the chloramphenicol
acetyl transferase gene (CAT), followed by transfection of the
construct into various cell types and measurement of CAT activity.
The CAT constructs will also be co-transfected with the LTR cDNA
expression vectors in order to test for possible effects of the ORF
translation product on transcription. MMTV antigen expressing
tumor cell Lines other than mammary tumor cells will be analyzed
for the presence of amplified MMTV proviruses. If amplified
proviruses are detected then restriction endonuclease analysis will
be performed in order to determine the presence of alterations in
the LTRs of the proviruses. Such alterations will then be studied
in more detail with the aim of understanding at a molecular level,
factors which determine tissue tropism.
Effective start/end date12/31/893/31/90


  • Genetics
  • Molecular Biology


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